DiMethyl-Proline DMSO -DiMethyl-SulfOxide DOSY -Diffusion Ordered SpectroscopY DPD -Direct Probability

Keywords: NMR, Metabolomics, PCA, Chirality, Ordering Media AP -DPD -Additive Potential -Direct Probability

In this Ph.D thesis NMR spectroscopy was applied in two fields of research: in the metabolic study of food matrices and natural extracts, and in the structural, orientational and conformational study of small flexible chiral molecules of pharmacological interest. The molecular profile of foods constitutes a "fingerprint" for the studied biosystem and can be used for its unique identification. In this context NMR spectroscopy has been used combining traditional pulse sequences to chemometrics tools with the aim of characterize the metabolic composition of foods and natural extracts and monitoring changes in the metabolic profile during industrial processing phases.

Since the biological activity of a food is a non trivial function of that of each individual metabolite and depends both on the configuration of the chiral centers, if it is a chiral species, and on the spatial arrangement of the atoms within the molecule, conformational studies and configurational discriminations of these bioactive flexible species must be performed in parallel with the characterization of the metabolome itself. In particular, NMR study in weakly ordering chiral media is carried out on pairs of enantiomers of pharmacological interest, by combining both traditional and more sophisticated pulse sequences to theoretical models for the data analysis. Titolo : Spettroscopia di Risonanaza Magnetica Nucleare (NMR) applicata allo studio di materiali bioattivi di origine naturale di interesse farmacologico e nutraceutico Parole Chiave : NMR, Metabolomica, PCA, Chiralità, Mezzi Orientanti Sommario : In questa tesi di dottorato la spettroscopia NMR è stata applicata in due ambiti di ricerca: nello studio metabolico di matrici alimentari ed estratti naturali, e nello studio strutturale, orientazionale e conformazionale di piccoli enantiomeri flessibili di interesse farmacologico.

Il profilo molecolare degli alimenti costituisce una "impronta digitale" per il biosistema studiato e può essere utilizzato per la sua identificazione univoca. In questo contesto è stata utilizzata la spettroscopia NMR combinando esperimenti tradizionali con strumenti chemiometrici con l'obiettivo di caratterizzare la composizione metabolica degli alimenti e degli estratti naturali e monitorare le variazioni del profilo metabolico durante le fasi di lavorazione industriale.

Poiché l'attività biologica di un alimento è funzione di quella di ogni singolo metabolita, e dipende sia dalla configurazione dei centri chirali, se si tratta di una specie chirale, sia dalla disposizione spaziale degli atomi all'interno della molecola, studi conformazionali e discriminazioni configurazionali di queste specie flessibili bioattive devono essere eseguiti parallelamente alla caratterizzazione del metaboloma stesso. In particolare, lo studio NMR in mezzi chirali debolmente orientanti è stato condotto su coppie di enantiomeri di interesse farmacologico, combinando sequenze di impulsi sia tradizionali che più sofisticate a modelli teorici per l'analisi dei dati.

unique identification. In this context NMR spectroscopy has been used combining traditional pulse sequences to chemometrics tools aiming to characterize the metabolic composition of foods and natural extracts and monitoring changes in the metabolic profile during industrial processing phases. In the first study presented the methodology is applied to the characterization of the metabolic profile of Calabrian orange and clementine juices. The main goal of this study has been to evaluate the possibility to follow any metabolic variations that the fresh juice undergoes during the industrial thermal process in order to obtain concentrated juices and evaluate the possibility to use this technique as a quality control tool. The same methodology is then adopted for the characterization of Calabrian apple juices coming from different cultivars in order to evaluate if it is possible to distinguish the different varieties of fruits based on their proton spectrum. The third study carried out in the metabolomic field has been the characterization of Cannabis Sativa extracts (hemp seeds oil and inflorescences extract) and the evaluation of the differences within the metabolomic profiles obtained with different extraction technique. The strategy is subsequently extended to the characterization and quantification of the main metabolites of bergamot essential oil, and to the investigation of the possibility of discriminating fraud and sophistications to which this high value extract can be subjected. The latest study in this field is the application of HR-MAS NMR (High Resolution -Magic Angle Spinning Nuclear Magnetic Resonance spectroscopy) in the characterization of the organic fraction of municipal solid waste and in the monitoring of the variations in terms of composition of the organic materials subjected to oxidation treatments. The attention was then shifted to the exploration of the conformational behaviour in solution of chiral bioactive molecules. Indeed, since the biological activity of a given metabolite depends both on the configuration of the stereogenic centers, if it is a chiral species, and on the spatial arrangement of the atoms within the molecule, conformational studies and configurational discriminations of these bioactive flexible species must be performed in parallel with the characterization of the metabolome itself. In particular, NMR study in weakly ordering chiral media was applied to a pair of enantiomers characterized by the presence of only one torsional angle and then extended to a more complex species with anti-inflammatory activity. The species (RS)-4-fluoro-αmethylbenzyl alcohol ((RS)-4FMBOH) is studied as a simple example of chiral molecule in II order to explore the molecular conformational equilibria and let emerge differences between the torsional distribution curves and orientational order parameters associated to each enantiomer. Since chirality become a crucial problem when dealing with bioactive species, the second study presented is the case of the thiaprofenic acid. Thiaprofenic acid is a more complex species respect to 4FMBOH, also chiral, and only one enantiomer is active as anti-inflammatory drug. The main goal of this study was to understand if the adopted methodology could furnish conformational information in solution of complex flexible chiral bioactive species hence allowing to increase the knowledge in the understanding of the mechanism of action of the drugs and the different biological activity of the enantiomers.

III

Sommario

In questo progetto di dottorato la spettroscopia NMR è stata applicata in due ambiti di ricerca: nello studio metabolico di matrici alimentari e nello studio strutturale, orientazionale e conformazionale di piccoli enantiomeri flessibili di interesse farmacologico. In campo metabolomico, il profilo molecolare degli alimenti costituisce una "impronta digitale" per il biosistema studiato e può essere utilizzato per la sua identificazione univoca. E' in questo contesto che è stata utilizzata la spettroscopia NMR, combinando i dati sperimentali con strumenti chemiometrici, con il duplice obiettivo di caratterizzare la composizione metabolica di alimenti ed estratti naturali e di monitorare le variazioni del profilo metabolico durante le fasi di lavorazione industriale. Nel primo studio presentato la metodologia è stata applicata alla caratterizzazione del profilo metabolico dei succhi di arancia e clementina Calabresi.

L'obiettivo principale di questo studio è stato quello di valutare la possibilità di seguire eventuali variazioni metaboliche che il succo fresco subisce durante il processo termico industriale che porta all'ottenimento di succhi concentrati e valutare la possibilità di utilizzare questo approccio come strumento per un controllo di qualità. La stessa metodologia è stata poi adottata per la caratterizzazione dei succhi di mela Calabrese provenienti da diverse cultivar con l'obiettivo di valutare se è possibile distinguere le diverse varietà di frutti in base al loro spettro protonico. Il terzo studio svolto in ambito metabolomico è stato la caratterizzazione degli estratti di Cannabis Sativa (estratti di olio di semi e infiorescenze). Questo studio ha permesso di valutare le differenze nei profili metabolici degli estratti ottenuti con differenti tecniche di estrazione. La strategia è stata successivamente estesa alla caratterizzazione e alla quantificazione dei principali metaboliti presenti dell'olio essenziale di bergamotto e all'esplorazione della possibilità di discriminare frodi e sofisticazioni a cui questo estratto molto pregiato può essere sottoposto. L'ultimo studio eseguito in questo ambito di ricerca ha previsto l'applicazione della spettroscopia HR-MAS NMR (High Resolution -Magic Angle Spinning Nuclear Magnetic Resonance spectroscopy) alla caratterizzazione della frazione organica dei rifiuti solidi urbani e al monitoraggio delle variazioni in termini di composizione dei materiali organici sottoposti a trattamenti ossidativi. L'attenzione è stata poi spostata verso lo studio del comportamento conformazionale in soluzione di molecole bioattive chirali. Infatti, poiché l'attività biologica di un dato metabolita dipende sia dalla configurazione del centro chirale, se si tratta di una specie chirale, sia dalla disposizione spaziale degli atomi all'interno della molecola, studi conformazionali e discriminazioni configurazionali di queste specie flessibili bioattive devono essere eseguiti parallelamente alla caratterizzazione del metaboloma stesso.

IV

In particolare, lo studio NMR in mezzi chirali debolmente orientanti è stato applicato inizialmente a una coppia di enantiomeri caratterizzati dalla presenza di un solo angolo torsionale e poi estesa ad una specie più complessa con attività anti-infiammatoria. La specie (RS)-4-fluoro-α-metilbenzil alcool ((RS)-4FMBOH) è stata studiata come esempio semplice di molecola chirale per esplorare gli equilibri conformazionali molecolari e far emergere differenze nelle curve di distribuzione torsionale e nei parametri d'ordine orientazionale dei due enantiomeri. Poiché la chiralità diventa un problema cruciale quando si tratta di specie bioattive, il secondo studio affrontato riguarda l'acido tiaprofenico, una specie più complessa di cui un solo enantiomero è attivo come farmaco antinfiammatorio. L'obiettivo principale di tale studio è stato capire se la metodologia adottata potesse fornire informazioni tali da accrescere la comprensione del meccanismo d'azione dei farmaci e della diversa attività biologica degli enantiomeri.

V

Resumé

Dans cette thèse de doctorat, la spectroscopie RMN est appliquée dans deux domaines de recherche: dans l'étude métabolique des matrices alimentaires, et dans l'étude structurale, orientationnelle et conformationnelle de petits molecules chirales flexibles d'intérêt pharmacologique. Dans le domaine métabolomique, le profil moléculaire des aliments constitue une «empreinte» du biosystème étudié et peut être utilisé pour son identification. Dans ce contexte, la spectroscopie RMN a été utilisée en combinant des séquences d'impulsions traditionnelles à des outils de chimiométrie dans le but de caractériser la composition métabolique d'aliments et d'extraits naturels, et de suivre l'évolution du leur profil métabolique lors de phases de traitement industriel. La première étude présentée est la caractérisation du profil métabolique de jus d'orange et de clémentine de Calabre. L'objectif principal de cette étude est d'évaluer la possibilité de suivre les variations métaboliques subies par le jus frais au cours du processus thermique industriel afin d'obtenir des jus concentrés et d'évaluer la possibilité d'utiliser cette technique comme outil de contrôle de la qualité. La même méthodologie a ensuite été adoptée pour la caractérisation de jus de pomme de Calabre provenant de différents cultivateurs afin d'évaluer la possibilié de distinguer les différentes variétés de fruits du jus en fonction de leur spectre RMN proton. La troisième étude réalisée dans le domaine métabolomique est la caractérisation des extraits de Cannabis Sativa (huile de graines de chanvre et extraits d'inflorescences) et l'évaluation des différences dans les profils métabolomiques obtenus après différentes techniques d'extraction. La quatrième étude présentée dans ce cadre est la caractérisation et la quantification des principaux métabolites de l'huile essentielle de bergamote, et l'exploration de la possibilité de discrimination de fraudes et adulterations auxquelles cet extrait de grande valeur peut être soumis. Enfin la dernière étude est l'application de la RMN HR-MAS (High Resolution -Magic Angle Spinning Nuclear Magnetic Resonance spectroscopy) dans la caractérisation de la fraction organique de déchets solides municipaux et dans le suivi de leurs variations en termes de composition des matières organiques lors qu'ils sont soumis à traitements d'oxydation. L'attention s'est ensuite portée sur l'étude conformationnelle d'espèces flexibles en solution. En effet, puisque l'activité biologique d'un aliment est une fonction non triviale de celle de chaque métabolite individuel, et qu'elle dépend à la fois de la configuration des centres stéréogène, s'il s'agit d'une espèce chirale, et de la disposition spatiale des atomes au sein de molécule, des études conformationnelles et configurationnelles de ces espèces flexibles bioactives doivent être réalisées en parallèle de la caractérisation du métabolome lui-même. En particulier, l'étude RMN dans des milieux chiraux faiblement orientés est appliquée à une paire d'énantiomères VI caractérisé structurellement par la présence d'un seul angle de torsion. L'espèce (RS)-4-fluoroα-méthylbenzyl alcool ((RS)-4FMBOH) a été étudiée comme un exemple simple de molécule chirale afin d'explorer les équilibres conformationnels moléculaires et de faire émerger des différences dans les courbes de distribution de torsion et l'ordre d'orientation paramètres des deux énantiomères. La chiralité constitue un problème crucial lorsqu'il s'agit d'espèces bioactives; c'est pourquoi la deuxième étude traite le cas de l'acide thiaprofénique, une espèce chirale complexe du point de vue conformationnel dont un seul énantiomère est actif comme médicament anti-inflammatoire. L'objectif principal de l'étude présentée est de comprendre si la méthodologie adoptée peut fournir des informations permettant d'accroître les connaissances dans la compréhension du mécanisme d'action des médicaments et des différentes activités biologiques des énantiomères. codification (c). If one spin moves from a position Z1 to Z2 during the spatial encoding, as shown by the circle and dashed line in blue, the spin generates a phase memory. This leads to incomplete refocusing after the application of the second gradient field pulse (d) and diffusion information can be obtained.

1.10

Spin-Echo (SE) pulse sequence (zggpse Bruker standard pulses sequence).

2.38

Histograms representing the quantitative results obtained for each metabolite in the different samples: pure BEO and adulterated ones.

2.39

Principal Component Analysis (PCA) of pure BEO (100BEO) and adulterated BEO with 5, 10 and 30% of OEO (95BEO:5OEO, 90BEO:10OEO and 70BEO:30OEO respectively). The score plot shows the three PCs: PC1, PC2 and PC3, with their respective variation. R2X(PC1) = 39.9%, R2X(PC2) = 23.5% and R2X(PC3) = 12.3%.

2.40

Biplot of PCA carried out on NMR spectra of pure and adulterated BEO samples showing how variables affected the two PCs: PC2 and PC3.

2.41

Experimental steps, oxidative reaction conditions, and NMR samples collected for each process the initial sample of the organic fraction of municipal solid waste (OFMSW) was subjected to.

2.42

HR-MAS 13 C-{ 1 H} NMR spectrum of Sample 0 (in blue) compared with CP-MAS 13 C NMR spectrum of OFMSW sample reported in the literature (in black) (Pichler,et al,200).

2.43

HR-MAS 13 C NMR spectrum of Sample 0 and signal assignment. In the box, the comparison with the CP-MAS spectrum of starch in the same region is shown (Tang & Hills, 2003).

2.44

Relative distribution in the assigned chemical shift regions of the four group of carbons (aliphatic, heteroatoms-linked, unsaturated and carbonylic) for the Samples 0, 1, 2 and 3.

2.45

Relative distribution in the assigned chemical shift regions of the four group of carbon (aliphatic, heteroatoms-linked, unsaturated and carbonylic) for each samples of process A (Sample 4_3h, Sample 4_6h, Sample 4_9h and Sample 4_12h) and comparison with the corresponding values of Sample 0.

2.46

Hhistogram representing the values of integrals, and their errors, of the four kind of carbons (aliphatic, heteroatoms-linked, unsaturated and carbonylic) for each samples of process B (Sample 5_3h, Sample 5_6h, Sample 6_8h, Sample 5_9h, Sample5_12h ) and comparison with the corresponding values Sample 0 and Sample 3.

3.1

In figure are shown the signals relating to the interactions between a pair of nuclei i and j in the three phases: A) achiral isotropic phase, where only the indirect coupling and the isotropic chemical shifts are present, B) achiral anisotropic phase, which allows us to observe the anisotropic chemical shift interactions and direct coupling, in addition to the interactions of isotropic origin, and C) the chiral anisotropic phase in which the anisotropic interactions that appear to be different for the two enantiomers are visible.

3.2

Structure, atomic labelling and torsional angle of (RS)-4FMBOH.

3.3

1D 1 H spectra recorded at 298 K for the samples R-aniso (in red) and S-aniso (in blue). In the enlargement the signals of the methyl group (H8) of the two enantiomers are reported.

3.4

2D SERF spectrum recorded at 298 on S-4FMBOHaniso sample where the offset of the selective pulse was set at 8. The total coupling between the three equivalent protons of the methyl group is easily read on the 2D map by measuring the distance between the triplet spots as shown by the red sign.

3.5

2D 1 H-1 H J-resolved spectra recorded at 298 K on S-4FMBOHaniso sample. Enlarged maps show projections corresponding to the columns centered on the chemical shifts of H7, H2, H3.

3.6

2D 13 C-1 H HetSERF spectrum of R-4FMBOHaniso sample. The offset of the selective pulse was set at H2 on the proton channel. Enlarged map shows C3 and C5 carbon signals.

3.7

Comparison of experimental and calculated RDCs for both the enantiomers R-4FMBOH in orange and S-4FMBOH in blue.

3.8

Comparison of experimental (solid line) and calculated (dot line) probability distribution function (𝑃 𝑖𝑠𝑜 ({𝜙 }) and 𝑃 𝑡ℎ𝑒𝑜 ({𝜙 }) obtained for both the enantiomers R-4FMBOH (in orange) and S-4FMBOH (in blue).

3.9

Comparison of experimental (solid line) and calculated (dot line) probability distribution function (𝑃 𝑖𝑠𝑜 ({𝜙 }) and 𝑃 𝑡ℎ𝑒𝑜 ({𝜙 }) obtained for both the enantiomers R-4FMBOH (in orange) and S-4FMBOH (in blue).

3.10

The 𝜙-dependent behavior of order parameters of (R) and (S) 4FMBOH.

3.11

Structure, atomic labelling and torsional angle of (RS)-TA.

3.12

1D 13 C spectrum recorded at 298 K for the sample RS-TAiso. In the red box is reported the enlargement of the C3 signal.

3.13

2D SERF spectrum recorded at 298 on RS-TAaniso sample where the offset of the excitation selective pulse was set at 2 and the offsets of the refocusing selective pulse at 2 and 3. The total coupling between the selected protons is easily read by extracting the column and by measuring the distance between the line of the resulting two quadruplets as shown in the enlargement in the red box.

3.14

2D 13 C-1 H HetSERF spectrum of RS-TAaniso sample. The offset of the selective pulse was set at 2 on the proton channel. Enlarged map shows the spectrum obtained by extrapolating the column corresponding to the C5 signal and the TC5-H2 can be easily measured.

3.15

Comparison of experimental (solid line) and calculated (dot line) probability distribution function (𝑃 𝑖𝑠𝑜 ({𝜙 }) and 𝑃 𝑡ℎ𝑒𝑜 ({𝜙 }) obtained for both the enantiomers R-4FMBOH (in orange) and S-4FMBOH (in blue).

3.16

Comparison of experimental probability distribution function (𝑃 𝑖𝑠𝑜 ({𝜙 }) obtained for the S-TA (in red) and S-Ketoprofene (in green).

A1

1D 1 H NMR spectrum (Bruker pulse sequence: zg) recorded on -pinene in CDCl3 (9.4 T).

A2

13 C-{ 1 H} NMR spectrum (Bruker pulse sequence: zgdc) recorded on -pinene in CDCl3 (11,7 T).

A3

1D 1 H NMR spectrum (Bruker pulse sequence: zg) recorded on -terpinene in CDCl3 (9.4 T).

3.2

Experimental dipolar couplings 𝐷 𝑖𝑗 𝑜𝑏𝑠 determined by the NMR analysis of R and S 4FMBOH dissolved in PBLG/CDCl3 (the estimated experimental error affecting the data is ±0.5) and calculated dipolar couplings 𝐷 𝑖𝑗 𝑐𝑎𝑙𝑐 determined by AP-DPD approach (the estimated experimental error affecting the data is ±0.2), are reported in the table together with all the output parameter obtained by performing the conformational study (i.e. potential, structural and orientational parameters).

3.3

Details about the preparation of the different samples of TA studied.

3.4

Experimental dipolar couplings 𝐷 𝑖𝑗 𝑜𝑏𝑠 determined by the NMR analysis of R-TA and S-TA dissolved in PBLG/CDCl3 (the estimated experimental error affecting the data is ±0.5) and calculated dipolar couplings 𝐷 𝑖𝑗 𝑐𝑎𝑙𝑐 determined by AP-DPD approach (the estimated experimental error affecting the data is ±0.2), are reported in the table together with all the output parameter obtained by performing the conformational study (i.e. potential, structural and orientational parameters).

Introduction Générale

Chapter 1. Nuclear Magnetic Resonance spectroscopy (NMR):

theory and applications

General Conclusions

Conclusioni Generali

Conclusions Générale

Appendix I

List of publications

List of poster

List of oral communications

General introduction

Over the years, NMR spectroscopy has earned a prominent position in different sectors of science and technology and it is today an incomparable tool in the study of a large number of chemical, physical and biological systems. In this context, an important part is represented by the application of NMR spectroscopy for the study of complex matrices such as foods and food extracts. Of great interest is the use of this technique also for the study of nutraceutical compounds, substances which, in addition to having a simple nutritional value, have positive effects on the well-being and health of people. Specifically, on one side, it is of fundamental interest to identify nutraceutical species and recognize their presence within a given food; on the other side, it is very important to investigate the spatial arrangement that the biologically active molecules adopt in solution in order to establish the relationships between configuration/conformation and bioactivity. In the context of food sciences, the characterization of foods is a very challenging topic. To pursue this objective from the literature, the most suitable approach appears to be metabolomics (Kell & Oliver, 2016), a scientific discipline capable of identifying and quantifying the different metabolites that determine and characterize the bio-system studied. Metabolomics is based on the global identification of a large number of metabolites present in a complex matrix such as a food and natural extract. The metabolic profile obtained constitutes a "fingerprint" for the studied biosystem and can be used for its unique identification. Indeed, the identification of metabolites or metabolite patterns is able to diversify a given food and discriminate its geographical, cultivar or processing characteristics, compared to others. The most common techniques employed for determining the metabolic profile of food products are Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy (NMR) assisted by multivariate statistical analysis tools. (Ebrahimi et al, 2017;Keun, 2018).

As stated above, in this Ph.D thesis the attention is focused on the use of NMR spectroscopy.

Today, the NMR technique is an important tool in a wide range of metabolic applications ranging, for example, from anti-sophistication controls to food quality analyses or to the determination of geographical origin, factors that are difficult to certify with other techniques and always most requested (Karabagias et al, 2018).

On the other hand, the biological activity of a food is due to its metabolic profile and, in particular, its overall bio-activity is a non trivial function of that of each individual metabolite.

The activity of a given metabolite depends both on the configuration of the chiral centers, if it is a chiral species, and on the spatial arrangement of the atoms within the molecule. This arrangement may or may not facilitate the establishment of a series of intramolecular interactions which determine the conformations that the molecule can assume in the reaction environment or within biological systems. This is due to the fact that, referring to the "keylock" model, a certain enzyme is able to distinguish both a pair of enantiomers and the different conformations that a molecule can assume, therefore each thermally accessible conformer plays an important role in molecular recognition (Boehr et al, 2019). From this it can be deduced how the conformational equilibria of bioactive species play a fundamental role in carrying out certain biological activity and knowing these equilibria is essential, first of all to understand the mechanism of action of the studied species, then for the rational design of analogous compounds capable of causing the same biological response. The techniques to be used to perform a structural and conformational analysis of species in solution are very few and, in this context, Nuclear Magnetic Resonance spectroscopy represents a powerful tool. In particular, the progress in NMR methods, based on the development of sophisticated pulse sequences, allows the study of such compounds in solution both in the isotropic and anisotropic solvents.

Of particular importance is the use of partially ordered phases in which the target molecule is dissolved and is affected by the ordering action of the phase by appropriately orienting itself with respect to the magnetic field of NMR (Canet, 1996;Burnell & de Lange, 2003). The anisotropy guaranteed, for example, by a liquid crystalline phase, allows to obtain information relating to interactions whose existence is strictly connected to the spatial arrangement of the observed nuclei. In fact, the NMR spectra of samples in these phases are extremely rich in information as they are governed by interactions not only of an isotropic but also anisotropic nature. In this context, the residual dipolar couplings (RDCs) which depend on the distances between magnetically active nuclei and on the orientations of the internuclear vectors with respect to the external magnetic field, assume particular importance and, for this reason, represent a probe sensitive to long-range interactions between portions of the molecule also spatially distant from each other. In the case of flexible molecules, since the internal torsional motions interconvert the possible conformers faster than those of the NMR experiment, it is necessary to resort to theoretical models in order to correctly interpret and convert the RDCs and to have access to structures, orientations and conformations of the species under examination (Di Pietro et al, 2018).

In this context this Ph.D project take place. Indeed, the overall goal of this Ph.D is to explore these fields of application of NMR spectroscopy, combining both traditional and more sophisticated pulse sequences to chemometrics tools and theoretical models for the data analysis. Specifically, the NMR methodologies have been applied in the metabolic study of food matrices, with the aim of using them in the traceability of a food product and in monitoring the metabolic profile during the processing and storage phases to evaluate any transformations to which the food can be subjected, in the characterization of the metabolic composition of natural extracts and for the structural, orientational and conformational study of small flexible enantiomers of pharmacological interest.

The first chapter of this thesis describes the project, its main objectives and it deals with the theoretical aspects of the faced topics. Indeed, the chapter starts with a brief overview of the NMR spectroscopy basic principles then it continues by giving a general description of metabolomics science. The different scientific approaches available in metabolomics are presented together with the main analytical platforms employed in this kind of studies focusing the attention on the NMR spectroscopy pulse sequences. Then the most common pre-processing and processing tools needed to perform the data analysis are presented in addition to a brief introduction to the chemometric tool adopted, i.e. the Principal Component Analysis (PCA).

The first chapter ends with the presentation of the NMR methodology used to obtain conformational and configurational information on a flexible solute dissolved in partially ordered chiral media by using the experimental residual dipolar couplings. Particularly, an overview of the basic tools of NMR in partially ordered media is given by focusing on the lyotropic-like liquid crystalline phase used in this project (PBLG/CDCl3). Then the pulse sequences able to furnish the needed RDCs are presented together with the theoretical model adopted that allow to obtain the structural, orientational and conformational information from the experimental observables.

The second chapter focuses on the NMR-based metabolomics application investigated in this Ph.D, in particular it starts with the characterization of the metabolic profile of Calabrian orange and clementine juices. The main goal of this study is to evaluate the possibility to follow any metabolic variations that the fresh juice undergoes during the industrial thermal process in order to obtain concentrated juices and evaluate the possibility to use this technique as a quality control tool. The second example presented is the application of NMR in the characterization of the metabolic profile of Calabrian apple juices coming from different cultivars in order to evaluate if it is possible to distinguish the different variety of fruits on the basis of their protonic spectrum. The chapter continues with a preliminary study carried out on Cannabis Sativa In particular, the technique is used as an innovative and non-destructive method able to characterize the chemical composition of the organic fraction of municipal solid waste and verify if it represents a good tool in the monitoring of the variations in terms of composition of the organic materials subjected to oxidation treatments.

In the third chapter the attention is shifted to the conformational study of flexible species. In particular, since the interaction of each bioactive molecule with endogenous ligands and receptors is dependent on the configuration and conformations of the flexible species involved, in this section the attention focused on the possibility of distinguishing a pair of enantiomers on the basis of their experimental conformational probability curves. The NMR study in weakly ordering chiral media was then applied to a pair of enantiomers characterized by the presence of only one torsional angle. The species (RS)-4-fluoro-α-methylbenzyl alcohol ((RS)-4FMBOH) was studied as a simple example of chiral molecule in order to explore the molecular conformational equilibria and let emerge differences in the torsional distribution curves and orientational order parameters of the two enantiomers. Since chirality become a crucial problem when dealing with bioactive species, the second study presented in this last chapter is the case of the tiaprofenic acid. Tiaprofenic acid is a more complex species of which only one enantiomer is active as anti-inflammatory drug. The main goal of the study presented is to understand if the adopted methodology can furnish conformational information in solution of complex flexible chiral bioactive species hence allowing to increase the knowledge in the understanding of the mechanism of action of the drugs and the different biological activity of the enantiomers.

Introduzione generale

Nel corso degli anni la spettroscopia NMR si è guadagnata una posizione di rilievo in ogni settore delle scienze e della tecnologia e costituisce oggi uno strumento ineguagliabile nello studio di un gran numero di sistemi chimici, fisici e biologici. (Ebrahimi et al, 2017;Keun, 2018). Come detto precedentemente, in questa tesi di dottorato l'attenzione è stata focalizzata sull'uso della spettroscopia NMR. Oggi la tecnica NMR è uno strumento importante in un'ampia gamma di applicazioni metaboliche che vanno, ad esempio, dai controlli anti-sofisticazione, ad analisi di qualità degli alimenti o alla determinazione di provenienza, fattori difficilmente certificabili con altre tecniche e sempre più richiesti (Karabagias et al, 2018).

Un alimento possiede una determinata attività biologica che è funzione del suo profilo metabolico e, in particolare, la sua bio-attività complessiva è funzione di quella di ogni singolo metabolita. L'attività di un dato metabolita dipende sia dalla configurazione dei centri chirali, qualora essi siano presenti all'interno della sua struttura, sia dalla disposizione spaziale degli atomi all'interno della molecola. Tale disposizione può favorire o meno l'istaurarsi di una serie di interazioni intramolecolari che determinano le conformazioni che la molecola può assumere nell'ambiente di reazione ovvero all'interno dei sistemi biologici. Ciò si deve al fatto che, facendo riferimento al modello "chiave-serratura", un determinato enzima è in grado di distinguere sia una coppia di enantiomeri sia le diverse conformazioni che una molecola può assumere; pertanto, ogni conformero termicamente accessibile gioca un ruolo importante nella ricognizione molecolare (Boehr et al, 2019). Da ciò si deduce come gli equilibri conformazionali delle specie bioattive abbiano un ruolo fondamentale nello svolgere determinate azioni biologiche e la conoscenza di tali equilibri è indispensabile in primo luogo per comprendere il meccanismo d'azione delle specie studiate, in secondo luogo per la progettazione razionale di composti analoghi in grado di scatenare la medesima risposta biologica. Le tecniche a cui far ricorso per eseguire un'analisi strutturale e conformazionale di specie in soluzione sono ben poche e in questo contesto la spettroscopia di risonanza magnetica nucleare rappresenta un potente mezzo di cui servirsi. In particolare il progresso nelle metodiche NMR, basato sullo sviluppo di sequenze di impulsi sofisticate, permette lo studio di tali composti in soluzione sia in fase isotropa che anisotropa. Di particolare rilevanza è il ricorso alle fasi parzialmente ordinate nelle quali la molecola target può essere disciolta e risentire dell'azione ordinante della fase orientandosi opportunamente rispetto al campo magnetico proprio dell'NMR (Canet, 1996;Burnell & de Lange, 2003). L'anisotropia garantita, ad esempio, da una fase liquido cristallina o da una fase gel, permette di ottenere informazioni relative ad interazioni la cui esistenza è strettamente connessa alla disposizione spaziale dei nuclei osservati. Infatti, gli spettri NMR di campioni in tali fasi, sono estremamente ricchi di informazioni in quanto governati dalle interazioni non solo di natura isotropa ma anche anisotropa. In questo contesto assumono particolare rilevanza le "residual dipolar couplings" (RDCs) le quali dipendono dalle distanze tra nuclei magneticamente attivi e dalle orientazioni dei vettori internucleari rispetto al campo magnetico esterno; per tale ragione, esse rappresentano un "probe" sensibile ad interazioni a lungo raggio di porzioni della molecola anche spazialmente distanti tra loro. Nel caso delle molecole flessibili, poiché i moti interni torsionali interconvertono i possibili conformeri in tempi più rapidi rispetto a quelli dell'esperimento NMR, risulta essere necessario ricorrere a modelli teorici in modo da interpretare correttamente le RDCs ed aver accesso ad aspetti strutturali, orientazionali e conformazionali delle specie in esame (Di Pietro et al, 2018).

È in tale contesto che il presente progetto di dottorato si sviluppa. Nello specifico, l'obiettivo generale della ricerca condotta è stato l'applicazione della spettroscopia NMR nei campi di ricerca appena descritti combinando l'utilizzo di sequenze di impulsi sia tradizionali che più sofisticate a strumenti chemiometrici e modelli teorici per l'analisi dei dati. In particolare, la spettroscopia NMR è stata applicata allo studio metabolico di matrici alimentari con l'obiettivo di utilizzare le informazioni ottenute nella caratterizzazione e nella tracciabilità di un prodotto alimentare, nel monitorare il profilo metabolico durante le fasi di lavorazione e per lo studio strutturale, orientazionale e conformazionale di piccoli enantiomeri flessibili di interesse farmacologico.

Nel primo capitolo di questa tesi è riportata una descrizione del progetto, i suoi obiettivi principali e illustrati gli aspetti teorici relativi agli argomenti affrontati. Nello specifico, il capitolo inizia con una breve panoramica dei principi di base della spettroscopia NMR quindi prosegue fornendo una descrizione generale della metabolomica. Sono poi presentati i diversi approcci scientifici comunemente adottati in metabolomica insieme alle principali piattaforme analitiche impiegate in questo tipo di studi focalizzando l'attenzione sulla spettroscopia NMR e sulle sequenze di impulsi comunemente utilizzate. Vengono poi presentati gli strumenti di processing spettrale e di elaborazione dei dati oltre ad una breve introduzione alla tecnica chemiometrica utilizzata in questa tesi, ovvero l'analisi delle componenti principali (PCA). Il primo capitolo si conclude con la presentazione della metodologia NMR utilizzata per ottenere informazioni conformazionali e configurazionali relative a enantiomeri flessibili disciolti in mezzi chirali parzialmente ordinati utilizzando gli accoppiamenti dipolari sperimentali (RDC).

In particolare, viene fornita una panoramica delle metodiche NMR in mezzi parzialmente orientanti concentrandosi sulla fase cristallina liquida liotropica utilizzata in questo progetto (Ebrahimi et al, 2017 ;Keun, 2018). En se référant au profil métabolique de l'aliment étudié, il est possible d'extrapoler des données pertinentes afin de réaliser, par exemple, un contrôle anti-adulteration, une analyse de la qualité de l'aliment ou une détermination de l'origine, facteurs difficilement contrôlables par d'autres techniques et de plus en plus exigées par les contrôles qualité (Karabagias et al, 2018).

(PBLG/CDCl3
Un aliment a une certaine activité biologique fonction de son profil métabolique et, en Chapter 1.

Nuclear Magnetic Resonance spectroscopy (NMR):

theory and applications

Introduction

Nuclear Magnetic Resonance (NMR) spectroscopy is one of the most important techniques in modern chemistry thanks to its many applications in different fields of research [START_REF] Williams | Modern NMR Approaches to the Structure Elucidation of Natural Products[END_REF]Keun, 2018;Bell & Pines, 2019;[START_REF] Cobas | NMR Signal Processing, Prediction, and Structure Verification with Machine Learning Techniques[END_REF][START_REF] Liu | Application of Low-Field NMR to the Pore Structure of Concrete[END_REF][START_REF] Luchinat | Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency[END_REF][START_REF] Colnago | Low Field, Time Domain NMR in the Agriculture and Agrifood Sectors: An Overview of Applications in Plants, Foods and Biofuels[END_REF][START_REF] Li | Recent Advances in the Application of Metabolomics for Food Safety Control and Food Quality Analyses[END_REF][START_REF] Nagel | Application of 1 H proton NMR Relaxometry to Building Materials -A Review[END_REF]. Beginning in 1950s when the first ethanol spectrum was published, a series of theoretical discoveries and technical improvements led to the enhancement of our ability to fully explore the nature of molecular structure [START_REF] Arnold | Induction Signals from Organic Compounds[END_REF][START_REF] Morris | Modern NMR Techniques for Structure Elucidation[END_REF][START_REF] Williams | Modern NMR Approaches to the Structure Elucidation of Natural Products[END_REF]. It did not take long for NMR to be applied to increasingly complex systems and this made it gaining ground over the years in different discipline such as biology, pharmacology, chemistry, medicine or food sciences [START_REF] Hatzakis | Nuclear Magnetic Resonance (NMR) Spectroscopy in Food Science: A Comprehensive Review[END_REF][START_REF] Crook | Quantitative NMR-Based Biomedical Metabolomics: Current Status and Applications[END_REF][START_REF] Fuertes-Martin | Human Serum/Plasma Glycoprotein Analysis by 1 H-NMR, an Emerging Method of Inflammatory Assessment[END_REF]. This fast development originated from the fact that NMR spectroscopy is a versatile technique that offers a wide range of informations that, in some situations, no other techniques are able to provide. For example in biology, NMR represents the only method for the analysis of biomacromolecules in aqueous solution. In particular, it allows to perform dynamics studies and permits to determine hydrogen bonding, hydratation state, weak molecular interactions and ionization of the biological species under physiological conditions [START_REF] Wüthrich | NMR Studies of Structure and Function of Biological Macromolecules (Nobel Lecture)[END_REF]Palmer III, 2004;[START_REF] Foster | Solution NMR of Large Molecules and Assemblie[END_REF][START_REF] Gossert | NMR in Drug discovery: A Practical Guide to Identification and Validation of Ligands Interacting with Biological Macromolecules[END_REF]. These constitute not an exhaustive list of the abilities of this spectroscopic technique presents: as a non-destructive, multinuclear, multiparametric and non-invasive, NMR represents one of the most sophisticated and powerful method in the chemical-physical analysis of substances whether synthetic or natural origin, ranging from simplest molecules (aminoacids, sugars, organic acids) to the more complex systems (polymers, liquid crystals, foods, natural extracts, biological fluids and tissues) [START_REF] Bubb | NMR Spectroscopy in the Study of Carbohydrates: Characterizing the Structural Complexity[END_REF][START_REF] Di Pietro | A Combined LX-NMR and Molecular Dynamics Investigation of the Bulk and Local Structure of Ionic Liquid Crystals[END_REF][START_REF] Gramatyka | Metabolic Changes in Mice Cardiac Tissue After Low-Dose Irradiation Revealed by 1 H NMR Spectroscopy[END_REF]. Among the various fields of application, in this Ph.D. thesis topics concerning two research fields, in which the NMR technique is now recognized as essential in obtaining experimental informations, will be addressed: the NMR-based metabolomic study of natural extracts and the structural and conformational analysis of small chiral flexible molecules of natural origin and/or of pharmacological interest.

Above all, NMR spectroscopy has demonstrated over the last years a unique potential in the analysis of complex mixtures and, within this framework, metabolomics born as a new approach in the metabolites determination [START_REF] Idle | Metabolomics[END_REF]Keun, 2018;[START_REF] Emwas | NMR Spectroscopy for Metabolomics Research[END_REF] . The use of NMR in the study of these small endogenous molecules produced by an organism, has been emerging both in the research field and as quality control tool [START_REF] Wishart | Metabolomics: Application to Food Science and Nutrition Research[END_REF][START_REF] Farag | Comparative Mass Spectometry and Nuclear Magnetic Resonance Metabolomic Approaches for Nutraceuticals Quality Contro Analysis: A Brief Review[END_REF]. Specifically NMR study of natural materials includes the characterization and quantification of hundreds of components in order to achieve a better understanding of the complex mixture composition and, indirectly, of the underlying biological mechanisms [START_REF] Weckwerth | Metabolomics: From Pattern Recognition to Biological Interpretation[END_REF].

At the biological level, the activity of a given bioactive molecule depends both on the configuration of the stereogenic centers, if they are present in its chemical structure, and on the spatial arrangement of the atoms constituting the molecule. This spatial arrangement affects the establishment of intramolecular interactions responsible for the conformations that the molecule can assume in the biological reaction environment. The determination of the relative configuration of a bioactive compound and of its conformational characteristics in solution is therefore of considerable help to understand the physicalchemistry properties associated with it and to establish the relationships between 3D-structure and bioactivity (Boehr et al, 2009). In this context, NMR methodologies constitute a valid experimental tool to explore both the structure and the conformational distribution of a bioactive molecular species in solution, including those molecules of pharmaceutical interest that perform their function in a physiological environment. Specifically, the combination of NMR spectroscopy with the use of partially ordered solvent offers the possibility of measuring anisotropic experimental parameters, such as Residual Dipolar Couplings (RDCs), which, treated with appropriate theoretical models, are able to provide information on structure, orientation, conformation and configuration that a flexible solute experiments in solution (Emsley & Lindon, 1975;[START_REF] Gschwind | Residual Dipolar Coupling-A Valuable NMR Parameter for Small Organic Molecules[END_REF]Di Pietro et al, 2018).

From a practical point of view, for structural NMR studies on pure materials in solution, a series of sophisticated pulse sequences can be required, together with the standard experiments, for a better 3D-structural elucidation and experimental parameters extraction [START_REF] Williams | Modern NMR Approaches to the Structure Elucidation of Natural Products[END_REF][START_REF] Berger | Twenty-five Years of SERF: The Progress of an NMR Pulse Sequence and its Application[END_REF]. On the contrary, in the case of metabolomics approach, few routine NMR experiments are typically needed and from these a variety of informations may be obtained treating the spectroscopic data with statistical methods [START_REF] Arani'bar | Metabolomic Analysis Using Optimized NMR and Statistical Methods[END_REF][START_REF] Emwas | NMR Spectroscopy for Metabolomics Research[END_REF].

The purpose of this chapter is to present a quick overview of the NMR Spectroscopy basic principles highlighting its application to the two research fields covered in this thesis.

Therefore, the theoretical principles of NMR spectroscopy will be discussed in section 1.2, while the metabolomic approach via NMR and the NMR methodology used to obtain conformational and configurational information on a flexible solute dissolved in partially ordered chiral media, will be briefly described in sections 1.3 and 1.4 respectively.

Principles of NMR spectroscopy

The first observation of nuclear magnetic resonance is dated 1939, at what time Isidor Rabi studied the deflection of a stream of hydrogen when it is sent through an homogeneous magnetic field and subjected to a radio frequency electromagnetic radiation [START_REF] Kellogg | The Magnetic Moments of the Proton and the Deuteron. The Radio frequency Spectrum of H2 in Various Magnetic Fields[END_REF]. Rabi won the Nobel Prize in physics for his work. However, it was only in 1945 that NMR in condensed matter was demonstrated thanks to the study on water performed by Felix Bloch at

Stanford and thanks to the study on paraffine carried out by Edward Mills Purcell at the Massachusetts Institute of Technology, independently and concomitantly [START_REF] Bloch | Nuclear Induction[END_REF][START_REF] Purcell | Resonance Absorption by Nuclear Magnetic Moments in a Solid[END_REF]. The efficiency of NMR in chemistry was not realized until 1950s when the so-called shieldig interaction and indirect spin-spin coupling were evidenced [START_REF] Ramsey | Interactions between Nuclear Spins in Molecules[END_REF]. During the following years, improvements in instrumentation and resolution have made it possible to study more complex substances and this has led to obtaining more complicated spectra whose interpretations could not be carried out by discussing the simple shielding and indirect spin-spin interactions. Great strides were made in understanding the origin of these complex spectra thanks to the advent of the quantum chemistry treatments of spin interactions that, coupled to symmetry considerations and to the development of appropriate computer programs, helped in the analysis of these intricate profiles [START_REF] Becker | A Brief History of Nuclear Magnetic Resonance[END_REF].

In order to understand the various applications that NMR has today and, in particular, the ones that are presented in this Ph.D. thesis, it is first necessary to introduce the theoretical aspects of NMR. Note that our intent is not to deeply explore the theoretical basis of NMR spectroscopy but we want to underline the fundamental concepts essential to understand the source of such informative technique applied to the purposes of this thesis. With this purpose, in the following sections the quantum mechanical approach to nuclear magnetic resonance spectroscopy is discussed.

Nuclear Spin Hamiltonian

The quantum state of an atomic ensemble is described by a wave function and by an

Hamiltonian operator which contains all the interactions involving the system. In order to understand NMR phenomena, only the nuclear contribution to the Schrödinger equation can be considered thanks to the possibility to separate the time-scales for nuclear and electronic motion (spin Hamiltonian hypothesis) [START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF].

When a nuclear spin system is placed within the magnet of a NMR spectrometer, physical interactions take place. The kind of interactions existing are the so called "external spin interactions" involving nuclear spins and the magnetic fields generated by the instrument, and "internal spin interactions" concerning the nuclear spins only. In particular, the external spin interactions are given by the interaction between nuclear spins and a) the strong homogeneous static magnetic field 𝐵 0 produced by a superconducting solenoid; b) the radio frequency oscillating field 𝐵 𝑅𝐹 (t) generated by a radio frequency coil; c) the magnetic field gradient 𝐵 𝑔𝑟𝑎𝑑 (r,t) produced by gradient coils. The first listed is a time-independent interaction, the biggest interaction in amplitude experienced by the sample and it is responsible for the so called Zeeman effect which removes the degeneracy of spin energy levels. The interaction with radio frequency radiation is a time-dependent interaction responsible for the population variation of spin states in thermal equilibrium, as predicted by the Boltzmann distribution, and is, therefore, at the basis of the transitions between nuclear energy levels. Finally, the interaction between the spins and the magnetic gradient field is both a time dependent interaction and a position coordinate dependent interaction. Unlike the other interactions, the latter does not always occur:

it takes place only if field gradients are used in the pulses sequence. This is the case of more sophisticated pulses sequences such as those used in order to obtain self-diffusion data (vide infra) [START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF].

Concerning the internal spin interactions, they have both electric and magnetic nature. These kind of interaction are time-independent and are (Canet, 1996;[START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF]:

-Shielding interaction: this term represents the indirect magnetic interaction between the nuclear spins and the external magnetic field through the participation of the electrons.

-Indirect spin-spin coupling interaction (scalar J-coupling): this term represents the indirect magnetic interaction between nuclear spins, through the bonding electrons.

-Direct spin-spin coupling interaction (dipolar coupling): it represents the direct magnetic interaction between a couple of nuclear spins.

-Quadrupolar coupling: it represent the electric interaction of spin greater than ½ with the surrounding electric fields through its quadrupole moment.

Static NMR interactions

Considering all the interactions presented, it is possible to separate the time-independent contribution from the time-dependent one and the total static Hamiltonian operator can be written as a sum of the different parts (Emsley & Lindon, 1975):

𝐻 ̂= ∑ 𝐻 𝑍 ̂ 𝜆 + 𝐻 𝜎 ̂+ 𝐻 𝐽 ̂+ 𝐻 𝐷 ̂+ 𝐻 𝑄 ̂ (1.1)
where 𝐻 𝑍 ̂, 𝐻 𝜎 ̂, 𝐻 𝐽 ̂, 𝐻 𝐷 ̂ 𝑒 𝐻 𝑄 ̂ are the Hamiltonians operators respectively modelling the "Zeeman" (Z) interaction, the chemical shielding (σ) effect, the indirect spin-spin coupling (J)

or scalar coupling, the dipolar (or direct) coupling (D) and, for nuclei with spin greater than 1/2 the quadrupolar interaction (Q). Since the term 𝐻 𝑍 ̂ is the major, the other contributions are considered as perturbative corrections to the Zeeman energy levels.

Zeeman interaction

The 2I + 1 orientations that a nuclear spin I can assume are all random at the same energy when no external perturbation is applied. This degeneracy is removed if a magnetic field is applied:

in this situation the orientation of the spins can be either "parallel" to the field (the most stable configuration) or "antiparallel" (the less stable configuration). The splitting between the two obtained levels can be calculated as the opposite of the scalar product involving both interacting vector, i.e. the external magnetic field (𝐵 0 ⃗⃗⃗⃗ ) and the nuclear magnetic moment (𝜇 ). From the quantum meccanics point of view the resulting Hamiltonian has the following form:

𝐻 𝑍 ̂= -𝜇̂ 𝐵 0 = -ħ𝛾 𝐵 0 𝐼 ̂𝑍 = ħ𝜔 0 𝐼 ̂𝑍 (1.2)
were 𝜇̂ = ħ𝛾 𝐼 ̂𝑍 is the nuclear magnetic moment, ħ = ℎ/2𝜋, with h the Planck's constant, 𝛾 is the magnetogyric ratio, 𝐼 ̂𝑍 is the Cartesian spin angular momentum operator along the Z-axis, 𝐵 0 is the static magnetic field aligned with Z-axis in the laboratory frame and 𝜔 0 = 𝛾 𝐵 0 is the Larmor frequency of the spin I. Since in a NMR experiment a number i of nuclear spins are involved, the equation (1.2) can be written as (Canet, 1996;Emsley & Lindon, 1975;[START_REF] Dybowski | Zeeman Interaction in Nuclear Magnetic Resonance[END_REF]:

𝐻 𝑍 ̂= -∑ 𝜇 𝑖 𝐵 0 𝑖 = -ħ ∑ 𝛾 𝑖 𝐵 0 𝐼 ̂𝑖,𝑍 𝑖 = ħ ∑ 𝜔 𝑖,0 𝑖 𝐼 ̂𝑖,𝑍 (1.3)

Internal interactions

The internal spins interactions are terms describing the electric or magnetic interaction between the nuclear spins that constitute the analyzed spins system. In order to derive and understand the equations relating to the energies involved in these interactions, lets consider a generic interaction 𝜆. From a quantum mechanical point of view, the 𝐻 𝜆 ̂ operator in the Cartesian representation can be described by the product (Canet, 1996;Emsley & Lindon, 1975;[START_REF] Dybowski | Zeeman Interaction in Nuclear Magnetic Resonance[END_REF]:

𝐻 𝜆 ̂= 𝑈 ̂𝜆𝑇 • 𝑇 𝜆 ̌• 𝑉 𝜆 ̂ (1.4)
where 𝑈 ̂𝜆𝑇 and 𝑉 𝜆 ̂ are the vector operators representing the two interacting quantities and 𝑇 𝜆 ̌ is a second rank tensor describing the 𝜆-th interaction. Thanks to the secular approximation (or high field approximation), due to the direction of the magnetic field that causes the quantization along Z-axis of the laboratory reference frame, all the components of the 𝑇 𝜆 ̌ tensor can be considered negligible with respect to that direct along the field (TZZ). This component is made up as follows (Emsley & Lindon, 1975):

𝑇 𝑍𝑍 = 1 3 𝑇𝑟(𝑇) + 2 3 ∑ 1 2 (3 𝑐𝑜𝑠 𝜃 𝛼𝑧 𝑐𝑜𝑠 𝜃 𝛽𝑧 -𝛿 𝛼𝛽 )𝑇 𝛼𝛽 𝛼𝛽 (1.5)
where Z is referred to the laboratory reference frame (x,y,z) in which the measures are carried out, 𝑇𝑟(𝑇) is the trace of the thensor 𝑇 𝜆 ̌, α, β, γ are the labels of the molecular reference frame in which the interactions occur and 𝑇 𝛼𝛽 represents the components of the interaction tensor 𝑇 𝜆 ̌ expressed in the molecular reference frame. If the molecules undergo rapid molecular motion, the continuous change in the direction of the α, β and γ axes with respect to the fixed laboratory axes causes different 𝜃 𝛽𝑧 and 𝜃 𝛼𝑧 during time. This makes the measured interaction a motionally averaged value. For this reason the previous equation can therefore be written as:

〈𝑇 𝑍𝑍 〉 = 1 3 𝑇𝑟(𝑇) + 2 3 ∑ 1 2 〈(3 𝑐𝑜𝑠 𝜃 𝛼𝑧 𝑐𝑜𝑠 𝜃 𝛽𝑧 -𝛿 𝛼𝛽 )𝑇 𝛼𝛽 〉 𝛼𝛽 (1.6)
in which the brackets, 〈. . . 〉, indicate the average of the aforementioned motions. Note that in (1.6) 𝑇 𝛼𝛽 is actually also considered as an averaged value due to the vibrational and rotational internal motions that cause a fluctuation in time of the term. When it is possible to separate internal motions from reorentational ones, the equation (1.6) can be written as:

〈𝑇 𝑍𝑍 〉 = 1 3 𝑇𝑟(𝑇) + 2 3 ∑ 1 2 〈(3 cos 𝜃 𝛼𝑧 cos 𝜃 𝛽𝑧 -𝛿 𝛼𝛽 )〉𝑇 𝛼𝛽 𝛼𝛽 (1.7)
Taking on:

𝑆 𝛼𝛽 = 1 2 (3 cos 𝜃 𝛼𝑧 cos 𝜃 𝛽𝑧 -𝛿 𝛼𝛽 ) (1.8)
in which 𝑆 𝛼𝛽 are the elements of the Saupe order matrix that expresses the orientation of the laboratory Z axis in the chosen molecular system. The Saupe order matrix, due to the properties deriving from the director cosines, is a symmetric traceless tensor, therefore only five components are useful in order to describe it according to the molecular symmetry of the solute.

When the matrix is expressed within the Principal Axis System (PAS), only two of the components need to be known [START_REF] Emsley | NMR Methods of Studying Orientational Order in the Liquid Crystalline and Isotropic Phases of Mesogenic Samples[END_REF]Burnell & De Lange, 2003).

Taking into account the equation (1.8), the equation (1.7) can be rewritten as:

〈𝑇 𝑍𝑍 〉 = 1 3 𝑇𝑟(𝑇) + 2 3 ∑ 𝑆 𝛼𝛽 𝑇 𝛼𝛽 𝛼𝛽 (1.9)
In a solvent in which Brownian motion occurs, the second term is equal to zero, therefore the 𝑇 𝑍𝑍 is constituted only by the component of isotropic origin:

〈𝑇 𝑍𝑍 〉 = 1 3 𝑇𝑟(𝑇) = 𝑇 𝑖𝑠𝑜 (1.10)
Consequently the term containing 𝑆 𝛼𝛽 is the representative component of the interactions of anisotropic origin.

In an ordered system, such as in the case of weakly ordering media, the anisotropic component is not null hence the expression for the 𝑇 𝑍𝑍 is the following:

〈𝑇 𝑍𝑍 〉 = 𝑇 𝑖𝑠𝑜 + 𝑇 𝑎𝑛𝑖𝑠𝑜 (1.11)
Experimentally, the generic term 〈𝑇 𝑍𝑍 〉 corresponds to the NMR quantities σ iso , σ aniso , 𝐽 𝑖𝑗 𝑖𝑠𝑜 , 𝐽 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 , 𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 and, νQ, which are obtained through the spectra. In the isotropic phase, from a spectrum it is possible to extract only the value of the chemical shift, 𝜎 𝑖𝑠𝑜 , and of the scalar coupling, 𝐽 𝑖𝑗 𝑖𝑠𝑜 , since the dipolar and quadrupolar tensors are intrinsically traceless and, consequently, do not affect directly the isotropic spectra. Contrariwise, in systems in which a certain degree of orientational order is present, the terms of anisotropic origin such as 𝜎 𝑎𝑛𝑖𝑠𝑜 , 𝐽 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 , 𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 and, for I > ½ the quadrupolar splitting, become predominant in the spectra. The various interactions are discussed in more detail in the following paragraphs in order to understand how each single part contributes to the total Hamiltonian.

-

Shielding interaction

The mechanism of the shielding interaction can be seen as a two step process. First the external magnetic field induces current in the electrons surrounding the nuclei. were 𝜎 𝑖,𝑍𝑍 is the ZZ-component of the tensor 𝜎 ̌.

Since both shielding and Zeeman contributions represent the interaction between the nuclear magnetic dipole moment and the static magnetic field, it is common to combine the two parts into an unique contribution and the resulting Hamiltonian has the following form (Canet, 1996;[START_REF] Dybowski | Zeeman Interaction in Nuclear Magnetic Resonance[END_REF]:

𝐻 𝛿 ̂= -∑ 𝛾 𝑖 𝐵 0 2𝜋 (1 -𝜎 𝑖,𝑍𝑍 )𝐼 ̂𝑖,𝑍 𝑖 = -∑ 𝜔 𝑖,0 𝑖 (1 -𝜎 𝑖,𝑍𝑍 )𝐼 ̂𝑖,𝑍 (1.13)
In equation 1.11 we expressed the possibility to write 𝜎 𝑖,𝑍𝑍 as a sum of the two elements, one of isotropic nature and one anisotropic. Applying it to the shielding Hamiltonian, the equation 1.13 becomes:

𝐻 𝛿 ̂= -∑ 𝛾 𝑖 𝐵 0 2𝜋 (1 -𝜎 𝑖 𝑖𝑠𝑜 -𝜎 𝑖 𝑎𝑛𝑖𝑠𝑜 )𝐼 ̂𝑖,𝑍 𝑖 (1.14)
where, according to the equations 1.9 and 1.10, 𝜎 𝑖 𝑖𝑠𝑜 and 𝜎 𝑖 𝑎𝑛𝑖𝑠𝑜 can be expressed as:

𝜎 𝑖 𝑖𝑠𝑜 = 1 3 𝑇𝑟(𝜎 ̌) = 1 3 (𝜎 𝑎𝑎 + 𝜎 𝑏𝑏 + 𝜎 𝑐𝑐 ) (1.15) 𝜎 𝑖 𝑎𝑛𝑖𝑠𝑜 = 2 3 ∑ 1 2 〈(3 cos 𝜃 𝛼𝑧 cos 𝜃 𝛽𝑧 -𝛿 𝛼𝛽 )𝜎 𝛼𝛽 〉 𝛼𝛽 (1.16)
In the spectra, shielding interaction results in different resonating frequencies for nuclei with different electronic environment and this effect is commonly known as chemical shift. As already introduced, chemical shift is an intramolecular interaction, but it must be emphasized that it have also significant intermolecular component. Indeed the same nucleus in a molecule resonate at different frequencies on the basis of the different solvent in which the species is dissolved [START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF]. Also the chemical shift do (strongly) depends on the temperature, the concentration, and the purity of the sample.

-Indirect spin-spin coupling

The indirect spin-spin interaction is given by the magnetic interaction between pairs of nuclei, through the bonding electrons. In this case the interacting quantities are two spins 𝐼 𝑖 ̂ and 𝐼 𝑗 ̂ and the type of interaction is expressed by the second-rank tensor 𝐽 𝑖𝑗 ̌, so the general form of the Hamiltonian is the following (Canet, 1996):

𝐻 𝐽 ̂= ∑ 𝐼 𝑖 ̂• 𝐽 𝑖𝑗 ̌• 𝐼 𝑗 î<𝑗 (1.17)
Resolving the equation, taking into account the secular approximation, the only terms remaining are (Emsley & Lindon, 1975): (1.20) the equation 1.18 can be written as (Emsley & Lindon, 1975;Canet, 1996):

𝐻 𝐽 ̂= ∑
𝐻 𝐽 ̂= ∑ 𝐽 𝑖𝑗 𝑖𝑠𝑜 {𝐼 𝑍𝑖 ̂𝐼𝑍𝑗 ̂+ 1 2 (𝐼 𝑖 + ̂𝐼𝑗 - ̂+ 𝐼 𝑖 - ̂𝐼𝑗 + ̂)} 𝑖<𝑗 + ∑ 𝐽 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 {𝐼 𝑍𝑖 ̂𝐼𝑍𝑗 ̂-1 4 (𝐼 𝑖 + ̂𝐼𝑗 - ̂+ 𝐼 𝑖 - ̂𝐼𝑗 + ̂)} 𝑖<𝑗 (1.21)
With, again, the isotropic and anisotropic contributions respectively equal to:

𝐽 𝑖𝑗 𝑖𝑠𝑜 = 1 3 𝑇𝑟(𝐽 𝑖𝑗 ̌) = 1 3 (𝐽 𝑎𝑎 + 𝐽 𝑏𝑏 + 𝐽 𝑐𝑐 ) (1.22) 𝐽 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 = 2 3 ∑ 1 2 〈(3 cos 𝜃 𝛼𝑧 cos 𝜃 𝛽𝑧 -𝛿 𝛼𝛽 )𝐽 𝛼𝛽 〉 𝛼𝛽 (1.23)
From the spectral point of view, the J-coupling for isotropic system can be easily obtained measuring the distance expressed in Hertz (Hz) between the consecutive lines of the multiplet that constitutes the signal and the value obtained is independent of the applied magnetic field, unlike the resonating frequencies. From the spectra it is possible to obtain the absolute value of the J-coupling, but this observable has a sign. It may be positive or negative on the basis of different aspect: for a couple of nuclei i and j connected by one chemical bond, Jij is positive if the two nuclei have the same sign of magnetogyric ratios [START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF]. For long-range Jcoupling the sign is variable and depends on the molecular geometry parameters and many other factors. It can be obtained using specific pulse sequences or through spectral simulation programs [START_REF] Parella | Long-Range Proton-Carbon Coupling Constants: NMR Methods and Applications[END_REF]. For similar molecular fragment the sign is the same and for this reason the literature can also help in the Jij sign assignment. The J-anisotropies have proven to be small enough to be neglected in the case of nuclear interaction 1 H-1 H and 1 H-13 C as will be shown in the Total spin-spin coupling section, while, in the case of coupling involving nuclei other than 1 H, they are not simple to be determined [START_REF] Vaara | Spin-spin coupling tensors as determined by experiment and computational chemistry[END_REF].

-

Direct dipolar coupling

Each nuclear spin generates a small magnetic field according to the direction of the spin magnetic moment and a second nuclear spin can interact with this magnetic field. As J-coupling his interaction is mutual: each spin experiences the field generated by the other. This kind of coupling does not involve however the bonding electron or the surrounding electric cloud, it is a through-space interaction and for this reason this interaction is strictly connected to the threedimensional spatial arrangement of nuclei and hence the measure of these couplings do provide experimental informations on structural, conformational and orientational properties of a molecule. The energy accounting for the classical interaction between two magnetic moments 𝜇 𝑖 ⃗⃗⃗ and 𝜇 𝑗 ⃗⃗⃗ is expressed as (Burnell & de Lange, 2003):

𝐸 = 𝜇 𝑖 ⃗⃗⃗ • 𝜇 𝑗 ⃗⃗⃗ 𝑟 𝑖𝑗 3 - 3(𝜇 𝑖 ⃗⃗⃗ • 𝑟 𝑖𝑗 ⃗⃗⃗ )(𝜇 𝑗 ⃗⃗⃗ • 𝑟 𝑖𝑗 ⃗⃗⃗ ) 𝑟 𝑖𝑗 5 (1.24)
where 𝑟 𝑖𝑗 ⃗⃗⃗ is the vector connecting both involved nuclei.

The quantomechanical form of this kind of interaction is given by (Burnell & de Lange, 2003;Emsley, 2007):

𝐻 𝐷 ̂= 2 ∑ 𝐼 𝑖 ̂• 𝐷 𝑖𝑗 ̌• 𝐼 𝑗 î<𝑗 = ∑ 𝜇 0 4𝜋 ℎ𝛾 𝑖 𝛾 𝑗 4𝜋 2 𝑟 𝑖𝑗 5 [𝑟 𝑖𝑗 2 𝛿 𝛼𝛽 -3𝑟 𝑖𝑗,𝛼 𝑟 𝑖𝑗,𝛽 ] 𝑖<𝑗 𝐼 𝛼𝑖 ̂𝐼𝛼𝑗 ̂ (1.25)
where 𝜇 0 is the permeability in vacuum and 𝑟 𝑖𝑗,𝛼 is the 𝛼 component of 𝑟 𝑖𝑗 ⃗⃗⃗ .

Adopting the secular term, the Hamiltonian operator associated to this interaction can be written as (Canet, 1996):

𝐻 𝐷 ̂= 2𝐷 𝑖𝑗,𝑍𝑍 {𝐼 𝑍𝑖 ̂𝐼𝑍𝑗 ̂-1 4 (𝐼 𝑖 + ̂𝐼𝑗 - ̂+ 𝐼 𝑖 - ̂𝐼𝑗 + ̂)} (1.26)
where:

2𝐷 𝑖𝑗,𝑍𝑍 = 𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 = - 1 2 𝜇 0 ℎ𝛾 𝑖 𝛾 𝑗 16𝜋 3 𝑟 𝑖𝑗 3 (3𝑐𝑜𝑠 2 𝜃 𝑖𝑗 -1) (1.27)
In isotropic liquid, the secular parts of the intramolecular coupling is averaged to zero by Brownian motion and, due to the fact that the dipolar coupling tensor is traceless, this interaction does not effect the spectra. Anyway, even in isotropic solvent dipole-dipole longrange interaction exists, this contribution does not induce spectral splittings, but in some case, using particular pulse sequences, it provides useful information on the molecular structure and conformation. NOESY and ROESY experiments make use of this principle for instance [START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF]. In weakly orienting media, as lyotropic liquid crystals used in this thesis, where there is a preferential molecular orientation, the intramolecular dipole-dipole interaction is not completely averaged to zero therefore it can indirectly be obtained from the spectra in the total spin-spin coupling are measured.

-Total spin-spin coupling

In a weak orienting solvent, the total splitting observed within the lines of a signal originated by the interaction between a couple of spins i and j, is given by both the indirect and dipolar interactions. From the quantomechanical point of view this is obtained by adding the two equations, (1.21) and (1.26), and a total coupling Hamiltonian can be defined as is shown below (Emsley & Lindon, 1975): Sn, 199 Hg which contribution was found to be small but significant [START_REF] Vaara | Spin-spin coupling tensors as determined by experiment and computational chemistry[END_REF]. In the case of nuclear interaction 1 H-1 H, 1 H-13 C and 1 H-19 F the ratio

𝐻
𝐽 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜
has proven to be approximately 10 -3 so that 𝐽 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 is small enough to be neglected [START_REF] Vaara | Spin-spin coupling tensors as determined by experiment and computational chemistry[END_REF]. In such cases the total spin-spin coupling measurable from the spectra can be expressed as [START_REF] Emsley | Nuclear Magnetic Resonance of Liquid Crystals[END_REF]:

𝑇 𝑖𝑗 = |𝐽 𝑖𝑗 𝑖𝑠𝑜 + 2𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 |(non -equivalent nuclei) (1.32) 𝑇 𝑖𝑗 = 3|𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 |(equivalent nuclei) (1.33)
On the other hand, in the case of much more ordered systems, as for example in the case in which thermotropic liquid crystals are used as ordering solvent, the situation is much more complex and the previous equations are no longer valid. In fact, these systems impart a very high degree of order to the solute dissolved in them and the interactions that take place influence the spectra generating second order signals from which it is not possible to obtain information by simple inspection. In order to extract the coupling constants from these spectra, which are much larger in magnitude than those obtained from the weakly ordering phases, it is necessary to use spectral simulation programs [START_REF] Di Pietro | Smettic Order Parameters via Liquid Crystal NMR Spectroscopy: Application to a Partial Bilayer Smectic A Phase[END_REF].

-Quadrupolar interaction

Nuclei with spin moment higher than ½ possess an electric quadrupole moment due to the asymmetric distribution of charge in the nucleus. This quadrupolar moment can interact strongly with the electric field gradient generated by the electron clouds and this interaction results in a contribution to the Hamiltonian given by (Canet, 1996;Emsley, 2007):

𝐻 𝑄 ̂= ∑ 𝐼 𝑖 ̂• 𝑄 𝑖 ̌• 𝐼 𝑖 î = ∑ 𝑞 ̃𝑖,𝑍𝑍 4𝐼 𝑖 (2𝐼 𝑖 -1) [3𝐼 𝑍𝑖 ̂𝐼𝑍𝑖 ̂-𝐼 𝑖 (𝐼 𝑖 + 1)] 𝑖 (1.34)
The term 𝑞 ̃𝑖,𝑍𝑍 can be expressed as:

𝑞 ̃𝑖,𝑍𝑍 = 𝑒𝑄 𝑖 𝑉 ̃𝑖,𝑍𝑍 ℎ (1.35)
where 𝑒𝑄 𝑖 is the nuclear electric quadrupole moment of the nucleus i, and 𝑉 ̃𝑖,𝑍𝑍 is the partially averaged component of the electric field gradient tensor measured at the nucleus and in the direction of the static magnetic field 𝐵 0 ⃗⃗⃗⃗ . The tensor 𝑄 𝑖 ̌ is purely anisotropic, which means that the values of 𝑞 ̃𝑖,𝑍𝑍 are particularly useful for probing the orientational order in partially ordered system. The relationship between 𝑞 ̃𝑖,𝑍𝑍 and 𝑞 ̃𝑖,𝛼𝛽 , the components in a molecular frame, is given by:

𝑞 ̃𝑖,𝑍𝑍 = 𝑞 𝑖,𝑎𝑎 [𝑆 𝑎𝑎 + 1 3 𝜂 𝑖 (𝑆 𝑏𝑏 -𝑆 𝑐𝑐 )] (1.36)
where 𝑞 𝑖,𝑎𝑎 > 𝑞 𝑖,𝑏𝑏 > 𝑞 𝑖,𝑐𝑐 conventionally, and 𝜂 𝑖 = (𝑞 𝑖,𝑏𝑏 -𝑞 𝑖,𝑐𝑐 )/𝑞 𝑖,𝑎𝑎

Time-dependent NMR interactions

Among the NMR interactions, the one taking place between the nuclei and the radio frequency oscillating field and the one between the nuclei and the magnetic gradient field, are timedependent interactions and needed a time-dependent quantomechanical treatment.

-Spins -gradient field interaction

The aim of high resolution NMR spectroscopy is to have the static magnetic field as uniform as possible across the measurement time and volume, in order to get narrower peaks and a better spectral resolution. However, there are some situations where field variations over volume and time can be beneficial and this variation can be obtained using pulsed field gradients (PFG).

Indeed, under PFG, NMR frequencies depends on the position of spins and for this reason position-encoding can be achieved preserving chemical shift resolution. The effect of the PFG depends on the direction along which the field strength changes. In particular the interaction of a spin Ii, located in a spatial position r(x,y,z), with a generic field gradient is given by: 𝐻 ̂𝑖𝑔𝑟𝑎𝑑 (𝒓, 𝑡) = -𝛾 𝑖 𝐺 𝑥 (𝑡)𝑥𝐼 ̂𝑖𝑧 for gradient 𝐺 𝑥 along x axis To note that spin operator is always 𝐼 ̂𝑖𝑧 and it is independent from the gradient direction. This is true because the static magnetic field is direct along the z-axis and is much more stronger than the gradient fields, for this reason the other components can be ignored in first approximation [START_REF] Kay | Field Gradient Techniques in NMR Spectroscopy[END_REF][START_REF] Parella | Pulsed Field Gradients: A New Tool for Routine NMR[END_REF][START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF]. The way the PFG can be used to encode spins for their displacement will be treated in the 1.3.2.2 section.

-Spins -radio frequency field interaction When a radio frequency (rf) field characterized by a strength 𝐵 𝑅𝐹 , an angular frequency 𝜔 𝑅𝐹 and a phase  is applied on the x axis, it produces an oscillating magnetic field that can be represented as [START_REF] Smith | The Hamiltonians of NMR. Part II[END_REF][START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF]:

𝐵 𝑅𝐹 ⃗⃗⃗⃗⃗⃗⃗ (𝑡) = 2𝐵 𝑅𝐹 𝑐𝑜𝑠(𝜔 𝑅𝐹 𝑡 +  )𝑒 𝑥 ⃗⃗⃗ (1.40)
The rf oscillates along the x axis and it can be seen as an element rotating on the xy plane. The effect of this field is normally much less intense than the static magnetic field B0. Nevertheless it is possible to treat quantum mechanically this interaction as we did for the Zeeman interaction. Thus the resulting Hamiltonian used to describe the influence of the applied coherent radio frequency on the spin has the following form: [START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF].

𝐻 𝑅𝐹 ̂(𝑡) = -∑ 𝜇 𝑖

Metabolomics approach

A biological organism is considered today as an integrated system due to its extreme complexity: it can be seen as a sum of individual parts that interact with each other. The behavior of each biological component (molecules, cells, organs) and their interactions are explored by the system biology approach (van der Greef et al, 2004). This is an interdisciplinary holistic methodology requiring the use of chemical, biochemical, physical and physiological methods in order to understand and measure the response of an organism subjected to different conditions [START_REF] Choi | Metabolomics: What You See Is What You Extract[END_REF]. Among the discipline commonly applied in this field of research, in recent years omics sciences emerged [START_REF] Karahalil | Overview of Systems Biology and Omics Technologies[END_REF]. The objective of omics sciences is to identify, characterize and quantify all biological molecules directly involved in the structure, function, and dynamics of a cells, tissues, or organisms. More specifically the omics sciences that allow to analyze the complete sets of genes, RNA, proteins and metabolites are respectively genomics, transcriptomics, proteomics and metabolomics and are arranged in the so called "omics cascade" (Figure 1.1) [START_REF] Berry | The integration of "omic" disciplines and systems biology in cattle breeding[END_REF][START_REF] Patti | Metabolomics: the Apogee of the Omics Trilogy[END_REF]. Although a detailed investigation of the entire omics cascade occupies an essential position in the system biology, metabolome is therefore most predictive of phenotype than other sets of genes, RNA and proteins. This is true because, while genes and proteins are respectively subjected to regulatory epigenetic processes and post-translational modifications, metabolites are the ultimate response of an organism genetic alterations, diseases and environmental influences.

Indeed, while the genome is almost invariant throughout the lifespan of an individual, the metabolome may change as an effect of lifestyle, stress and on the basis of pathologies. The possibility to correlate the phenotype to the metabolomic profiles in a simpler way than comparing with genomic, transcriptomic and proteomic profiles, put metabolomics at the end point of the omics cascade (Figure 1.1) [START_REF] Fiehn | Metabolomics-the Link Between Genotypes and Phenotypes[END_REF][START_REF] Muthubharathi | Metabolomics: Small Molecules that Matter More[END_REF]. Metabolomics has grown rapidly in the last decade and the increasing number of paper published in this field of research makes it an emerging tool for the study of natural and biological complex mixtures (Figure 1.2). Indeed, during the last years, the study of metabolome have been successfully applied to multiple research fields including disease diagnostics [START_REF] Bujak | Metabolomics for Laboratory Diagnostics[END_REF], biomarker discovery [START_REF] Diaz-Beltran | Human Plasma Metabolomics for Biomarker Discovery: Targeting the Molecular Subtypes in Breast Cancer[END_REF], toxicology [START_REF] Olesti | Approaches in Metabolomics for Regulatory Toxicology Applications[END_REF] and food science [START_REF] Li | Recent Advances in the Application of Metabolomics for Food Safety Control and Food Quality Analyses[END_REF]. The basis of most of these applications has the common aspect that alterations in metabolism due to functional responses of a biological system (e.g. mutations, aging, diet, exercise, or life style) result in variations in the groups of metabolites that form characteristic patterns and represent the so-called metabolome of the system itself. Indeed, the metabolome can be defined as the complete set of small molecules having a weight lower than 1500Da, such as lipids, amino acids and sugars [START_REF] Muthubharathi | Metabolomics: Small Molecules that Matter More[END_REF]. The size and the composition of the metabolome varies depending on the organism and/or the sample studied. For example, it is estimated that plant kingdom contains between 200000 and 1 million of distinct metabolites [START_REF] Wang | The Structure and Function of Major Plant Metabolite Modifications[END_REF] while 4500 different low molecular weight species have been detected in human urine [START_REF] Miller | Real Time Health Monitoring Through Urine Metabolomics[END_REF]. In the following sections the different experimental approaches adopted in metabolomics will be introduced, focusing the attention on the NMR-based metabolomic approach that represents the core of this Ph.D. thesis.

Different experimental approaches in metabolomics

The idea that each organism might have a typical metabolic profile that could be reflected in each of its biological fuids was conceived by Roger Williams in the late 1940s when he tried to associate characteristic metabolic patterns of urine and saliva with schizophrenia [START_REF] Gates | Quantitative metabolic profiling based on gas chromatography[END_REF]. However, it was only in 1998 that the term "metabolome" was coined by

Stephen Oliver by analogy to the term "genome" [START_REF] Williams | Biochemical Institute Studies IV. Individual metabolic patterns and human disease: An exploratory study utilizing predominantly paper chromatographic methods[END_REF][START_REF] Oliver | Systematic functional analysis of the yeast genome[END_REF] and, although its birth can be dated back to the mid-1900s, even up to today metabolomics is regarded as an emerging field [START_REF] Krastanov | Metabolomics-The State of Art[END_REF]. Indeed, it is a field of research in continuous development and for this reason it is often difficult to find an univocal and general nomenclature to name the different approaches used in this research area. Currently, three main approaches are used for metabolomic investigations that in literature are called in different ways, for example "fingerprinting", "global metabolite profiling" or "untargeted profiling" are commonly used in order to design the same kind of metabolomic method [START_REF] Krishnan | Metabolite Fingerprinting and Profiling in Plants Using NMR[END_REF][START_REF] Patti | Metabolomics: the Apogee of the Omics Trilogy[END_REF][START_REF] Wolfender | Current Approaches and Challenges for the Metabolite Profiling of Complex Natural Extracts[END_REF]. With the aim of avoid confusion in the reader, the three metabolomics approaches in this manuscript are called "targeted analysis", "fingerprinting" and "profiling" and they are defined as follows [START_REF] Fiehn | Metabolomics-the Link Between Genotypes and Phenotypes[END_REF]. The first approach is the targeted analysis: it consists in monitoring a group of metabolites previously selected on the basis of known metabolic pathways that are undoubtedly associated with the condition of interest [START_REF] Roberts | Targeted Metabolomics[END_REF]. These metabolites must be unequivocally assigned and quantified in the samples studied. The second approach is the fingerprinting [START_REF] Wolfender | Current Approaches and Challenges for the Metabolite Profiling of Complex Natural Extracts[END_REF]: it is defined as an untargeted method because it consists in the rapid comparison and classification of samples in order to analyze metabolites comprehensively and systematically, without the need to identify and quantify single species. The goal of this kind of investigations is to perform an unbiased analysis in order to compare patterns of unknown metabolites that change in response to environmental modifications and, thus, to obtain informations without trying to get qualitative or quantitative details. These kind of informations are acquired treating chemical data with statistical tools (see section 1.3.2.4.) and they allow to discover new biomarkers and unknowns compounds that have the potentiality to lead to new insights. All the above described characteristics of fingerprinting make it an hypothesis-generating approach and a discovery method that is commonly used in different fields, particularly in medical research. The last methodology is the metabolic profiling: this is the oldest and most established metabolomics approach and for this reason is considered the precursor of metabolomics itself [START_REF] Wolfender | Current Approaches and Challenges for the Metabolite Profiling of Complex Natural Extracts[END_REF]. It focuses on studying a group of metabolites with the aim of identifying and quantifying them. In addition to the qualitative and quantitative analyses, a statistical study can also be performed. In this case, known metabolites sets are feed into statistical data analysis tools to investigate how they contribute to group separation between samples. Contrary to fingerprinting, profiling is based on the demonstration of specific hypotheses by studying the behavior of the targeted metabolites. From a qualitative point of view, the set of metabolites studied with the profiling approach is typically predetermined by the scientific question underlying the study but it is also limited to the prior knowledge of the metabolites known for the studied matrix and available in databases. Indeed, usually the characterization step is performed by comparing the data obtained from the analysis with those reported in literature or deposited in the so-called "libraries". Libraries are databases containing chemical information about the single species previously studied and deposited, thus allowing to help, confirm and validate suspected metabolites in natural and biological complex matrices analyzed. The greater is the number of metabolites deposited in the libraries, the greater will be the comprehensiveness of the studies conducted on new complex mixtures. Hence the importance to discovery and characterize new molecules possessing a biological activity and involved in the organisms metabolism. Molecular fingerprintings are essential in building these libraries which, in turn, are essential in profiling studies of new mixtures, demonstrating that the various approaches to metabolomics, even if they seem very different from each other, they are instead closely linked. Indeed, the combination of targeted analysis, metabolic profiling and fingerprinting lead to the realization of metabolomics itself and this contributes to our biological understanding both in a mechanistic as well as a predictive manner. In Figure 1.3 the general scheme of the different approaches used in metabolomic is reported. Regardless of the type of approach to be adopted, a metabolomics study need to follow a specific workflow in order to achieve adequate and reliable results. The starting point of the metabolomics study is the existence of a scientific problem that lead to the formulation of an idea or a hypothesis to be proved. The next steps in the workflow consist of experimental design, sample collection, preparation and storage. These points are crucial in any type of adopted approach. For example, a correct sampling procedure, in terms of number of collected samples and their representativeness of the entire matrix, is critical when looking for statistical information able to capture the main features of the organism metabolome. In the literature, several protocols are available that can be applied [START_REF] Ralli | NMR Spectroscopy Protocols for Food Metabolomics Applications[END_REF]. However , if a standardized procedure does not exist for the problem to be addressed, a preliminary procedure for the design, implementation and validation of a new protocol should be performed. The next steps consist in the data acquisition and elaboration. In particular in the elaboration step the statistical

NMR based metabolomics

The data acquisition step in the metabolomics workflow can be carried out using different analytical platforms. The possibility to select proper instrumentation and combine different techniques allow to obtain a more comprehensive metabolomic study of the analysed system [START_REF] Oldiges | Metabolomics: current state and evolving methodologies and tools[END_REF]. Infrared spectroscopy (IR) [START_REF] Ellis | Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy[END_REF][START_REF] Seli | Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization[END_REF][START_REF] Aernouts | Mid-Infrared Spectrometry of Milk for Dairy Metabolomics: A Comparison of Two Sampling Techniques and Effect of Homogenization[END_REF], gas chromatography (GC) [START_REF] Solanky | NMR-Based Metabonomic Studies on the Biochemical Effects of Epicatechin in the Rat[END_REF], liquid chromatography (LC) [START_REF] Groussac | Improved protocols for quantitative determination of metabolites from biological samples using high performance ionic-exchange chromatography with conductimetric and pulsed amperometric detection[END_REF], mass spectrometry (MS) [START_REF] Castrillo | An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry[END_REF][START_REF] Ramautar | CE-MS for metabolomics: developments and applications in the period 2012-2014[END_REF] and nuclear magnetic resonance (NMR) [START_REF] Daykin | Nuclear Magnetic Resonance Spectroscopic Based Studies of the Metabolism of Black Tea Polyphenols in Humans[END_REF] are the techniques commonly used in metabolomics. Among them, NMR spectroscopy [START_REF] Duarte | NMR metabolomics of human blood and urine in disease research[END_REF] and MS coupled with chromatographic techniques (GC-MS and LC-MS) [START_REF] Kopka | Current challenges and developments in GC-MS based metabolite profiling technology[END_REF][START_REF] Ma | Ultrafast polyphenol mtabolomics of red wines using MicroLC-MS/MS[END_REF] are the most popular techniques because they have proven to be powerful and complementary analytical approaches allowing to obtain a complete characterization of the metabolome [START_REF] Bingol | Recent Advances in Targeted and Untargeted Metabolomics by NMR and MS/NMR Methods[END_REF]. Although MS is the most sensitive technique allowing to detect traces of metabolites in the sample (pM level), NMR spectroscopy offers many interesting advantages over MS [START_REF] Emwas | NMR Spectroscopy for Metabolomics Research[END_REF]. Indeed, NMR is a non-destructive, a unbiased method that allows the identification and quantification of the more aboundant metabolites present in the studied samples without any kind of sample pretreatment or chemical derivatization. Isotropic samples can be studied but also solid or semisolids, the latter are performed using specific equipment as solid-state NMR (ssNMR) [START_REF] Kruk | NMR Techniques in Metabolomics Studies: A Quick Overview on Examples of Utilization[END_REF] and High-Resolution Magic-Angle sample Spinning NMR (HRMAS-NMR) [START_REF] Ritota | Metabolomic Characterization of Italian Sweet Pepper (Capsicum annum L.) BY Means of HRMAS-NMR Spectroscopy and Multivariate Analysis[END_REF] respectively. The possibility to record multinuclear spectra (such as 1 H, 13 C, 15 N, and 31 P) and New study plans measure correlations using multidimensional NMR methods, are other strengths of this spectroscopic technique. Additionally, NMR spectroscopy is highly automatable and reproducible, and allow to detect and characterize compounds that are less detectable using other techniques, such as the highly polar compounds that are not easily identified using LC-MS [START_REF] Emwas | NMR Spectroscopy for Metabolomics Research[END_REF]. Moreover, NMR can be applied on biofluids or intact tissues, and this make NMR ideal for real-time metabolites study of living cells. For example, the application of this non-invasive technique for the study of plasma [START_REF] Kumar | An Elaborative NMR Based Plasma Metabolomics Study Revealed Metabolic Derangements in Patients With Mild Cognitive Impairment: A Study on North Indian Population[END_REF], serum [START_REF] Nuñez-Gil | Serum Metabolomic Analysis Suggests Impairment of Myocardial Energy Production in Takotsubo Syndrome[END_REF], cerebrospinal fluid [START_REF] Lin | 1 H NMR metabolomic profiling of human cerebrospinal fluid in aging process[END_REF], saliva [START_REF] Kim | Effects of Acute Heat Stress on Salivary Metabolites in Growing Pigs: An Analysis Using Nuclear Magnetic Resonance-Based Metabolomics Profiling[END_REF] and urine [START_REF] Yang | Novel Metabolic Signatures of Prostate Cancer Revealed by 1H-NMR Metabolomics of Urine[END_REF] allow to obtain useful information for the diagnosis of different diseases such as cancer [START_REF] Yang | Novel Metabolic Signatures of Prostate Cancer Revealed by 1H-NMR Metabolomics of Urine[END_REF], diabetes [START_REF] Amor | Nuclear Magnetic Resonance-Based Metabolomic Analysis in the Assessment of Preclinical Atherosclerosis in Type 1 Diabetes and Preeclampsia[END_REF] or kidney disorders [START_REF] Hunter | NMR-Based Metabolomics Associated With Chronic Kidney Disease in Humans and Animals: A one Health Perspective[END_REF]. The major drawback of NMR spectroscopy is its sensitivity, it can identify metabolites present in millimolae, whereas nanomolar concentrations can be detected using only very high fields instruments and cryoprobes [START_REF] Emwas | NMR Spectroscopy for Metabolomics Research[END_REF].

NMR based metabolomics in natural extract analysis

All the advantages discussed in the last section make NMR-based metabolomics an important tool in different fields of research and, among them, foods analysis holds a prominent place. In this field, the determination and certification of food quality represents a crucial problem for the researchers due to the increasing demand from both industry and consumers that progressively demand safe, healthy and high-quality food products [START_REF] Li | Recent Advances in the Application of Metabolomics for Food Safety Control and Food Quality Analyses[END_REF]. The essential characteristics of a food are connected to the metabolites present in it: each food has its own and unique set of metabolites that represents a fingerprint of the system and can be identified and characterized. The metabolome of non-processed foods, such as fruits or vegetables, can be divided in two components, a first part is composed by molecules owning a certain nutritional value, a second part represents a small fraction of the entire metabolome and it is composed by bioactive molecules offering additional health benefits beyond basic nutrition [START_REF] Kumar | Metabolomics-Assisted Biotechnological Interventions for Developing Plant-Based Functional Foods and Nutraceuticals[END_REF]. This last portion is composed by phytochemicals commonly called nutraceutic compounds that can be extracted from the natural species and added to other processed foods in order to increase the health benefit of the resulting functional foods [START_REF] Siró | Functional Food. Product Development, Marketing and Consumer Acceptance-A Review[END_REF]. Furthermore, natural extracts that are rich in these healthy substances, additionally to being used in the food industry, can also have important applications in pharmacology and medicine such as, for example, the case of essential oils or other extracts obtained from some plants [START_REF] Pisseri | Essential Oils in Medicine: Principles of Therapy[END_REF][START_REF] Rupasinghe | Industrial Hemp (Cannabis sativa subsp. sativa) as an Emerging Source for Value-Added Functional Food Ingredients and Nutraceuticals[END_REF]. A variety of parameters, such as geographic origin, pedoclimatic characteristics of the production area, agronomic practices, can alterate the metabolome of these foods and this change can be easily detected and studied through metabolomics analyses and, in particular, using NMR spectroscopy [START_REF] Masetti | NMR Tracing of Food Geographical Origin: The Impact of Seasonality, Cultivar and Production Year and Data Analysis[END_REF]. The possibility to perform reproducible analyses on natural extracts, assess the quality and authenticity of foods, monitoring food processing and storage in a fast way and with a minimal sample preparation make NMR a powerful tool in this research field. Indeed, recently NMRbased metabolomics coupled with chemometric analysis has been applied to obtain metabolic profiles of various foods including honey [START_REF] Schievano | An NMR-Based Metabolomic Approach to Identify the Botanical Origin of Honey[END_REF], oils [START_REF] Lioupi | Virgin Olive Oil Metabolomics: A Review[END_REF][START_REF] Ancora | Sensitivity of Proton NMR Relaxation and Proton NMR Diffusion Measurements to Olive Oil Adulterations with Vegetable Oils[END_REF], milk [START_REF] Gómez-Gallego | Human Breast Milk NMR Metabolomic Profile across Specific Geographical Locations and Its Association with the Milk Microbiota[END_REF], wine [START_REF] Gougeon | 1 H NMR Metabolomics Applied to Bordeaux Red Wines[END_REF], fresh fruit and their juices [START_REF] Tang | NMR-Based Analysis of Pomegranate Juice Using Untargeted Metabolomics Coupled with Nested and Quantitative Approaches[END_REF] and also other natural substances such as the case of Eugenia jambolana, Withania somnifera and Aegle marmelos herbal extracts [START_REF] Chauthe | Quantitative NMR: An Applicable Method for Quantitative Analysis of Medicinal Plant Extracts and Herbal Products[END_REF] or African tree Anogeissus leiocarpus extract [START_REF] Hubert | Identification of Natural Metabolite in Mixture: A Pattern Recognition Strategy Based on 13 C NMR[END_REF].

The main contribution of this Ph.D. thesis is to show the application of NMR-based metabolomics in natural extract sciences and the next section of the thesis will briefly introduce the tools commonly applied in the NMR-based metabolomics and used in this work.

NMR pulse sequences for metabolites characterization

As already introduced in section 1.2, NMR spectroscopy is based on the study of the behavior of molecules by observing different isotopes of nuclei when are placed in a magnetic field and are exposed to radio frequency radiations. Because of the high natural abundance of 1 H isotope (99.9%), hidrogen nucleus is largely employed in NMR based metabolomic studies. Since the molecules that make up the metabolome are generally organic species, together with the 1 H isotope, other commonly observed nuclei are the 13 C isotope of carbon atom, whose natural abundance is about 1.1%, and 15 N, having natural aboundance of 0.4%, that is present in biological molecules such as nucleic acids and amino acids. In this Ph.D. project we focused our attention on the 1 H and 13 C nuclei on which both one-dimensional and two-dimensional NMR experiments have been acquired. For NMR experiments on these nuclei, different pulse sequences already encoded in the acquisition programs are generally used even if, at times, it is necessary to modify and/or combine them appropriately. It should be noted that the literature is full of pulse sequences, some of them are simple and other are more sophisticated. In the following sections some of the existing pulse sequences will be shown, in particular only the simplest and most representative of the phenomenon to be described are presented, without going into details beyond this thesis project.

One-Dimensional (1D) NMR Spectroscopy

One-dimensional 1 H NMR spectra are the most useful in metabolomic studies due to its characteristics to be very fast and highly automatable. Indeed, 1D 1 H NMR collection times for a single spectrum last few minutes. Additionally, 1D 1 H NMR spectra contain a large amount of informations able to lead to the identification of almost 50-100 metabolites at a time [START_REF] Emwas | NMR Spectroscopy for Metabolomics Research[END_REF]. An important step for this kind of experiment is the choice of the most adequate pulse sequences. The simplest and more used 1D pulse sequence consists in two main steps: the preparation step in which a 90° excitation pulse is performed, and the detection step that allows to collect the free induction decay signal (FID) (Figure 1.5A). However, some sample conditions make it necessary to use more sophisticated pulse sequences. Since in metabolomics field most of the 1D 1 H NMR spectra are acquired on natural and/or biological samples which therefore contain water, solvent suppression pulses sequences are an important tool that cannot be avoided when solvent replacement methods, such as lyophilization, cannot be performed.

Different water suppression pulse sequences are designed in order to lead to a better suppression of the solvent peak and some of them are simpler than others. Two different sequences have been used in this thesis, the first is the simplest one and consists of a 90 ° pulse followed by a presaturation pulse centered on the solvent signal (O1). This type of experiment allows an acceptable signal suppression that in some case may result not adequate. The second sequence used consists in collecting the first increment of the 2D 1 H-1 H-nuclear Overhauser effect spectroscopy (NOESY) experiment. This kind of experiment allows to record 1D 1 H spectra with good water suppression without extending too much the time required for the analyses and for this reason it has become the predominant approach used in NMR-based metabolomics [START_REF] Mckay | How the 1D-NOESY Suppresses Solvent Signal in Metabonomics NMR Spectroscopy: An Examination of the Pulse Sequence Components and Evolution[END_REF] In spite of the advantages that 1 H NMR spectroscopy possesses, there are a few limitations that have to be take into account. The most important limitation of the 1 H NMR spectra is the spectral width which, being narrow (∼15 ppm), leads inevitably to overlapping peaks in the spectra of complex mixture which makes it difficult to assign peaks and quantify metabolites.

Signal overlapping is much less pronounced if nuclei with a wider spectral window are observed. This is the case of 13 C, the second most studied nucleus, wich is characterized by a larger chemical shift dispersion (∼250 ppm). Although the spectral window is quite large, in order to obtain more readable spectra which are not affected by 1 H-13 C J-coupling, experiments are often recorded observing the 13 C nuclei and decoupling the proton nuclei, in order to obtain spectra composed by only singlets ( 13 C-{ 1 H} NMR). This decoupling procedure is carried out using the classic 1D pulse sequence reported in Figure 1.5A and by adding on a second channel a pulse able to saturate the 1 H nuclei. This pulse can be added in different point of the sequences: for example, it can last for all the duration of the experiment (preparation and detection) (Figure 1.5B) or only during the detection (Figure 1.5C). In this last case the nOe effect is avoided and, using appropriate delays between scans, a quantitative 13 C spectra is obtained [START_REF] Giraudeau | Improvement Of The Inverse-Gated-Decoupling Sequence For A Faster Quantitative Analysis Of Various Samples By 13 C NMR Spectroscopy[END_REF]. Anyway, all these procedures result in an increment in resolution but, the low natural abundance of 13 C coupled with the low sensitivity, limit the application of 13 C NMR spectroscopy in metabolomics field.

Two-Dimensional (2D) NMR Spectroscopy

In addition to using higher fields for which it is essential to possess a specific instrumentation, a commonly used method to increase the spectral resolution and resolve overlapping peaks is to use two-dimensional spectroscopic techniques. This kind of experiments have the potentiality to detect and identify more metabolites than any one-dimensional experiment at the cost of a significant increase of the experimental time. 2D experiments commonly used in metabolomics are correlation experiments: 1 H Correlation SpectroscopY (COSY) 1 H-13 C Heteronuclear Single Quantum Coherence Spectroscopy (HSQC), Heteronuclear Multiple-Quantum Correlation (HMQC) and Heteronuclear Multiple-Bond Correlation Spectroscopy (HMBC). Other important 2D experiments that can be used in metabolomics are 1 H-1 H and 1 H-13 C J-resolved (J-Res) experiments, able to provide well-resolved information about multiplicity and coupling patterns. experiment for structural elucidation [START_REF] Aue | Two-Dimensional Spectroscopy. Application to Nuclear Magnetic Resonance[END_REF]. The simplest COSY pulse sequence consists in four steps: a preparation step consisting in a 90° rf pulse followed by a variable evolution step (t1). Then a second 90° pulse is performed during the mixing time and after that the measurement period time (t2) takes place during the detection step. The graphical representation of this pulse sequence is reported in Figure 1.6. This sequence is performed many times with a range of incremented delays for t1 and, by carrying out the Fourier Transform on both dimensions of the detected FIDs, the COSY map is obtained. The spectrum is composed by the frequencies of the 1 H nucleus on both axes (F2, the horizontal dimension and F1, the vertical one), the diagonal peaks correspond to the peaks of the 1D NMR experiment while the cross peaks are the informative part of the 2D map because they result from the magnetization tranfer from one nucleus to another one through Jinteractions and for this reason indicates couplings between nuclei. Each coupling gives two symmetrical cross peaks, one above and one below the diagonal and are centered at the chemical shifts of the two interacting nuclei. COSY-90 is the most common COSY experiment (Figure 1.5) but sometimes different degree of excitation are used in order to improve some characteristics of the experiment. For example, the use of a 45° improves intensities and streamlines the diagonal with only a slight loss of signal-to-noise ratio, allowing to match, in the case of large molecules, cross-peaks near the diagonal in a simpler way. However, in some cases a 60° mixing pulse is recommended for optimal sensitivity. Moreover, also in this case other more complex pulse sequences exist. Some of them consist in the use of quantum filters based on the use of coherence selection method such as phase cycling or pulsed field gradients that are applied. The ultimate goal of these more sophisticated sequences is to obtain cleaner spectra with fewer artifacts and with cross peaks not obscured by the diagonal peaks. As can be deduced, there are numerous variations of the COSY NMR experiment and the choice of the most appropriate experiment depends on the system to be studied, the desired resolution and the accuracy of the experimental information to be obtained. Anyway, since the COSY experiment is relatively simple, easy to perform and to interpret, makes it very useful in metabolomics allowing the identification of both known and unknown metabolites [START_REF] Xi | Automated Screening For Metabolites In Complex Mixtures Using 2D COSY NMR Spectroscopy[END_REF].

Heteronuclear Correlation Spectroscopy (HSQC, HMQC and HMBC)

In addition to the homonuclear case, correlation spectroscopy can also be used for measuring heteronuclear correlations allowing to obtain informations which facilitate the structure determination or the metabolites identification. This kind of experiment is used in order to assign the signal in the spectrum of a nucleus I ( 15 N and 13 C for example) once the spectrum of one nucleus S is known ( 1 H generally). The 2D spectrum obtained from these experiments has the 1 H spectrum displayed on the horizontal dimension (F2 axis) and the less sensitive nucleus spectrum on the vertical dimension (F1 or indirect axis). The correlation spectrum does not contain diagonal peaks such as in the case of COSY, and all the cross-peaks shown in the 2D map indicate the correlations between the two involved nuclei. The most used heteronuclear correlation experiments in metabolomics are HSQC, HMQC and HMBC that involve the 13 C and 1 H nuclei. 1 H-13 C HSQC is an experiment commonly used to correlate the chemical shift of directly J-coupled spins ( 1 J). In addition to the correlation information, it is possible to enhance signal of lower sensitivity nucleus by transferring the nuclear spin polarization from the more sensitive nucleus, 1 H, to the less sensitive one that is 13 C. Such transfer is possible thanks to the INEPT pulses block (Insensitive Nuclei Enhanced by Polarization Transfer) that is composed by a spin Echo (90°H(x)- -180°H(x) -) followed by a population inversion (180°C(x)--90°H(y),90°C(x)). This technique allows to increase the sensitivity of the less abundant nuclei and for this reason it is possible to significantly reduce the experimental times required to observe them. Even if these experimental times are lower than those required for the simple one-dimensional experiment, they remain long and dependent on the needed spectral resolution [START_REF] Bodenhausen | Natural Abundance Nitrogen-15 by Enhanced Heteronuclear Spectroscopy[END_REF]. Another 2D correlation experiment able to provide the same kind of information given by 1 H-13 C HSQC is the 1 H-13 C HMQC. It uses a different approach to transfer magnetization and this sometimes lead to much broader correlation peacks compared to those produce by HSQC experiment. This kind of sequence provides correlation between protons and scalar coupled carbon atom through the creation of heteronuclear multiplequantum coherence [START_REF] Bax | Correlation of Proton and Nitrogen-15 Chemical Shifts by Multiple Quantum NMR[END_REF]. Indeed, in this case the pulse sequence start with a 90° on the channel of the nucleus 1 H followed by time (JCH) in wich the in-phase magnetization of 1 H is converted into anti-phase magnetization. Then a second 90° is on channel 13 C in order to convert this anti-phase magnetization in a combination of zero-and double quantum coherence.

The coherence evolve during a time in which a second pulse of 180° on 1 H channel cancel the chemical shift evolution letting place to the only J-evolution. A last 90° pulse on 13 C channel returns the multiple quantum coherence to observable anti-phase H-spin magnetization and finally a second delay converts the anti-phase into an in-phase term (Mandal & Majumdar, 2003). Usually, during the acquisition on the proton channel, a broadband decoupling sequence is performed on the 13 C channel in order carry out a decoupling of the observed 13 C satellites.

A third commonly used correlation experiment is HMBC [START_REF] Bax | 1 H And 13 C Assignments From Sensitivity-Enhanced Detection Of Heteronuclear Multiple-Bond Connectivity By 2D Multiple Quantum NMR[END_REF]. A low-pass filter is used in order to detect only smaller J-coupling and eliminate the single bond correlation.

This kind of experiment allow to assign also signal of quaternary carbon atoms that are not detected using HSQC and HMQC, for this reason a combination of HMBC with HMQC or HSQC is commonly performed in metabolomics studies in order to perform a complete signal assignment and chemical structure elucidation [START_REF] Emwas | NMR Spectroscopy for Metabolomics Research[END_REF]. Also in this case there are many variants of each described experiments and in any case the pulse sequence must be chosen according to the problem to be faced. The graphical representation of the three pulse sequences described in this section are reported in Figure 1.7. Pulse sequences commonly used for the heteronuclear correlation spectroscopy: the 1 H-13 C HMQC pulse sequences is reported in the box A) (hmqcqf Bruker standard pulses sequence), 1 H-13 C HMBC is reported in B) (hmbclpndqf Bruker standard pulses sequence) and 1 H-13 C HSQC pulse sequences is reported in C) (hsqcph Bruker standard pulses sequence).

2D J-Resolved Spectroscopy (J-Res)

In metabolomics studies, as we have seen in the previous sections, samples containing a high number of metabolites are analyzed and studied. This situation determines a poor dispersion of signals in the proton spectrum which is crowded not only due to the number of species studied, but also to an extensive splitting of the signals due to scalar coupling. The J-Res is one of the first 2D NMR experiments performed and was designed in order to allow a separation of the two contributions, chemical shift and scalar coupling, in a two-dimensional map able to lead to a reduction of spectral crowding and an increase of metabolite specificity [START_REF] Parella | Current Developments in homonuclear and Heteronuclear J-Resolved NMR Experiments[END_REF].

Indeed, 2D J-Res separates coupling and chemical shift information into two orthogonal dimensions enabling multiplet analysis. The one-dimensional spectrum ( 1 H or 13 C) is shown on the F2 dimension, while the couplings related to each chemical shift can be read on the indirect dimension, F1, at the chemical shift value of the signal whose multiplicity is to be studied. The pulse sequences of the J-Res is composed by a simple Echo experiment (90°- -180°) (Figure 1.8). After a 90º pulse, transverse magnetization evolve during the mixing time, then a 180º pulse is performed in order to remove chemical shift evolution and letting only the couplings free to evolve. This sequence is performed many times with a range of incremented delays for  and this allows to separate the contribution of the coupling on the second dimension. It is a with proton decoupling (jresxh).

Diffusion studies in metabolomics

Diffusion studies have been performed for long time on binary and ternary solutions with the aim of obtaining physical-chemistry properties of the studied systems. More complex, but also very interesting, are the studies on multicomponents systems, such as the case of natural and biological extracts involved in metabolomics analyses. Specifically, from the NMR point of view, diffusion measurements are particularly suitable for complex samples analysis because they provide an effective way to separate the components of the mixture according to their diffusion coefficients [START_REF] Yemloul | Self-Diffusion Coefficients Obtained from Proton Decoupled Carbon-13 Spectra for Analyzing a Mixture of Terpenes[END_REF]. In order to understand how NMR spectroscopy provides this type of information and all the applications deriving from it, in this section the diffusion phenomenon will be briefly introduced and the NMR techniques commonly used in order to carry out this kind of study will be described. Notice that the overview that is provided in this section is absolutely not enough to carry out a complete discussion of the phenomenon, but, in our opinion, it appears to be sufficient for the purposes of understanding the work carried out and the experimental results obtained during this Ph.D.

Diffusion theory

In a substance composed by two or more species with different molecular shapes and whose concentrations vary within the material, there is tendency for mass to move. This microscopic process is called diffusion and results from random molecular motions. The best way to describe axis at constant temperature and pressure is described using the following equation:

𝐽 𝐴 𝑧 = -𝐷 𝐴 ∇ 𝐴 𝑍 (1.42)
The equation (1.42) is known as Fick's first law and it states that the diffusive flux of A in the z-axis direction is proportional to the concentration gradient along the z-axis ∇ 𝐴 𝑍 through the diffusion coefficient 𝐷 𝐴 also called diffusivity [START_REF] Cussler | Diffusion Mass Transfer in Fluid Systems[END_REF]. The magnitude of diffusivity is dependent from the mass and the shape of the molecule, temperature and viscosity of the solvent. To take into account these properties, the Stokes-Einstein relation is commonly used as a model for the diffusion in liquids. The relation is the equation first derived by Einstein in his Ph.D. thesis for the diffusion coefficient of a "Stokes" particle undergoing Brownian motion in a quiescent fluid at uniform temperature [START_REF] Miller | The Stokes-Einstein Law for Diffusion in Solution[END_REF][START_REF] Einstein | Investigations of the Theory of the Brownian Movement[END_REF][START_REF] Edward | Molecular Volumes and the Stokes-Einstein Equations[END_REF][START_REF] Kholodenko | Generalized Stokes-Einstein Equation for Spherical Particle Suspensions[END_REF][START_REF] Bird | Transport Phenomena[END_REF]. In particular Einstein has shown that the relation between molecular motion and diffusion in liquid may be expressed as function of time (t) and is proportional to the mean square of the deviation in a given direction (∆):

𝐷 = ∆ 2 ̅̅̅ 2𝑡 (1.43)
Assuming that the particles possess the same kinetic energy of gas molecules at a given temperature (T), the mean square of the deviation is:

∆ 2 ̅̅̅ = 2𝑅𝑇 𝑁 • 𝑡 𝐶 (1.44)
where R is the gas constant, N is the Avogadro's number and C is the so called frictional resistance of the molecule. Stokes has shown that for spherical particles C is proportional to the viscosity of the medium (𝜇) and to the radius of the diffusing particle (r):

𝐶 = 6𝜋𝜇𝑟 (1.45)
By making the necessary substitutions, and considering that 𝑅 𝑁 is equal to the Boltzmann constant kb, the diffusion equation for a species i, known as the Stokes-Einstein equation, is obtained:

𝐷 𝑖 = 𝑘 𝑏 𝑇 6𝜋𝑟 𝑖 𝜇 (1.46)
This relation has been subjected to modification during the years in order to obtain an equation able to take into account other characteristics of the system (the shape of the particle for example) [START_REF] Zwanzig | Modifications of Stokes-Einstein Formula[END_REF][START_REF] Kooijman | A Modification of the Stokes-Einstein Equation for Diffusivities in Diluite Binary Mixtures[END_REF][START_REF] Achuthan | A Modified Stokes-Einstein Equation for  Aggregation[END_REF][START_REF] Ohtori | Molecular size and Shape effects: Rotational Diffusion and the Stokes. Einsein-Debye Relation[END_REF]. However the Stokes Einstein relation is the one able to cover a wide range of conditions and allow to obtain a modelled diffusion coefficient that is not far from the experimental one [START_REF] Miller | The Stokes-Einstein Law for Diffusion in Solution[END_REF]Gaskell, 1984;[START_REF] Mason | Estimating the Viscoelastic Moduli of Complex Fluids Using the Generalizad Stokes-Einstein Equation[END_REF][START_REF] Harris | Communications: The fractional Stokes-Einstein Equation: Application to Water[END_REF][START_REF] Thakurathi | The Stokes-Einstein Equation and the Diffusion of Ferrocene in Imidazolium-Based Ionic Liquids Studied by Cyclic Voltammetry: Effect of Cation ion Symmetry and Alkyl Chain Length[END_REF].

Form the experimental point of view it is curious to note that an established and universally applicable technique for measuring diffusion coefficients does not exist. Indeed, unlike others transport properties for which standardized procedures are available, measurements of mass transfer studies have not a defined protocol. The main complication is due to the difficulty in the measuring point values of concentration, so different techniques are available to carry out this study and the existing experimental methods can be classified into direct and indirect methods [START_REF] Mehrer | Methods of Measuring Diffusion Coefficients in Diffusion in Solid Metals and Alloys[END_REF]. The direct methods evaluate the diffusion coefficient by measuring concentration of the diffusing species as a function of depth of penetration. Such methods include the wide variety of physicochemical methods like spectrophotometry. The disadvantages of direct methods are expensive, time consumption, many of them are systemintrusive and last but not least they are poor in accuracy. On the contrary indirect methods measure the changes of one of the system parameters that depend on the diffusion rate. These parameters could be the rate of pressure drop in a confined cell which is known as pressure decay method or magnetic field characteristics. The advantage of these methods is that they do not need to determine the change in composition. Among the possible indirect methods, Nuclear Magnetic Resonance emerges as a powerful tool able to lead to diffusion informations.

Diffusion ordered spectroscopy

The beginning of NMR in diffusion studies is dated 1950 when Hahn observed signal attenuation in spin echo experiments due to translational diffusion [START_REF] Hahn | Spin Echoes[END_REF]. Since then, several experiments were carried out: Carr and Purcell investigated this phenomenon modifying the pulse sequence used by Hahn [START_REF] Carr | Effect of Diffusion on Free Precession in Nuclear Magnetic Resonance Experiments[END_REF] but only in 1956 Torrey incorporated Fick's law into Bloch equations [START_REF] Torrey | Bloch Equations with Diffusion Terms[END_REF]. In 1965 Stejskal and Tanner used pulsed field gradient (PFG) experiment setting the basis of the modern complex experiments commonly used: DOSY (Diffusion Ordered SpectroscopY) [START_REF] Stejskal | Spin Diffusion Measurements: Spin Echoes in the Presence of a Time Dependent Field Gradient[END_REF][START_REF] Johnson | Diffusion Ordered Nuclear Magnetic Resonance Spectroscopy: Principles and Applications[END_REF]. This technique is also named "in tube chromatography" due to the possibility to virtually separate compounds in complex mixtures according to the molecular diffusion of each species. This is possible because DOSY experiments uses radio frequency pulses along with magnetic field gradients that encode spatial information. Indeed, when the transverse magnetization generated by a rf pulse is further submitted to a gradient pulse along the direction of the main magnetic field, the phase history of spins depends on both the rf field and the spin position with respect to the gradient field. If another gradient field is applied with the same intensity but opposite sign, the magnetization is refocused again. Due to molecular diffusion, magnetization is not perfectly refocused and this is the main cause of loss of coherence in the gradient echo. By measuring the amount of signal attenuation obtained using different gradient magnitude, diffusion of molecules can be studied and diffusion coefficient measured. The graphical representation of this phenomenon is reported in Figure 1.9.

Figure 1.9. Application of gradient pulses GZ after rf pulses. On the left is shown the sequence consisting of a 90° pulse followed by gradient field pulses along z with a length of . The NMR tube can be seen as composed by a series of plane that in the Figure are indicated using the ellipses. Before the experiment begins, the magnetization vectors within each plane are directed along z, the axis of the static magnetic field (a). After applying the first pulse, the magnetization along the sample precesses at the same rate and all the magnetization vectors are uniformly distributed on the plane (b). Afterward, the first gradient pulse twists the distribution of each magnetization in order to generate the spatial codification (c). If one spin moves from a position Z1 to Z2 during the spatial encoding, as shown by the circle and dashed line in blue, the spin generates a phase memory. This leads to incomplete refocusing after the application of the second gradient field pulse (d) and diffusion information can be obtained.

In order to better understand PFG based experiments, let us consider the simplest form of DOSY reported in Figure 1.10.

RF  G Z 90  a b c d Z a b c d Z 1 Z 2 FID 1 H 90 180 G Z  D
This pulses sequence is known by the name of spin-echo -SE-sequence and in this kind of experiment the magnetization is excited with a 90° pulse then it is uniformly dephased using a gradient pulse (having a magnitude G I and a duration of ). After a time of Δ from the 90 pulse, a 180° pulse is applied to invert the previously dephased magnetization than a second gradient pulse is applied in order to refocus the signal [START_REF] Carr | Effect of Diffusion on Free Precession in Nuclear Magnetic Resonance Experiments[END_REF][START_REF] Stejskal | Spin Diffusion Measurements: Spin Echoes in the Presence of a Time Dependent Field Gradient[END_REF][START_REF] Johnson | Diffusion Measurements by Magnetic Field Gradient Methods in Encyclopedia of Magnetic Resonance[END_REF]. The second gradient pulse can refocus the signals only if there are nuclei that have not moved longitudinally. If there is diffusion, the last gradient is not able to refocus the The attenuation of the intensity of the signal change according the following equation [START_REF] Johnson | Diffusion Ordered Nuclear Magnetic Resonance Spectroscopy: Principles and Applications[END_REF][START_REF] Johnson | Diffusion Measurements by Magnetic Field Gradient Methods in Encyclopedia of Magnetic Resonance[END_REF]:

𝐼 𝐼 = 𝐼 0 exp (-𝐷𝛾 2 𝐺 𝐼 2 𝛿 2 (∆ -𝛿 3 ⁄ )) × 10 -4 (1.47)
when 𝐼 𝐼 is the intensity (or the integral) of the considered signal, D is the diffusion coefficient,  is the gyromagnetic ratio, G I is the applied gradient and  and D are the diffusion gradient and the diffusion time. Using this equation the diffusion coefficient can be easily calculated from the experimental data. To notice that the shape of this equation is sigmoid like (Figure 1.12). In order to obtain an experimental attenuation that fits well this curve, and to calculate an accurate values of D, the two deltas' ( and D ) need to be appropriately chosen in a preliminary step consisting in the optimization of all the experimental parameters. The advantages of this easy pulse sequence are numerous. First of all, the possibility of recovering the maximum possible value of the signal. The disadvantages is due to the long time in which the magnetization is on the xy-plane. Indeed, in this condition the magnetization is subjected to the T2 relaxation and to the J-modulation effects. These two influences can lead to an important loss of signal that is particularly enhanced in strongly coupled spin systems [START_REF] Vold | Nuclear Magnetic Relaxation in Coupled Spin Systems[END_REF]. In order to avoid this kind of problem other pulse sequences were elaborate during the years. In particular were find out that a sequence containing more than one 90° pulse can generate different echoes. In particular the Stimulated Echo (STE) sequence (Figure 1.13) is of particular interest. It consists in three-90°-pulses sequence with a steady gradient that can generate echoes. The advantage of this sequence are due to the possibility to limit the evolution time for transverse magnetization so that J-modulation can be not very significant [START_REF] Woessner | Effect of Diffusion in Nuclear Magnetic Resonance SpinEcho Experiments[END_REF][START_REF] Tanner | Use of the Stimulated Echo in NMR Diffusion Studies[END_REF]. Main drawback of STE experiment is due to the possibility to obtain distorted signal caused by eddy currents induced by the gradient pulse, in particular due to the final gradient pulse [START_REF] Johnson | Diffusion Ordered Nuclear Magnetic Resonance Spectroscopy: Principles and Applications[END_REF]. The best ways to avoid the effect of eddy current are the use of shorter time between the pulse and to use modern instruments in which a peculiar radiofrequencies coil are designed or using widebore magnets. If the instrumentation does not allow to use this pulse sequence, LED sequences can be used (Longitudinal Eddy current Delay). This pulse sequence is obtained adding a fourth 90° pulse in order to keep the magnetization in the longitudinal direction while the eddy currents decay. After a fifth 90° pulse recall the magnetization and the FID is acquired [START_REF] Gibbs | A PFG NMR experiment for accurate diffusion and flow studies in the presence of Eddy currents[END_REF][START_REF] Johnson | Diffusion Ordered Nuclear Magnetic Resonance Spectroscopy: Principles and Applications[END_REF]. The LED sequence improves the quality of the spectra even if it still suffers from slowly decaying eddy current. Sometimes the addition of three gradient pre-pulse can enhance the effectiveness of the sequence but they can also create problems due to the introduction of additional heat. All of these illustrated sequences have pros and cons. The choice of the most adequate sequence is surely a function of the equipment available but also of the system studied. Indeed, when very small diffusion coefficients are involved, large gradient amplitudes are required to obtain adequate signal attenuation. As disturbances to the local field increase with the amplitude of the gradient pulse, the LED feature is often required. However, when only modest gradients are required and an efficient, well designed probe with active shielding is available, the STE sequence should be considered [START_REF] Johnson | Diffusion Ordered Nuclear Magnetic Resonance Spectroscopy: Principles and Applications[END_REF]. to the study of Hollis on aspirine, phenacetine and caffeine, so this technique can be considered as old as NMR itself [START_REF] Kumar Bharti | Quantitative 1 H NMR Spectroscopy[END_REF]. During the years there has been growing interest in quantitative NMR studies due to its numerous advantages and this also led to the fact that today qNMR is largely applied in NMR-based metabolomics. In fact, using this robust and validated methodology [START_REF] Malz | Validation of Quantitative NMR[END_REF], it is possible to obtain in a short time, an absolute and a relative quantification of several metabolites present in biological samples simultaneusly without the need of specific sample preparation and also without the need for separate the individual components. In order to perform a quantitative study, a known amount of an internal standard must be added to the sample. The characteristics that this species have to possess are the following: it must be available in a highly pure form, inexpensive, stable and chemically inert, non volatile and non hygroscopic. It must be soluble in the used NMR solvent and the NMR signals generated from this species have not to interfere with the signal associated to the target metabolite [START_REF] Pauli | Quantitative 1H NMR: Development and Potential of a Method for Natural Products Analysis[END_REF]. Once the sample has been prepared, the subsequent phases involve a first step of experimental design and parameter selection, than, after the data acquisition, a post-acquisition processing step leads to the desired quantitative results. In particular, in order to obtain integrals accurately and reproducibily proportional to the number of observed nuclei, the data acquisition parameters need to be carrefully chosen and oprimized.

First of all the shimming procedure need to be carefully performed in order to avoid signal distortion and inappropriate peack shapes. Also tuning and matching procedure need to be correctly completed because improper values may affect the effectiveness of the 90° pulse and this results in a variation in the intensity of the signal. Than relaxation delays (D1), one of the most important parameter, need to be set. It is the the delay between two consecutive FID accumulation: studies have shown that D1 must be set long enough in qNMR experiments to fully relax the nucleus and generate informative integrals. This is the reason why a preliminary T1 measurement is required in order to perform a quantitative study [START_REF] Yu | Quantitative Analysis of Flavonoids in Glycyrrhiza Uralensis Fisch by 1 H-qNMR[END_REF]. In addition, also the acquisition time (AQ) need to be long enough to avoid truncation of FID that can lead to inaccurate integral measurements. Usually D1 and AQ are chosen in order to satisfy the relation AQ+D1>5T1 when a 90° pulse is used. Also the receiver gain (RG) is an important parameter to set because it can cause baseline distortion if is too high or it causes a very low Signal-to-Noise ratio (S/N) if it is too low.

Once the FID has been acquired, data must be processed using appropriate post-acquisition processing techniques. The first step consist in the zero filling procedure in order to increase the spectral resolution: this is performed by processing the spectra by increasing the data point to be Fourier-transformed taking care not to truncate the acquired signal. Sometimes some weighting function need to be applied to the FID in order to enhance some features of the spectra. For example a typical function used in order to obtain a better S/N is the apodization by an exponential function (EM). By multiplying the FID with this function an improvement of the S/N is achieved, but also an increase in the line broadening is obtained. There are other functions that can be used as filter, however all of them should be used with some caution in order to obtain accurate quantitative information. If this kind of functions are not used in moderation, validation of the method need to be performed.

Once the FID has been Fourier-transformed, the obtained spectrum is phase and baseline corrected, the chemical shifts are calibrated to reference and then the selected signals are integrated. The relationship between the signals integrals and the concentrations of metabolite (x) and standard (st) is expressed by the following equation [START_REF] Kumar Bharti | Quantitative 1 H NMR Spectroscopy[END_REF]:

𝑀 𝑥 𝑀 𝑠𝑡 = 𝐼 𝑥 𝐼 𝑠𝑡 • 𝑁 𝑠𝑡 𝑁 𝑥 (1.48)
where M, I and N represent respectively the quantity in moles, the integral and the number of protons that generate the considered signal.

Some physical-chemistry properties can effect qNMR analysis and are needed to be taken into account. The pH, for example, is important because it may cause drift in chemical shift that can lead to overlapping signal in the spectrum but it can also cause change in relaxation time as ionic strength does too. It must be emphasized that in the case of aqueous samples, a prerequisite of an accurate quantification is the selection of a good water-suppression technique. Indeed, presaturation cause partial saturation of the signal and for this reason the quantification cannot be carried out and in this case WATER GATE sequence is preferable [START_REF] Singh | The Application of Absolute Quantitative 1 H NMR Spectroscopy in Drug Discovery and Development[END_REF]. It should be noted that, when we are dealing with very complex mixtures such as biological fluids or natural extracts, total or partial overlapping of signals can occur. In this case, before carrying out the integration, it is necessary to individuate the contributions due to each signal. This is done through a process called deconvolution which, by using certain functions and optimizing their parameters, it allows to reproduce the complex signal present in the spectrum and, once a good fitting has been obtained, it is possible to integrate the final optimized function. The algorithm that allow to perform this kind of procedure is implemented in most of the spectral analysis software. In this thesis, deconvolution was carried out using TopSpin, the software used for all the spectral processing.

All the step involved in the qNMR method are represented in the Figure 1.15. To note that, even if the proton quantitative NMR study is the most common, qNMR can be carried out by observing other nuclei. The limit of this procedure is linked to the low natural abundance of the species, the long relaxation delay and the possibility to have nOe effect.

Indeed heteronuclear experiment performed using a 1 H broad band decoupling cause distortion in the integral due to the nOe. This can be reduced by decoupling during the acquisition time and using very long values of D1. These criteria, linked to the recent progress in the development of high-field magnets and CryoProbe technologies, provide an opportunity to perform quantitative studies by observing nuclei different than proton in order to analyze samples in low concentrations in metabolomics and natural products field.

The described methodology represents the approach commonly adopted for the quantification via NMR spectroscopy. However, the use of a known amount of some internal calibration standard has several drawbacks. The T1 of the reference compound must be considered, for example. Also the sample recovery can be problematic due to the contamination of the sample by the standard. Last, the most important, there are a number of consideration to make in order to choose a suitable reference compound, as already discussed. An alternative technique called Akoka [START_REF] Akoka | Concentration Measurement by Proton NMR Using the ERETIC Method[END_REF]. The ERETIC method provides a reference signal, synthesized by an electronic device, which can be used for the determination of absolute concentrations.

Indeed, rather than use a standard compound, in ERETIC the spectrum is "spiked" using a signal generated on a free channel of the NMR spectrometer. The total NMR signal sampled by the receiver has a new frequency component added and a subsequent Fourier transform will yield a spectrum with a "synthetic" peak added in the spectrum. An initial calibration of the area of this eretic signal against a standard of known concentration then permits the use of this signal to quantify components of unknown concentration in complex mixture. The experiment is fast and there is any dependence on the relaxation times of the reference signal. Additionally, it is extremely easy to move the position of the eretic peak to a favourable position in the spectrum, away from analyte signals of interest. This approach is not restricted only to 1 H nucleus but can be easily extended to other nuclei and also to 2D experiments [START_REF] Michel | The Application of the ERETIC Method to 2D-NMR[END_REF]. Moreover, this methodology lays the foundations for the in vivo NMR quantitative study [START_REF] Marro | Quantitative In Vivo Magnetic Resonance Spectroscopy Using Synthetic Signal Injection[END_REF]. This method however requires a specific instrumentation, not available during this Ph.D thesis

Statistical analysis

NMR spectroscopy is a powerful tool in metabolomics application as has been largely proven in the previous sections. Indeed, thanks to NMR spectroscopy it is possible to recognize and quantify a number of species present in the samples studied. Anyway, the information contained in a single NMR spectrum is so large that a statistical analysis is mandatory if we want to study all the facets of the system. Actually, an NMR experiment records thousands of variables for every sample analysed providing a very complex dataset. Chemometrics and in particular multivariate data analysis, provide a valuable tool for the study of this data matrix in order to recover as much information as possible. This kind of approach can be chosen for different reasons, it can be performed in order to: a) make an exploratory analysis able to detect patterns and outliers; b) classify the samples and discriminate between groups and c) make a regression analysis in order to estimate relationship existing between dependent and indipendent variables and make predictions. In this work only the first approach has been used and in the next paragraphs the way the dataset is obtained from the experimental spectra is described and the approach used to perform the exploratory analysis of the data is briefly discussed.

Data processing for statistical analysis

The most used NMR experiment for the statistical treatment is the 1D 1 H NMR. Each spectrum is not readily suitable for the chemometric study as it is and raw data need to be transformed into an appropriate form for the subsequent statistical analyses. The preliminar spectral treatment needed is similar to the one commonly applied on the spectra for the quantitative study discussed in the section 1.3.2.3. Indeed, the FIDs of each 1D 1 H NMR experiment must be zero filled before to be Fourier-transformed in order to provide an high spectral resolution.

Then in the obtained spectrum phase and baseline must be properly adjusted and the chemical shifts need to be aligned by calibrating a known resonance of a reference signal. This procedure is done in order to avoid mismatching between different spectra that lead to errors during the construction of the data matrix and consequently misinterpretations of the results. The data matrix is a matrix in which the samples, also called object, are reported on the rows (p) and the variables, or features, on the columns (n). This matrix can be obtained following two procedure: a) it can be made using as variables the intensity of each points of the spectrum and b) dividing the entire spectrum into small regions which are integrated and the obtained integrals represent the variables. The first methodology is difficult to use because usually the spectra are recorded by sampling 16-32K points and this means that for each sample there are 16-32K variables, a really high number especially if compared with the number of samples studied which generally does not exceed a hundred. This methodology also represents a problem because by operating in this way even minimal fluctuations in the alignment of the spectra can be highlighted by the statistical analysis and therefore it is easier to make mistakes. Last, it represents a difficulty also for the calculators used for statistical study which take much longer to process matrices of this size. Indeed, the best way to perform this study is is to use the procedure described in b, commonly known as binning. Binning procedure consists of dividing the spectra into small regions called buckets (usually 0.04 ppm), which are large enough to include chemical shift variations [START_REF] Craig | Scaling and Normalization Effects in NMR Spectroscopic Metabonomic Data Sets[END_REF]. The integral of each bucket is then calculated providing an acceptable and significant number of variables. In addition, sometimes a variables selection can be made. Indeed, if a preliminary study has provided information on which variables are more informative than others, or if it is already known from the literature which metabolites play an important role in the process to be studied, then it is possible to select some regions to use as variables. Furthermore, if in the spectra there are regions characterized by a very low S/N ratio, one can also choose not to consider this part of the spectra and this leads to a further decrease in the size of the data. Once the number of variables has been chosen and the binning procedure completed, a normalization step is required before building the data matrix. Indeed, as already mentioned, metabolomic responses to external stimuli (aging, diet, exercise, or life style) are reflected in differences in metabolites concentration that is translate into variations in the intensity/integral of the signals in the spectra. However changes in signal intensities can be also attributed to differences in the amount of material analyzed and to dilution rather than changes in metabolic responses. Such differences, together with different relaxation times or variations in rf pulse calibration, can be corrected by normalization. Then the normalization step removes all these effects making spectra comparable and affected only by the metabolic responses variability [START_REF] Euceda | Preprocessing of NMR Metabolomics Data[END_REF]. Different kind of normalization can be performed, the most used being normalization to an internal standard, normalization to a particular reference peak or normalization total intensity or total area. In the works reported in this Ph.D. thesis the data have been normalized respect to the total area by setting the global integral of the each spectrum equal to 100. The integrals obtained are then arranged in the data matrix and, before carrying out the statistical treatment, the data matrix obtained must be further refined. Indeed, another important preprocessing step for NMR data is mean-centering and scaling procedures. Meancentering is a mathematical procedure which consists in subtracting the average value of each variable from each single value of the matrix. This allow to correcting displacement between existing metabolites in high and low concentrations, leading to values that vary around zero rather than around the average value, thus allowing to have a simplest model possible. Meancentering is commonly used in combination with scaling method. Scaling procedure, aims to balance signal area variances due to difference in aboundance of metabolites. Indeed, most aboundant metabolites can exhibit larger variation between samples. This large differences can mask variations in less aboundant metabolites that also possess biological importance. While normalization is independently performed on each sample, scaling operations depend on all samples in the dataset. The scaling operation can be executed by using different methodologies and in this work the autoscaling has been performed on the data matrix. Autoscaling is a method in which all the variable have unit variance. This is done by dividing each value in a column by the standard deviation after mean centering [START_REF] Craig | Scaling and Normalization Effects in NMR Spectroscopic Metabonomic Data Sets[END_REF][START_REF] Euceda | Preprocessing of NMR Metabolomics Data[END_REF]. At this point, the data matrix X, obtained for the p samples considering the n variables (p × n), can be used for the statistical treatment. The tool used in this Ph.D. thesis for the multivariate study is the Principal Component Analysis (PCA) that will be described in the following section. All

Principal Component Analysis (PCA)

The Principal Component Analysis (PCA) is an unsupervised method commonly used in metabolomics. Invented by [START_REF] Pearson | On lines and Planes of Closest Fit to Systems of Points in Space[END_REF][START_REF] Pearson | On lines and Planes of Closest Fit to Systems of Points in Space[END_REF] and rediscovered by Wold [START_REF] Wold | Principal Component Analysis. Chemometr[END_REF], PCA is a statistical procedure applied to the data matrix in order to perform a compression of a high-dimensional dataset [START_REF] Varmuza | Introduction to Multivariate Statistical Analysis in Chemometrics[END_REF][START_REF] Ren | Computational and Statistical Analysis of Metabolomics Data[END_REF] New variables (PCs) are uncorrelated in contrast to the original ones and arranged in decreasing order of variance. This means that the first PC accounts for as much of the variability in the data as possible so that each succeeding component accounts decreasing amounts of variance.

Therefore, the higher PCs can be discarded and keeping only the first PCs (e.g. three or less)

the number of variables is reduced without significant loss of information and data patterns can be more clearly visualised. Usually, to identify the number of components to be used, three criteria can be used: a) total variance share explained; b) scree-graph and c) eigenvalue one.

According to the first criterion, a number of PCs must be considered such that they take into account a sufficiently high percentage of total variance (at least 70%). The second criterion makes use of a graph called scree-graph containing eigenvalues as a function of the number of PCs. Since the eigenvalues are decreasing, the graph takes the form of a broken line with an always negative slope. By analyzing the graph, it will be possible to identify a point in which a sudden change in slope occurs, at which the number k of PCs to be considered is identified.

However, it may happen that the decrease of the eigenvalues is gradual and the graph does not show obvious jumps; in these cases it is necessary to use different methods (Cattel, 1966). The third criterion suggests considering all the PCs whose eigenvalue is greater than 1. This criterion derives from the fact that the eigenvalue of a PC is equal to its variance and that by operating on standardized variables these have unit variance. Therefore, it is decided to keep a PC only if it explains a share of total variance greater than that of a single variable. From a mathematical point of view PCA is done by executing the following steps [START_REF] Jolliffe | Principal Component Analysis[END_REF][START_REF] References Abdi | Principal Component Analysis[END_REF][START_REF] Paul | Methodological Analysis of Principal Component Analysis (PCA) Method[END_REF]:

1 Write the data matrix X (p × n) following the methodology discussed in the previous section.

2 Construct the covariance matrix C.

The covariance matrix is a matrix (n × n) that represents the variation of each variable respect to the others so it has the following form:

𝐶𝑜𝑣𝑎𝑟𝑖𝑎𝑛𝑐𝑒 𝑀𝑎𝑡𝑟𝑖𝑥 (𝑪) = [ 𝐶𝑜𝑣(𝑎, 𝑎) 𝐶𝑜𝑣(𝑎, 𝑏) 𝐶𝑜𝑣(𝑎, 𝑐) 𝐶𝑜𝑣(𝑏, 𝑎) 𝐶𝑜𝑣(𝑏, 𝑏) 𝐶𝑜𝑣(𝑏, 𝑐) 𝐶𝑜𝑣(𝑐, 𝑎) 𝐶𝑜𝑣(𝑐, 𝑏) 𝐶𝑜𝑣(𝑐, 𝑐) ] (1.50)
where each term is obtained using the following equation:

𝐶𝑜𝑣(𝑎, 𝑏) = 1 𝑛 -1 ∑(𝑎 𝑖 -𝑎 ̅)(𝑏 𝑖 -𝑏 ̅ ) 𝑛 𝑖=1 (1.51)
In this equation n is the number of the variables, ai and bi are respectively the values of the variables a and b for the i th object and 𝑎 ̅ and 𝑏 ̅ are their mean values.

Since 𝐶𝑜𝑣(𝑎, 𝑏) = 𝐶𝑜𝑣(𝑏, 𝑎), the covariance matrix is a symmetric matrix and, as variance measures the variation of a single variable, on the diagonal of the covariance matrix, variances of each variable are reported (𝐶𝑜𝑣(𝑎, 𝑎) = 𝑉𝑎𝑟(𝑎)).

When X is autoscaled, the mean values are zero and the variances are 1 so the covariance matrix is named correlation matrix.

From the matricial point of view, the covariance matrix is obtained from the matrix of the difference 𝑋 ̃ between the data matrix 𝑋 and the matrix of the means 𝑋 ̅ (see equation 1.52). In particular, the covariance matrix is found to be the square of the differences matrix as reported in equations 1.53

𝑿 ̃= 𝑿 -𝑿 ̅ (1.52) 𝑪 = 1 (𝑛 -1) 𝑿 ̃′𝑿 ̃ (1.53)
where n is the number of variables and (𝑛 -1) are the degree of freedom. In case the data matrix is centered, the means matrix is null and the two matrix, the differences and the data matrices, will be the same.

Eigenvalues 𝜆 𝑖 are then calculated according the classical relation:

𝑑𝑒𝑡(𝑪 -𝜆 𝑖 𝑬) = 0 (1.54)
Where E is the identity matrix.

The eigenvalue matrix Λ (n × n), whose diagonals elements are the eigenvalues λi (i = 1, ...,n), is easily obtained by diagonalizating C:

Last the eigenvectors zi, associated to each each λi, each of which has the form (z1, z2, z3, … , zn), are calculated as follows:

(𝑪 -𝜆 𝑖 𝑬)𝒛 𝒊 = 0 (1.56)

From the equation is derived the eigenvector matrix Z (n × n) for which is valid the following relations:

𝑪 = 𝒁 𝜦 𝒁 𝑻 (1.57)
This last equation corresponds to a rotation operation that defines a new space (eigenspace), which is orthogonal and for which the eigenvectors are the versors. The axes of the eigenspace defined by Z are the axes relative to the maximum directions variance, in decreasing order, and are called Principal Components (PCs). Each eigenvalue obtained in 3) represents the variance associated with the corresponding principal component and the sum of all eigenvalues is equals to the total variance of the data.

The matrix containing the coordinates of the objects in the new principal components space, 𝑻 (p × n), is then obtained by multiplying the eigenvector matrix Z by the original data matrix X, as shown below.

𝑻 = 𝑿 𝒁

(1.58)

𝜦 = 𝑑𝑖𝑎𝑔 𝑪 (1.55)
The points 4 and 5 produce two matrices called the scores, T, and the loadings, Z, that are able to furnish useful informations for the analyzed problem. Indeed, the rows of the scores matrix, T, correspond to the objects, that in this case are the samples (as in the original dataset matrix), whereas the columns are the PCs. The values in a given column represent the samples coordinates for that PCs axis. Plots of the scores for different pairs of PCs reveal which samples have similar properties: they appear as clusters on the two or three-dimensional plots. Instead, each column vector of the loadings matrix, Z, represents one PC and the elements of the vector relate that PC to the original variables that are the NMR data. The column plots have the same form as the NMR spectra and provide interpretable chemical information on the variables responsible for the clustering. Each term 𝑧 𝑖𝑗 of the loading matrix, Z, are the standardized linear coefficient of the linear combination reported in the equation 1.49. They have values between +1 and -1, and the sum of their squares is equal to 1, as summarized by the following relations.

-1 < 𝑧 𝑖𝑗 < +1

(1.59)

∑ 𝑧 𝑖𝑗 2 𝑛 𝑗=1 = 1 (1.60)
From a pratical point of view, PCA can be carried out using different softwares. In this Ph.D.

work, the software R (R Core Team, 2019) has been employed for the calculations and, in particular, the code used is reported in Figure 1.17.

Figure 1.17. Code used in order to perform the principal component analysis by using the software R.

To note that the PCA can be also performed in a faster way by using functions implemented in some R package such as the case of the function prcomp(). In our work the procedure reported in Figure 1.17 was performed together with the prcomp() command and, since the two methods led to the same solutions, in the experimental part of this thesis only the results obtained following the methodology shown in Figure 1.17 are reported.

> Data<-read.csv2("Data.csv",na.strings=999) #The file containing the data, saved with csv extension, is read by the software > Da<-data.matrix(Data) # The file just read is declared a data matrix > n<-nrow(Da) > Un<-matrix(1,n,n) # The matrix of means is calculated > Me<-Un%*%Da/n > P<-Da-Me # The differences matrix is obtained > R<-crossprod(P)/(n-1) # The correlation matrix is calculated > A>-eigen(R) # Eigenvalues and eigenvector are calculated > U>-Z%*%A[ [START_REF] All | A eview on Vermicomposting of Organi Wastes[END_REF]] # The matrix of the objects in the new principal components space is obtained > V<-cumsum((A$values)/sum(A$values))*100 # A vector containing the percentage of variance explained by each PCs is calculated

NMR in partially ordered media

The study of the molecular properties of flexible species such as structure and conformational equilibrium are knots that chemists and biologists have always tried to undo. Indeed, this is a crucial point when dealing with flexible molecules possessing biological or pharmacological activity. This is due to the fact that the spatial arrangement may favor the establishment of a series of intramolecular interactions which determine the conformations that the molecule can assume in the reaction environment. Referring to the "key-lock" model, a certain enzyme is able to interact with a substrate only if the latter has a "shape" that is complementary to the active site [START_REF] Segel | Enzyme Kinteics[END_REF]. Actually, this description greatly simplifies the process that occurs in living organisms; in fact the macromolecular ligand can distinguish different conformations of a molecule and therefore each thermally accessible conformer plays an important role in molecular recognition (Boehr et al, 2009). The possibility to carry out experimental study on the structural and conformational behavior, in solution, assumes particular importance as it allows to obtain information relating to bioactive species in conditions as close as possible to those in which they realize their biological activity. Although it is possible to use various experimental methods to obtain this kind of information in solid and gaseous states, the techniques to be used to perform a structural and conformational analysis of species in solution are few [START_REF] Kappe | Conformational Analysis of 4-Aryl-Dihydropyrimidine Calcium Channel Modulators. A Comparison of Ab Initio, Semiempirical and X-Ray Crystallographic Studies[END_REF][START_REF] Singh | Conformational Analysis and Vibrational Study of Daidzein by Using FT-IR And FT-Raman Spectroscopies and DFT Calculations[END_REF]. Among the different techniques available, NMR spectroscopy proved to be an analytical method of the highest value to investigate these issues.

Classical NMR strategies for the study of molecular structure and conformation in solution are based on scalar spin-spin couplings ( 3 J) measurement and Nuclear Overhauser Effect (NOE) measurement [START_REF] Karimi-Nejad | Conformation of Valine Side Chains in Ribonuclease T1 Determined by NMR Studies of Homonuclear and Heteronuclear 3J Coupling Constants[END_REF][START_REF] Farès | Conformational Investigations in Flexible Molecules Using Orientational NMR Constraints in Combination with 3J-Couplings and NOE Distances[END_REF]. However, this kind of parameters have short range nature and this cause limitations in obtaining information between distant atoms. In addition, the information obtained from the spectra are averaged over the different conformations and this represent a problem in the case of molecules possessing more than one conformers, as the biologically active species, since the estimation of a single conformer, even if the most stable, is not longer sufficient. The limits just discussed are overcome by the use of NMR spectroscopy in ordered media in which the target molecule can be dissolved and be affected by the ordering action of the phase. Indeed, the anisotropy guaranteed by a liquid crystalline phase or a gel phase, allows easily to obtain information relating to interactions whose existence is strictly connected to the spatial arrangement of the observed nuclei [START_REF] Gill | Constitutional, Configurational and Conformational Analysis of Small Molecules on the Basis of NMR Residual Dipolar Couplings[END_REF]. In fact, the NMR spectra of samples in these phases are extremely rich in information as they are governed by interactions not only of an isotropic but also anisotropic nature. Among the four anisotropic interactions, i.e. chemical shift anisotropy, indirect coupling anisotropy, dipolar coupling and quadrupolar interaction, the direct dipolar interaction is recognized as the one that provides more information on molecular structure, orientation and conformation (Emsley & Lindon, 1975;Canet, 1996;Burnell & de Lange, 2003). Indeed, as states in section 1.2, dipolar couplings depend on the distances between the interacting nuclei and on the orientations of the internuclear vectors with respect to the external magnetic field, for this reason, they represent a sensitive probe to long-range interactions of portions of the molecule also spatially distant from each other (Di Pietro et al, 2017;Liu et al, 2017).

In the last 50 years, NMR in strongly ordered media has been applied together with spectral simulation program, in order to study both the structure, order and dynamic of mesogenic molecules and to investigate the properties of the solutes dissolved therein. At the beginning this kind of study was performed on small rigid molecules in order to obtain very accurate structural and orientational parameters. The extension of this methodology to more complex and flexible molecules entails obtaining very complex spectra, due to the large number of dipolar couplings and their magnitude, thus restricting the application to highly symmetrical molecules with no more than 10 or 12 spins [START_REF] Luy | An Easy and Scalable Method for the Partial Alignment of Organic Molecules for Measuring Residual Dipolar Couplings[END_REF], Celebre et al, 2012[START_REF] Di Pietro | A Combined LX-NMR and Molecular Dynamics Investigation of the Bulk and Local Structure of Ionic Liquid Crystals[END_REF]. The need to study more complex systems (larger molecules or species characterized by the presence of more torsional angles), has made it necessary to develop new systems capable of inducing a weak order in the dissolved solute in order to obtain less complex spectra with characteristics similar to those of the first order from which it is possible to extract information by simple inspection. In general, what makes partially ordered media ideal is their ability to provide a matrix characterized by an "intermediate" order between the one typical of the solid phase, characterized by both a positional and orientational long-range order, and that of the liquid phase, characterized instead by the absence of these orders. This kind of phase is generally made up of molecules free to diffuse within it but which are organized into microdomains within which they are statistically aligned along a preferential direction called director 𝑑 ̂. In the absence of external forces, the director is not constant throughout the volume but, if it is subjected to the action of an external field of sufficient intensity, for example the magnetic field used in NMR spectroscopy 𝐵 0 ⃗⃗⃗⃗ , it induces in the phase an alignment and, consequently, an order of directors. This implies that the phase acquires a long-range statistical order which leads to greater stability of the system, stability linked to a reduction of the free energy G which is equal to:

∆𝐺 = - ∆𝜒|𝐵 0 ⃗⃗⃗⃗ | 2 (3𝑐𝑜𝑠 2 𝜃-1)

6

(1.61)

where

⊥ - = D    //
(commonly called "diamagnetic anisotropy") is the difference between the magnetic susceptibility of the anisotropic phase measured parallel and perpendicular to the director, and θ is the angle between the director and the magnetic field. The director will therefore align itself parallel to 𝐵 0 ⃗⃗⃗⃗ for positive values of Δχ and perpendicularly for negative Δχ (Burnell & de Lange, 2003).

Weakly ordering media

Historically, the first and most widespread alignment media used were thermotropic liquid crystals. In the early 1960s Saupe and Englert reported the first NMR spectrum of benzene dissolved in a partially ordering medium consisting of a thermotropic nematic liquid crystalline phase [START_REF] Saupe | High-Resolution Nuclear Magnetic Resonance Spectra of Orientated Molecules[END_REF]. The thermotropic liquid crystals induce a rather high order in the solutes dissolved in them and, therefore, the resulting spectra are composed by complex second order signals whose analysis requires the use of appropriate simulation programs (Celebre et al, 1994). This limits the use of these phases to molecules characterized by spin systems no larger than 10-12 nuclei. Hence the need to develop new systems capable of inducing a weak order in the solute dissolved therein in order to obtain less complex spectra and with characteristics similar to those of the first order. Currently, the weakly orienting systems used in NMR studies consist of lyotropic liquid crystalline mesophases and gelatinous phases (Emsley & Lindon, 1975;[START_REF] Luy | An Easy and Scalable Method for the Partial Alignment of Organic Molecules for Measuring Residual Dipolar Couplings[END_REF]. While the gels phases used are polymerbased, the liquid crystalline mesophases can be obtained starting from different species suitably dissolved in a compatible solvent. Indeed, it is possible to obtain a lytropic phase by using, for example, amphiphilic compounds [START_REF] Tjandra | Direct Measurement of Distances and Angles in Biomolecules by NMR in a Dilute Liquid Crystalline Medium[END_REF], viruses [START_REF] Zweckstetter | Characterization of Molecular Alignment in Aqueous Suspensions of Pf1 Bacteriophage[END_REF] or antibiotic particles [START_REF] Maltsev | Improved Cross Validation of a Static Ubiquitin Structure Derived from High Precision Residual Dipolar Couplings Measured in a Drug-Based Liquid Crystalline Phase[END_REF] in addition to the classical polymers (Aroulanda et al, 2001). To date, the most used systems, both gelatinous and liquid crystalline, are those with a polymeric matrix [START_REF] Schmidts | Perspectives in the Application of Residual Dipolar Couplings in the Structure Elucidation of Weakly Aligned Small Molecules[END_REF] but research in this field is constantly evolving as evidenced by the numerous works in the literature on new alignment media [START_REF] Lei | A Self-Assembled Oligopeptides as a Versatile NMR Alignment Medium for the Measurement of Residual Dipolar Couplings in Methanol[END_REF][START_REF] Leyendecker | Development of New Supramolecular Lyotropic Liquid Crystals and Their Application as Alignment Media for Organic Compounds[END_REF][START_REF] Schwab | Polyaspartates as Thermoresponsive Enantiodifferentating Helically Chiral Alignment Media for Anisotropic NMR Spectroscopy[END_REF]. Based on the conditions necessary for obtaining a liquid-crystalline phase, liquid crystals can be divided into two broad categories: thermotropic and lyotropic. Thermotropics are mesogenic compounds that exhibit mesophases as the temperature varies, while lyotropics are system formed by dissolving a mesogenic compound in a suitable solvent under appropriate conditions of concentration, temperature and pressure [START_REF] Priestley | Introduction to Lyquid Crystals[END_REF]. Both categories are often used; but, while thermotropic liquid crystals induce a rather high degree of order in the solute dissolved therein, lyotropic liquid crystals, instead, induce a weak order and the NMR spectra mostly retain the simplicity normally observed in isotropic solution (first-order features) [START_REF] Harris | Nuclear Magnetic Resonance Spectroscopy[END_REF][START_REF] Günther | NMR Spectroscopy, Basic Principles, Concepts, and Applications in Chemistry[END_REF]. Indeed, when the alignment of the solutes is sufficiently weak, the anisotropic interactions are averaged significantly but not entirely, and the size of dipolar couplings are usually comparable to the scalar couplings and can be observed as an additional contribution to the line splitting. For this reason, this class of liquid crystals phase is widely used for application to larger systems of lower symmetry, as bioactive molecules [START_REF] Rückert | Alignment of Biological Macromolecules in Novel Nonionic Liquid Crystalline Media for NMR Experiments[END_REF][START_REF] Bax | Weak Alignment Offers New NMR Opportunities to Study Protein Structure and Dynamics[END_REF]. Historically, the first lyotropic systems are composed by dissolving an amphiphilic molecules in water, but the need to study in these media also molecules that are not compatible with the aqueous environment has meant that over the years anisotropic systems consisting of a polymer and an organic co-solvent have been developed [START_REF] Alexandridis | Lyotropic Liquid Crystallinity in Amphiphilic Block Copolymers: Temperature Effects on Phase Behavior and Structure for Poly(ethylene oxide)-bpoly(propylene oxide)-b-poly(ethylene oxide) Copolymers of Different Composition[END_REF]Aroulanda et al, 2001).

Among the various weakly alignment media, that are suitable for this kind of study, the lyotropic phases based on the synthetic chiral homopolypeptides poly-γ-benzyl-L-glutamate (PBLG) dissolved in a suitable organic solvent was used in this Ph.D. as lyotropic phase .

1.4.1.1 Lyotropic liquid crystal based on PBLG PBLG (poly-γ-benzyl-L-glutamate) (Figure 1.18) is a chiral homopolypeptide that assumes liquid-crystalline characteristics in solution, with organic solvents, in concentrations between 12-25%wt, a range that can vary depending on the temperature, the nature of the solvent and also on the degree of polymerization of the used polymer (Sarfati et al, 2000). The mesophase obtained dissolving the polymer in a suitable organic solvent is called cholesteric and is characterized by the presence of chiral molecules which assume a preferential statistical orientation but do not enjoy any positional order [START_REF] Collings | Introduction to Liquid Crystals, Chemistry and Physics[END_REF].

Specifically, in this phase, the main chain that constitutes the fibers of the polymer adopts a rigid α-helix conformation from which the side chains of the residues branch off [START_REF] Papadopoulos | Self-Assembly and Dynamics of Poly(γ-benzyl L-glutamate) Peptides[END_REF]. More molecules of PBLG assume this form and are organized in a particular way: they form microdomains each of which has a director with a certain orientation. At a macroscopic level, a number of microdomains orient themselves so as to form a supramolecular helix of the directors as shoun in Figure 1.19.

A limitation of PBLG, and of homopolypeptides in general, is the impossibility of maintaining this structure with organic co-solvents that form strong hydrogen bonds with them destroying the -helix, such as DMSO or trifluoroacetic acid. Suitable organic solvents are CDCl3, CD2Cl2, DMF, THF and dioxane (Sarfati et al, 2000). In this Ph.D. thesis, deuterated chloroform, CDCl3, was used in order to preserve the PBLG structure and avoid interference in the proton spectra due to signals attributable to the organic co-solvent. When the PBLG/co-solvent solution is immerged in a magnetic field, such as that used in NMR spectroscopy, the supramolecular helix unwinds and, since the phase has a diamagnetic positive anisotropy, Δχ, the directors align parallel to the field 𝐵 0 ⃗⃗⃗⃗ [START_REF] Panar | Magnetic Ordering of Poly-γ-benzyl L-glutamate Solutions[END_REF]. The phase obtained behaves like a nematic phase, consisting of helical structures from which the side chains branch off as shown in Figure 1.20 [START_REF] Abe | Deuterium NMR Analysis of Poly(γ-benzyl L-glutamate) in the Lyotropic Liquid-Crystalline State: Orientational Orde of α-Helical Backbone and Conformation of the Pendant Side Chain[END_REF]. These create interstices between the various molecules within which the solute is positioned and weakly oriented.

Pulse sequences and spectral analysis

As stated in section 1.4.1, the use of weakly orienting media makes it possible to obtain, from samples in these phases, spectra that have characteristics mainly of the first order. In this type of spectra it is possible to extract the total coupling constants 𝑇 𝑖𝑗 (see equations 1.32 and 1.33) by simple inspection. In order to obtain a set of experimental parameters as large as possible, a series of 1D and 2D NMR pulse sequences was performed. First, spectral assignment of the peaks need to be performed, and this is done by using 1D 1 H, 13 C and 13 C-{ 1 H} spectra and 2D

1 H COSY, 1 H-13 C HSQC, 1 H-13 C HMQC and 1 H-13 C HMBC correlation experiments discussed in section 1.3.2.1. In order to conduct a conformational analysis starting from the experimental data it is necessary not only to obtain a large number of independent data, but also that they refer to torsional fragments of the molecule. Then different kind of experiments must be carefully performed. In this panorama, we perform the homonuclear and heteronuclear Jresolved experiments shown in section 1.3.2.1 to reach the goal. In addition, more sophisticated pulse sequences were also employed such as the selective SERF (Selective ReFocusing experiment) and HetSERF (Heteronuclear Selective ReFocusing experiment) which proved to be particularly useful to edit the long-range couplings between spins belonging to the different fragments of the molecule.

Selective refocusing experiment (SERF)

SERF experiment is an NMR pulse sequence for selectively measuring a 1 H-1 H coupling constant without interference from other couplings. This sequence was developed in 1995 by Thomas Fäcke and Stefan Berger as a selective version of the standard 2D homonuclear Jresolved [START_REF] Fäcke | SERF, a new method to measure H, H spin coupling constants in organic chemistry[END_REF]. In the years following the publication of this work, the sequence was not very successful and had only a few applications. Instead, after 10 years from its publication, many groups began to modify it and adapt it to the study of various problems and this has led to the fact that today there are about 35 variants of the sequence that are commonly applied [START_REF] Berger | Twenty-five Years of SERF: The Progress of an NMR Pulse Sequence and its Application[END_REF]. As stated in 1.3.2.1 section, from a J-resolved experiment, for each chemical shift the couplings related to the corresponding multiplet can be read on the indirect dimension, F1. This signal is a multiplet from which sometimes is difficult to extract useful informations. Replacing the first 90° pulse with a selective pulse (selective on the group of protons a) leaves the situation unchanged and the multiplicity is given by all the coupling interaction existing. However, by replacing also the 180° pulse with a selective 180° pulse (selective on the groups of protons a), only the selected protons will contribute to the signal and thanks to a second selective 180° pulse (selective on the groups of protons b), only the spin of the desired protons are refocused, allowing to reduce the multiplet structure to signal simpler in multiplicity from wich informations can be easily read. In particular, on the F1 dimension of the 2D map obtained, only the coupling between the two group of protons selected by the selective pulses can be observed. The used pulses are commonly named as soft pulses and they are made selective by reducing its intensity and increasing its duration. The shape of the soft pulses should be chosen properly in order to have the best selection and efficiency according to the pulse angle (90° or 180°) requested. The right value of this two parameter need to be carefully calibrated during the step of preparation of the experiment. In the Figure 1.21 the pulses sequence used in this thesis is shown together with a schematic example of the obtained SERF spectrum. Pulses sequence used the SERF experiment is reported on the left. On the right, an example of the SERF spectrum obtained using this sequence. The offset of the selective pulse was set at Ha on the proton channel and the offsets of the refocusing selective pulse at Hb.

Heteronuclear selective refocusing experiment (HetSERF)

HetSERF experiment was originally developed by Bax and Freeman as an efficient NMR method that allows to measure long-range 1 H-13 C coupling constant [START_REF] Bax | Long-Range Proton-Carbon-13 NMR Spin Coupling Constants[END_REF].

During the years it has been modified in different ways [START_REF] Nolis | IFSERF, An Isotope-Filtered SERF Experiment for the Precise Measurement of Proton-Proton Coupling Constants Between Chemically Equivalent Protons[END_REF][START_REF] Di Pietro | GET-SERF, A New Gradient Encoded SERF Experiment for the Trivial Edition of 1 H-19 F Couplings[END_REF] and, among the possible application, it has been used in order to extract also short-range couplings and total spin-spin couplings in solute dissolved in chiral liquid crystals (Farjon, et al., 2004). This pulses sequence is based on the 2D heteronuclear J-resolved experiment but involves a 180° selective proton pulse in order to selectively measure a particular coupling constant without interference from other couplings. The sequence used in this thesis is reported in Figure 1.22 and can be briefly described as follows. The pulse consists in a spin echo sequence (90--180) on the 13 C channel. As showed in heteronuclear J-resolved section, if all spin experience the effects of the 180° refocusing pulse, spin echoes obtained are modulated by spin coupling. This permits proton-carbon couplings to be separated from 13 C chemical shifts by using the second dimension showing the fine structure due to all the proton-carbon couplings. In the case of HETSERF experiment, a 180° selective pulse is applied on a proton while a 180° hard pulse is applied on 13 C. These two pulses refocus the 13 C chemical shifts and the couplings except the coupling between all the 13 C atoms of the molecule and the selected group of protons. The spin-spin coupling between the selected protons and the carbons then evolve. At the end of the sequence, the FID is observed on the 13 C channel under proton decoupling. The 2D HetSERF spectrum obtained after the two-dimensional Fourier transform, allows to measure the couplings of all carbon atoms present in the molecule, whose spectrum is reported on the orizontal (F2) dimension, with a selected proton or group of protons whose coupling is directly measurable on the vertical (F1) dimension at each chemical shift of the 13 C spectrum. Once all the needed experiments have been performed, it is necessary to extract the informations from the spectra obtained. As anticipated in paragraph 1.4, dipolar coupling constants, also known as Residual Dipolar Couplings (RDCs), are an excellent tool for the conformational study of flexible molecules. These observables can be experimentally obtained by using the equations 1.16 and 1.17. This implies that, in order to have access to the values of 𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 , for magnetically non-equivalent nuclei, it is necessary to carry out both NMR measurements in isotropic, to measure the 𝐽 𝑖𝑗 𝑖𝑠𝑜 , and in the orienting phase. Furthermore, the absolute values of both the 𝐽 𝑖𝑗 𝑖𝑠𝑜 and the 𝑇 𝑖𝑗 are obtained from the spectra, the determination of the sign of these parameters is not easy: in the worst case, only the magnitude of the two constants is known and, consequently, we can obtain four possible values of 𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 considering all the possible combinations of 𝑇 𝑖𝑗 and 𝐽 𝑖𝑗 𝑖𝑠𝑜 . If, on the other hand, the sign of 𝐽 𝑖𝑗 𝑖𝑠𝑜 is known (from data present in the literature for similar systems for example), there are two possible values of 𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 . Finding a reliable procedure that, for a given interaction, allows to identify among the various possibilities which is the correct 𝐷 𝑖𝑗 𝑎𝑛𝑖𝑠𝑜 , is of fundamental importance. In (Canet et al, 1995;[START_REF] Lesot | Measurement and Analysis of the Molecular Ordering Tensors of Two Enantiomers Oriented in a Polypeptide Liquid Crystalline System[END_REF]. In the case of 𝐷 𝐶𝐻 𝑛 (with n>1) and 𝐷 𝐻𝐻 𝑛 the situation is more complex and in most cases the correct value is determined through a trial and error process during the conformational analysis procedure. How it is possible to obtain the desired conformational information from the experimental observables, and what is the theoretical approach used to obtain this information, are the topics of the next paragraphs. where {𝜙} is the set of torsional angles that fix the structure of each conformer and Z and Ziso are normalization constants. 𝐷 𝑖𝑗,𝛼𝛽 ({𝜙})) are the components of the dipolar interaction tensor 𝐷 𝑖𝑗 ̌, expressed in the molecular reference system, when the molecule is in its 𝜙 conformation.

Regarding the term 𝑊({𝜙}), it can be written as:

𝑊({𝜙}) = ∫ 𝑒𝑥𝑝[-𝑈 𝑒𝑥𝑡 (𝛺, {𝜙})/𝑘 𝑏 𝑇] 𝑑𝛺 (1.63)
Where kb is the Boltzmann constant, T is the absolute temperature and 𝑈 𝑒𝑥𝑡 (𝛺, {𝜙}) represents the intramolecular external potential energy, dependent on both the conformation and the orientation. The terms 𝑆 𝛼𝛽 ({𝜙}) are the orientational order parameters of the solute that depend on its conformation and, finally, the term 𝑃 𝑖𝑠𝑜 ({𝜙}) is the conformational distribution function, lacking of the possible effects due to the orientational order of the mesophase and represents the real target in conformational studies. Observing the relationship 1.62 it can be noted how the NMR observables are related to the product between the conformational probability and the orientational parameters. Since it is not possible to experimentally separate the two contributions, a theroetical model must be adopted to deal with the problem of the interdependence between orientational and conformational motion.

1.4.4 Theoretical approach for conformational analysis Among the various models available for the treatment of these problems, in the present Ph.D.

thesis the AP-DPD model was used [START_REF] Celebre | Exploiting the Information Content of Dipolar Couplings: Determination of the Temperature Dependence of the Inter-Ring Twist Angle of Biphenyl Dissolved in Uniaxial Mesophases[END_REF]. It consist of the combination of two models: the Additive Potential (AP), to treat the orientational interactions [START_REF] Emsley | A Theory of Orientational Ordering in Uniaxial Liquid Crystals Composed of Molecules with Alkyl Chains[END_REF], and the Direct Probability Description (DPD), for the description of the 𝑃 𝑖𝑠𝑜 ({𝜙}) (Celebre et al, 2003). In particular in the AP model the orientational interaction energy, 𝑈 𝑒𝑥𝑡 (𝛺, {𝜙}), can be described through an expansion in spherical harmonics (Emsley, 2007):

𝑈 𝑒𝑥𝑡 (𝛺, {𝜙}) = -𝜀 2,0 {𝜙}𝐶 2,0 (𝛺) -𝜀 2,2 {𝜙}𝐶 2,2 (𝛺) (1.64)
where 𝐶 𝑝,𝑚 (𝛺) are the modified spherical harmonics and 𝜀 𝑝,𝑚 ({𝜙}) are the conformationdependent components of the solute-solvent interaction tensor.

The peculiarity of the AP approach is the possibility to express the interaction tensor 𝜀 𝑝,𝑚 ({𝜙})

as the sum of conformation-independent contributions, each of which representative of a rigid fragment j in which the molecule can be decomposed (Celebre & Longeri, 2003):

𝜀 2,𝑚 ({𝜙}) = ∑ ∑ 𝜀 2,𝑝 𝑗 𝐷 𝑝,𝑚 2 ({𝛺 𝜙 𝑗 }) 𝑗 𝑝
(1.65) when ({𝛺 𝜙 𝑗 }) is the set of Eulerian angles that describe the orientation of the j-th fragment compared to a system of axes fixed on a rigid molecular fragment. 𝐷 𝑝,𝑚 2 ({𝛺 𝜙 𝑗 }) represents the elements of the Wigner rotation matrix, which correlate the principal axes of 𝜀 2,𝑝 𝑗 with the molecular reference system. Actually, the 𝜀 2,𝑝 𝑗 are unknown quantities whose values can be adjusted to obtain the best possible agreement with the experimental data.

As for the 𝑃 𝑖𝑠𝑜 ({𝜙}), following the DPD approach, it is possible to model it directly in terms of Gaussian functions: as a sum if the conformations are dependent or as product if they are independent (Celebre et al, 2003). Thus, for a molecule with d independent rotors and nj minima for the j-th rotor, the general form for 𝑃 𝑖𝑠𝑜 ({𝜙}) becomes: (1.66)

𝑃 𝑖𝑠𝑜 ({𝜙}) = ∏ ( ∑ 𝐴
where 𝜙 𝑖,𝑗,𝑘,…𝑑 0 are the maximum angles in the conformational probability function and 𝐴 𝑖,𝑗,𝑘,…𝑑 their relative weight. The amplitude of each Gaussian is given by ℎ 𝑖,𝑗,𝑘,…𝑑 which represents the width at half height of the curve (Celebre et al, 2003).

From the practical point of view, all these calculation are implemented in the AnCon package (Pileio, 2005), developed in the LXNMR S.C.An. laboratory, where the conformational studies of this Ph.D. thesis were performed. The calculations are carried out by fitting a set of calculated dipolar couplings (obtained by a trial set of orientational, geometrical, and potential parameters) against the experimental ones, while iterating on a pertinent number of unknowns until their optimized values. Such unknowns are the orientational parameters, represented by the set of the chosen (𝜀 2,𝑝 𝑗 ), the potential parameters(𝐴 𝑖,𝑗,𝑘,…𝑑 , 𝜙 𝑖,𝑗,𝑘,…𝑑 0 and ℎ 𝑖,𝑗,𝑘,…𝑑 ) and, eventually, the geometrical parameters as bond distances and angles. This procedure is carried out several times by varying the iteration parameters until the mean square deviation between the N constants calculated and observed is minimal:

𝑅𝑀𝑆 = ∑ √ ( (𝐷 𝑖𝑗 𝑜𝑏𝑠 -𝐷 𝑖𝑗 𝑐𝑎𝑙𝑐 ) 2 𝑁 ) 𝑁 𝑖,𝑗
(1.67)

The strategy just discussed is showed in a flowchart in Figure 1.23. To note that the number of experimental observables must be large enough to obtain the most precise conformational description possible. Even in the case where the number of experimental data is large, to determine the complete structure and conformational distribution it is necessary to have a geometry for the most stable conformers and a good estimate of the potential energy surface.

Input Parameters

• Geometrical parameters (bond distances and angle). • Orientational parameters (solute-solvent interaction tensors ). • Potential parameters needed to model the conformational probability in therms of Gaussian functions ( , A, h).

Calculation Procedures

• Calculation of a set of from the trial set of orientational, geometrical and conformational parameters.

• Comparison between and .

fitted against iterating on a proper number of unknowns ( , , A, h) and eventual geometrical parameters.

Iteration Procedures

Output Parameters

Optimised values for the orientational, potential and geometrical parameters

Chapter 2.

Metabolic analysis of natural matrices

Introduction

During the last decades, researchers, industries, and consumers have grown their interest towards nutraceuticals due to their proven healthy effect and fashionable character. Searches for new compounds and new sources together with the need to find new information on their activity, are nowadays problematics most encountered and, in this framework, metabolomics offers an unique tool able to face this kind of study. Indeed, the metabolome of foods and natural extracts is composed by secondary, or specialized, metabolites, which, in addition to being specific and essential compounds for the survival of plants in environmental interactions, also possess a variety of biological activities that make these metabolites candidates as health promoting factors. In this panorama both untargeted and targeted metabolomics analysis can be performed in order to study biological systems by obtaining simultaneous qualitative and quantitative analyses of the metabolites of a given biological system under a specific set of conditions [START_REF] Valentino | NM -Based Plant Metabolomics in Nutraceutical esearch: An Overview[END_REF]. Although several analytical techniques can be used for metabolomics analysis, NMR Spectroscopy has proven to possess undeniable advantages as shown in section 1.3. In this chapter, some NMR-based metabolomics application are presented. In this chapter, some NMR-based metabolomic applications are presented.

Specifically, sections 2.2 and 2.3 show the NMR profiles of Calabrian citrus fruits and apples respectively; in section 2.4 the metabolic characterization of the natural extracts of Cannabis Sativa inflorescences and its oil, obtained from the seeds, is treated; while in section 2.5 the NMR profiling of the essential oil of Calabrian Bergamot is reported together with an evaluation study of its authenticity. Finally, in the last part of this chapter, section 2.6, an NMR study, based on the HR-MAS methodology, carried out on the organic fraction of municipal solid waste is presented.

NMR-based metabolomics analysis of Calabrian citrus fruit juices and its application to industrial process quality control

Citrus fruits are among the major horticultural crops grown worldwide and they represent the most traded from the international point of view. This is due to their popularity that arise from their flavour, smell, juiciness of the pulp and to the fact that they contribute to human diet through a variety of healthy organic substances [START_REF] Musumeci | Citrus Fruits and Their Flavonoids in Inflammatory Bowel Disease: an Overview[END_REF]Okwu & Emenike, 2006;[START_REF] Liu | History, Global Distribution and Nutritional Importance of Citrus Fruits[END_REF][START_REF] Silalahi | Anticancer and Health Protective Properties of Citrus Fruit Components[END_REF][START_REF] Sicari | Bioactive Compounds and Antioxidant Activity of Citrus Juices Produced from Varieties Cultivated in Calabria[END_REF]. In Italy, thanks to the favourable climate, the production of oranges, lemons, mandarins, clementines and bergamot is concentrated in the southern regions of the state and, in particular, about 29% of the national citrus production came from Calabria, where several varieties of native citrus are cultivated [START_REF] Proto | is Assessment of epetitive Movements in the Citrus Fruit Industry[END_REF][START_REF] Scuderi | Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) Italian Citrus Production[END_REF]. Citrus fruits are commonly consumed during winter season as fresh fruit, but they are also subjected to industrial processes in order to obtain concentrate juices to be placed on the market throughout the year. The industrial process to obtain concentrated juices to be used, once diluted, in the preparation of citrus-based drinks, represented a turning point in the commerce of a product which is commonly subject to alteration and degradation over time, both during transport and storage steps [START_REF] Petrotos | Osmotic Concentration of Liquid Foods[END_REF][START_REF] Orellana-Palma | Elaboration of Orange Juice Concentrate by Vacuum-Assisted Bloc Freeze Concentration[END_REF][START_REF] De Oliveira | Trac ing the Degradation of Fresh Orange Juice and Discrimination of Orange Varieties: An Example of NMR in Coordination with Chemometrics Analyses[END_REF]. Among the citrus fruits, the orange juice is a fruit which request is constantly growing and this lead to an increase of the commercial value of the product that can be traduced in an increase in adulteration attempts [START_REF] Grumezescu | The Science of Beverages[END_REF][START_REF] Moore | Development and Application of a Database of Food Ingredient Fraud and Economically Motivated Adulteration from 1980 to 2010[END_REF][START_REF] Ebeler | Authentication of Food and Wine[END_REF]. In this context, one of the problems related to citrus juices commerce is represented by the quality and traceability assessment of the food. Indeed, this topic is a challenge in the food industry, as consumers are increasingly demanding safe, healthy and high-quality food for both fresh and processed products [START_REF] Grumezescu | The Science of Beverages[END_REF][START_REF] Opara | Traceability in Agriculture and Food Supply Chain: A eview of Basic Concepts, Technological Implications, and Future Prospects[END_REF]Van Rijswijk & Frewer, 2008).

Consequently, controlling the industrial processes to which the fruits are subjected is of fundamental importance: the commercial value of the final product and its acceptability by consumers is closely linked to the organoleptic and nutraceutical characteristics that the concentrated juice must preserve from fresh fruit during the industrial treatment. On the other hand, it is well known that the essential characteristics of a food product are connected to the metabolites and the levels of metabolites in a biological system can change based on both genetic and environmental factors or after industrial treatments. For this reason, the study of metabolites allows to tackle the problems connected to the quality of a food product also during the transformation and conservation processes. In this sectiont an NMR spectroscopic study, coupled to statistical treatments, is reported in order to explore the metabolomic profile of Calabrian orange and clementine fresh juices coming from the Plains of Gioia Tauro, and evaluate any metabolic variations during the industrial thermal process of concentrated juices by analysing samples taken at three different processing steps (squeezing, pasteurization and concentration).

Samples preparation and experimental procedure

The samples investigated in this study were provided by the Calabrian industry "Gioia Succhi srl" (San Ferdinando, Reggio Calabria, Italy) which uses Calabrian citrus fruits in the production of fresh juices and concentrates. Three industrial processes that lead to a concentrated product starting from fresh squeezed juice were investigated. Two industrial procedures concern the treatment of orange juice (process A and process B), while the third concerns the processing of mixed orange and clementine juice (process C). A single industrial treatment consists of three phases, squeezing, pasteurization and concentration. For each industrial treatment considered, material samples were collected during all the manufacturing phases directly at the production site: for the process A and C the material samples were collected during all the three phases, for a total of 24 samples (12 for each process, 4 for each step), while for process B material samples were collected only for squeezing and concentration phases (for a total of 8 samples, 4 for each step). Note that the two processes A and B, although both involve orange juice, differ in the starting juices because they come from different batches of oranges. The citrus fruits used in all the processes came from the Plain of Gioia Tauro and from different cultivars of the plain. For the sake of clarity, it is necessary to emphasized that we considered real industrial treatments in which the fruits employed do not belong to a single cultivar but are mixed cultivar fruits. After the collection, all the samples were frozen and stored at -18°C. From each collected sample, four samples for NMR measurements were prepared by using the procedure reported in literature [START_REF] Cuny | Fruit Juice Authentication by 1H NM Spectroscopy in Combination with Different Chemometrics Tools[END_REF]. Therefore, the squeezed and pasteurized samples were defrosted and centrifuged at 6000 rpm for 30 minutes while the concentrated samples were diluted with distilled water (1:1) before the centrifugation. In the samples studied there are metabolites which chemical shifts is sensitive to the pH of the sample, in order to obtain comparable results between the data obtained from this study and the data reported in literature, the pH of the supernatant of each centrifuged sample has been adjusted to a value of 2.9-3.0 by adding microliter amounts of 1M NaOH or 1M HCl solutions. 500 μL of this surnatant were inserted into 5-mm NMR-tube and mixed with 150 μL of D2O and to 50 μL of an aqueous solution containing 0.16%wt sodium azide (NaN3) and 0.11%wt 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TMSP) [START_REF] Rinke | An 1H-NMR-Technique for High Throughput Screening in Quality and Authenticity Control of Fruit Juice and Fruit Juice Raw Materials -SGF-Profiling[END_REF]. The sodium azide was used to suppress microorganism activity, D2O was used as the field frequencies lock signal and TMSP as internal referencing of chemical shifts. No additional treatment was necessary. On each samples obtained following this procedure, NMR spectra were recorded at room temperature on a 500 MHz Bruker Avance spectrometer (11.74 T) (Bruker, Fällanden, Switzerland) equipped with a 5 mm TBO probe and a standard variable-temperature unit BVT-3000. Once obtained the spectra, all the data processing steps were carried out using TopSpin 3.6 software (Bruker BioSpin, Rheinstetten, Germany) (TopSpin, 2018).

Signal assignments

The metabolites were assigned on the basis of the 1D 1 H and 13 C-{ 1 H} NMR experiments, 2D

homo and heteronuclear correlation NMR spectra ( 1 H COSY and 1 H-13 C HMQC) and by comparison with published data (Le [START_REF] Gall | Discrimination between Orange Juice and Pulp Wash by 1H Nuclear Magnetic esonance Spectroscopy: Identification of Mar er Compounds[END_REF][START_REF] Alves Filho | 1H NM Spectroscopy and Chemometrics Evaluation of Non-Thermal Processing of Orange Juice[END_REF][START_REF] Ludwig | Birmingham Metabolite Library: a Publicly Accessible Database of 1-D 1H and 2-D 1H J-resolved NMR Spectra of Authentic Metabolite Standards (BML-NM )[END_REF]. 1D NMR spectra were Fourier transformed, phased, baseline corrected and aligned using the TMSP signal as reference. 13 C-{ 1 H} NMR spectra were filtered with 1 Hz line broadening before Fourier-transformed. The 1 H and the 13 C-{ 1 H} NMR spectra obtained from squeezed fresh orange juice are reported in Figure 2.1. In the proton spectrum three regions are clearly distinguished: the first region goes from 0 to 3 ppm and contains the frequencies of the aliphatic protons belonging to organic acids and free amino acids; from 3 to 6 ppm there is the most complex region constituted by the signals of the sugars that are overlapped on each other; and, lastly, the third region, that goes from 6 to 8 ppm, is the aromatic region in which there are weak signals due to phenolic metabolites. In order to assign each signal to the corresponding metabolites, in addition to the classical onedimensional 1 H and 13 C-{ 1 H} NMR spectra, 2D correlation experiments 1 H COSY and 1 H-13 C HMQC were employed. The two correlation spectra were processed using a sine filter and a qsine filter on both dimensions, F1 and F2, respectively on COSY and HMQC experiments before Fourier-transformed and the obtained 2D maps are reported in Figure 2.3 and 2.4. In order to show the methodology adopted for the signal assignment, the case of citric acid, a simple molecule, is discussed as example. Citric acid, which numbered structure is reported in Figure 2.2, is an organic compound that naturally occurs in citrus fruits and is an intermediate in the citric acid cycle in the metabolism of all aerobic organisms. This substance is used in food in order to reduce acidity and improve taste. Because it is one of the stronger edible acids, the dominant use of citric acid is as a flavoring and preservative in food and beverages, especially soft drinks and candies [START_REF] Sawant | Fungal Citric Acid Production Using Waste Materials: A Mini-eview[END_REF]. were recognized thanks to the COSY experiment. In particular, the Figure 2.3a shows the COSY spectrum in which the correlation spots relating to the considered groups of protons are highlighted in the enlargement. Thanks to the correlations found in the 2D map, it has been The 1 H COSY experiments were acquired employing pre-saturation sequence for water signal suppression and using a SW of 9 ppm on both dimensions, 2K data points, 4 scans and 512 increments. In red is reported the enlargement of the region 2.80 -3.15 ppm on both dimensions; B) region ranging from 2.80 to 3.15 ppm of the 500 MHz 1 H NMR spectrum recorded on squeezed orange juice in D2O (298K).

To confirm this assignment, it is possible to see how in the 2D 1 H-13 C HMQC spectrum the same carbon atom signal is correlated to the two protons H3a and H3b (or H5a and H5b) signals, having different chemical shifts (Figure 2.4).

H3b(H3a) + H5b(H5a) H3a(H3b) + H5a(H5b) H3b(H3a) + H5b(H5a) H3a(H3b) + H5a(H5b) 2 J H3a-H3b = 2 J H5a-H5b 2 J H3a-H3b = 2 J H5a-H5b The 1 H-13 C HMQC spectra were recorded employing pre-saturation sequence for water signal suppression and using a SW of 9 ppm ( 1 H) and 200 ppm ( 13 C), 2K datapoints, 256 scans and 16 experiments. In red is reported the enlargement of the region (F1: 39.5-46.5 ppm; F2: 2.50-3.25ppm).

Connectivity information obtained from 2D spectra together with the use, as guidelines, of the chemical shift data reported in the literature (Le [START_REF] Gall | Discrimination between Orange Juice and Pulp Wash by 1H Nuclear Magnetic esonance Spectroscopy: Identification of Mar er Compounds[END_REF][START_REF] Alves Filho | 1H NM Spectroscopy and Chemometrics Evaluation of Non-Thermal Processing of Orange Juice[END_REF][START_REF] Ludwig | Birmingham Metabolite Library: a Publicly Accessible Database of 1-D 1H and 2-D 1H J-resolved NMR Spectra of Authentic Metabolite Standards (BML-NM )[END_REF], allowed to assign the resonances of twenty-three metabolites. In the Table 2.1 the experimental informations obtained from the NMR spectra relating to fresh squeezed orange juice are summarized. Figure 2.5 shows the expanded regions of 1 H-NMR spectra with the relevant chemical shifts associated to the various recognized metabolites. In order to highlight possible variations in the metabolite composition deriving from the industrial production of concentrate juice, spectra recorded on pasteurized and concentred samples were compared with the squeezed one. Figure 2.6 shows the 1 H-NMR spectra of the three samples and, with the exception of ethanol, whose signals (1.17 ppm and 3.65 ppm) are present in squeezed and pasteurized samples while they are absent in the concentrated ones, no further differences in terms of profile stand out. The amount of information in each spectrum is so great that, for any systematic comparison of a large number of samples, the need of multivariate analysis is mandatory. The comparison between the squeezed orange juice and the squeezed clementine and orange juice 1 H-NMR spectra is reported in Figure 2.7. As can be seen, no essential difference in metabolites NMR signals were detectable by simple inspection, except for the aromatic region which remain largely unassigned due to the poor signal-to-noise ratio. In particular, in this part of the spectrum, the phlorin signals have different intensity: in orange juice they are easily confused with the noise, on the contrary in the clementine and orange juice they are more visible. According to some papers, phlorin represents a marker compounds because it is present in the peel of the oranges while the juice is poor in this. For this reason, the phlorin concentration can be used in order to evaluate the authenticity of the juice discriminating between authentic juice and pulp wash (Le [START_REF] Gall | Discrimination between Orange Juice and Pulp Wash by 1H Nuclear Magnetic esonance Spectroscopy: Identification of Mar er Compounds[END_REF][START_REF] Louche | Determination of Phlorin as Peel Marker in Orange (Citrus Sinensis) Fruits and Juices[END_REF]. The presence of an appreciable concentration of phlorin in sample C is somewhat outlandish because, according to the study by Louche et al., clementine juice does not contain this compound [START_REF] Louche | Determination of Phlorin as Peel Marker in Orange (Citrus Sinensis) Fruits and Juices[END_REF][START_REF] Louche | Phlorin Screening in Various Citrus Species and Varieties[END_REF], then, probably, during the squeezing phase of sample C, an appreciable amount of phlorin is transferred from orange peel into the juice.

Multivariate Analysis

The 1 H NMR spectra used for the multivariate analysis were segmented in rectangular bucket of fixed 0.05 ppm each of which represents a variable in the statistical study.

The preliminar step that must be performed before the Principal Components Analysis consists in a variable selection. In this work, it was achieved by choosing the NMR regions of characteristic compounds that most contribute to fruit juices: sugar and acid regions.

Furthermore, the ethanol signal and phlorin signal were added in order to evaluate the ethanol variation during the industrial process and the phlorin content in different fruit juice. The phenolic region was not considered for the bucketing due to the poor signal-to-noise ratio. Thus, regions selected for the PCA were 0.50-4.00 ppm and 6.10-6.25 ppm. Once the spectral region were chosen, the bucketing procedure was performed defining the integration range on the first spectra, the integration regions were saved, and the other spectra were bucketed automatically using the saved list. The integrals were normalized and exported in an excel file for the construction of the data matrix to use in the statistical treatment performed on R software (R Core Team, 2019). A dataset of 32 samples and 69 variables has been obtained. The PCA score plot obtained for the selected regions of the data summarizes the relationships between the 32 samples. The cumulative percentage of explained data variance with the three first PC is above 90% (68.2%, 14.0% and 7.8% for PC1, PC2 and PC3 respectively), so data loss is negligible. In order to better evaluate the effect of the thermal processing on the various production phases, pasteurization process leaves the metabolic profile of the orange juice unchanged, while, in the case of sample C, it seems that there is a sort of pre-concentration of the squeezed juice already during the pasteurization process so that the metabolic profile of pasteurized juice is closer to the concentrated one. Since in all the investigated concentrate production processes the same procedure has been applied, this difference in pasteurization step is probably due to variation in the water content of the fruits used. phases, for all the samples, the ethanol decreases, and the sugar component is the highest in the concentrate. This means that during heat treatment the profile of the metabolites does not vary but the concentration of amino acids and other aliphatic components decreases. This is clear in Figure 2.10B where the variables loadings of these metabolites are in the region of squeezed juices scores (negative values of PC2) to prove that the squeezed juices are richer in these substances than concentrated ones. Therefore, these data indicate that the thermal process induces a deterioration of the sensorial and nutritional value of the finished product and they are in agreement with the works reported in the literature regarding several thermal processes on juices obtained not from citrus fruits [START_REF] Petrotos | Osmotic Concentration of Liquid Foods[END_REF][START_REF] Gil-Izquierdo | Effect of Processing Techniques at Industrial Scale on Orange Juice Antioxidant and Beneficial Health Compounds[END_REF].

Nowadays, as an alternative to the thermal process it is possible to use other promising concentration techniques such as the reverse osmosis through the membranes [START_REF] Jesus | Orange (Citrus Sinensis) Juice Concentration by everse Osmosis[END_REF].

In order to confirm the consideration made on the clustering, a cluster analysis has been performed. Cluster Analysis was carried out in order to classify all samples studied using the same variables used for PCA. Classification was done using R software by Ward's method, which joins cases together in order to minimize variance within a cluster [START_REF] Varmuza | Introduction to Multivariate Statistical Analysis in Chemometrics[END_REF]. From the dendrogram obtained (Figure 2.11), samples studied can be classified into two main clusters at scale distance of 120, namely Cluster 1 (Orange and Clementine juices samples) and Cluster 2 (Orange juices samples). At scale distance of 60, orange juices, samples can be classified into two main clusters depending on the process (A and B). This confirms that different process can be discriminated on the different nature of initial fruits (geographical origins, cultivar and so on). It is interesting to find out that also cluster analysis shows the non-reproducibility of pasteurization step. Indeed, at scale distance between 5 and 15, the pasteurization process in orange and clementine juices is clusterized with the concentrated juice; on the contrary, in orange juices (process A) it is clusterized with the squeezed samples. There are many factors account for the difference in this clustering;

obviously, the nature of the citrus fruits but also the amount of water in the fruits represent an important parameter.

Conclusions

In this study NMR spectroscopy was applied in order to investigate the metabolic profile of Calabrian fresh orange juice and to follow any metabolic variations that the fresh juice undergoes during the industrial thermal process in order to obtain concentrated juices. Classic 1D pulse sequences together with 2D correlation experiments and literature data, were used and 23 low molecular weight compounds present in freshly squeezed orange juice were identified.

Soc3 Soc5 Soc2 Soc4 Poc1 Poc3 Poc2 Poc4 Coc1 Coc4 Coc2 Coc3 CoB1 CoB2 CoB3 CoB4 SoB4 SoB1 SoB2 SoB3 CoA2 CoA1 CoA3 CoA4 SoA2 SoA3 PoA3 SoA4 PoA1 SoA1 PoA2 PoA4
Dendrogram using Ward's method

Metabolic characterization of Calabrian apple juices

Apple is one of the most commonly eaten fruit in world and its juice is largely consumed [START_REF] Gunathilake | Flavonoids rich apple for healthy life[END_REF]. Indeed, both apples and apple juices are appreciated for their nutritional value, aroma, firmness, and health-promoting effects [START_REF] Harker | The case for fruit quality: an interpretive review of consumer attitudes, and preferences for apples[END_REF][START_REF] Ting | X-ray micro-computer tomographic method to visualize the microstructure of different apple cultivars[END_REF]. All these quality traits are ultimately based on the metabolic composition of the fruit itself, namely primary and secondary metabolites [START_REF] Cuthbertson | Utility of metabolomics toward assessing the metabolic basis of quality traits in apple fruit with an emphasis on antioxidants[END_REF]. Actually, while amino acids, sugars, organic acids respectively determine taste, sweetness and acidity, the polyphenolic compounds, mostly located in apple skins, are potential disease-preventing agents [START_REF] Boyer | Apple phytochemicals and their health benefits[END_REF][START_REF] Gerhauser | Cancer chemopreventive potential of apples, apple Juice, and apple components[END_REF][START_REF] Francini | Phenolic compounds in apple (Malus x domestica Borkh.): compounds characterization and stability during postharvest and after processing[END_REF][START_REF] Koutsos | Apples and cardiovascular health -is the gut microbiota a core consideration?[END_REF][START_REF] Pirlak | Determination of antioxidant activities of some apple cultivars[END_REF] and from these properties derive the applications of apples and its extracts. In fact, in addition to being consumed as fresh fruit or used as a row material for further preparations (ice cream, creams, cakes, candies and soft drinks), apple extracts are used as nutritional supplements [START_REF] Shoji | The Toxicology and Safety of Apple Polyphenol Extract[END_REF] and are studied for their antiallergic [START_REF] Kanda | Inhibitory Effect of Apple Polyphenol on Induced Histamine Release from RBL-2H3 Cells and at Mast Cells[END_REF],

antihypertensive (Balasuriya & Rupasinghe, 2012) Jeong et al, 2015) so that even the smallest variation of one of these factors can cause a variation on the concentration of some metabolites or cause the appearance of new ones usually not present. Therefore, each fruit cultivar has a unique and specific metabolic profile that can be used as potential marker for quality, origin and authenticity of fruit and fruit-derived foods [START_REF] Sobolev | Untargeted NMR-based methodology in the study of fruit metabolites[END_REF]. In literature, many analytical methodologies have been applied to determine specific fruits metabolites [START_REF] Seger | Analytical aspects of plant metabolite profiling platforms: current standings and future aim[END_REF][START_REF] Gören | Rapid quantitation of curcumin in turmeric via NMR and LC-tandem mass spectrometry[END_REF][START_REF] Lima | Metabolite profiling by UPLC-MSE, NMR, and antioxidant properties of Amazonian fruits: Mamey apple (Mammea Americana), Camapu (Physalis Angulata), and Uxi (Endopleura Uchi)[END_REF][START_REF] Mamat | GC-MS and LC-MS analyses reveal the distribution of primary and secondary metabolites in mangosteen (Garcinia mangostana Linn.) fruit during ripening[END_REF][START_REF] Tallapally | GC-MS based targeted metabolomics approach for studying the variations of phenolic metabolites in artificially ripened banana fruits[END_REF] and in particular apple metabolic profile has already been extensively characterized (Eisenmann et al, 2016, and references therein). In this section an untargeted NMR-metabolic analysis is reported in order to explore four different varieties of Calabrian apple juices, namely Golden Delicious, Royal Gala, Pink Lady and Fuji, coming from the Plains of Gioia Tauro, grown in the same Calabrian orchard, and harvested in the same period of the year, i.e., February 2020. Moreover, in order to discriminate among the samples coming from the different cultivars, the 1 H NMR spectra recorded on them have been combined

with chemometric analysis Principal Component Analysis.

It must be undeline that the study carried out presented in this section, is part of a project that involved different units of the Department of Chemistry and Chemical Technologies (CTC) of the University of Calabria together with the Institute on Membrane Technology (ITM) National

Research Council (CNR). The aim of the aforementioned project was the characterization of metabolites present in the food matrix and at the same time evaluate the possibility of using innovative methodologies based on membrane operations, such as ultrafiltration (UF) and nanofiltration (NF), to obtain from the squeezed apple juice, extracts rich in antioxidants that can be employed in food and pharmaceutical industries for the production of enriched foods and supplements.

Samples preparation and experimental procedure

Apple juices were supplied by "Gioia Succhi srl" (San Ferdinando, Reggio Calabria, Italy). The industrially processed apples were all grown in the same Calabrian orchard and they belonged to four different varieties: Royal Gala, Golden Delicious, Pink Lady, Fuji. Each apple variety, one at a time, was ground entirely (peel, flesh, and seeds) through a mill. Then, each sample was pasteurized to inhibit the tyrosinase action. Then, potassium metabisulfite (K2S2O5) was added as preservative followed by the addition of a pectolytic enzyme to degrade pectin and to reduce the viscosity of the juice so that it can be handled easily. Finally, the liquid part was separated from the pulp through a molecular sieve. Only the liquid part was transferred to aseptic bags and stored in the cold room at -18° C to be thawed for subsequent physico-chemical analysis. In particular, the samples for NMR analysis were prepared following the procedure reported in literature [START_REF] Belton | High-field proton studies of apple juices[END_REF][START_REF] Iaccarino | Ancient Danish apple cultivars -A comprehensive metabolite and sensory profiling of apple juices[END_REF]. Apple juices from the four varieties were first thawed at room temperature and centrifuged (4000 rpm for 20 minutes) to remove suspended matter. Since the chemical shifts of the metabolites are sensitive to pH it is necessary to operate at similar pH in order to obtain results comparable to each other and to the data reported in the literature. For this reason, the pH of the supernatant of each centrifuged sample was adjusted to a value of 3.25-3.38 by adding microliter amounts of 1M HCl solution.

The samples were then prepared by transferring about 0. and deuterium oxide (99.9 atom % D) were purchased from Sigma-Aldrich.

NMR spectra were recorded at 298K on a high-resolution Bruker Avance 500 MHz spectrometer (11.74 T) (Bruker, Fällanden, Switzerland) equipped with a 5 mm TBO probe and a standard variable-temperature unit BVT-3000. Once obtained the spectra, all the data processing steps were carried out using TopSpin 3.6 software (Bruker BioSpin, Rheinstetten, Germany) (TopSpin, 2018).

Signal assignments

The metabolites were assigned on the basis of the 1D 1 H and 13 C-{ 1 H} NMR experiments, 2D

homo and heteronuclear correlation NMR spectra, 1 H COSY and 1 H-13 C HMQC, and by comparison with published data [START_REF] Belton | High-field proton studies of apple juices[END_REF][START_REF] Vermathen | Investigation of different apple cultivars by high resolution magic angle spinning NMR. A feasibility study[END_REF][START_REF] Vermathen | Exploring high-resolution magic angle spinning (HR-MAS) NMR spectroscopy for metabonomic analysis of apples[END_REF]. The 1D 1 H and 13 C-{ 1 H} NMR spectra of the Golden Delicious juice are reported in Figure 2.12. The distribution of the signals in the protonic spectrum is not far from the one observed in the orange juices. Indeed, also in this case, three regions are clearly distinguished in the spectrum: the first region ranges from 0 to 3 ppm contains the frequencies of the aliphatic protons belonging to organic acids and free amino acids; the resonances appeared in the region between 3 and 5.6 ppm includes the signals of sugars; while the third region, from 5.6 to 10 ppm, is the aromatic region relating to phenolic metabolites. In order to assign each signal to the corresponding metabolites, in addition to the classical onedimensional 1 H and 13 C-{ 1 H} NMR spectra, 2D correlation experiments 1 H COSY and 1 H-13 C HMQC were employed. The two 2D spectra were processed using a sine filter and a qsine filter on both dimensions, F1 and F2, respectively on COSY and HMQC experiments before Fouriertransformed, the obtained 2D maps are reported in Figures 2.13 and 2.14 together with the enlargements of the spectral regions containing the correlation signals of the anomeric groups of -glucose and sucrose, shown as an example of the methodology adopted.

H 1 -glucose H 1 Sucrose H 1 Sucrose -H 2 Sucrose H 1 -glucose -H 2 -glucose Sucrose -glucose H 1 -glucose H 1 Sucrose H 1 Sucrose -C 1 Sucrose H 1 -glucose -C 1 -glucose
Thanks to one-dimensional NMR spectra and the 2D correlation experiments, 1 H COSY and 1 H-13 C HMQC, together with the data reported in the literature [START_REF] Belton | High-field proton studies of apple juices[END_REF][START_REF] Vermathen | Investigation of different apple cultivars by high resolution magic angle spinning NMR. A feasibility study[END_REF][START_REF] Vermathen | Exploring high-resolution magic angle spinning (HR-MAS) NMR spectroscopy for metabonomic analysis of apples[END_REF][START_REF] Tomita | A NMR-based, non-targeted multistep metabolic profiling revealed L-rhamnitol as a metabolite that characterised apples from different geographic origins[END_REF]) eleven metabolites were recognized by adopting the same methodology showed in 2.2.2 in both 1 H and 13 C spectra. In Table 2.2 are summarized the experimental information obtained from the NMR analysis of Golden Delicious apple juice. Once the metabolic profile for the Golden Delicious was determined, a comparison between the NMR spectra of the samples relating to the other apple varieties was possible. The comparison between proton spectra highlights that the metabolic profile is the same for all the four varieties of apples, as can be seen in In order to obtain more information and to evaluate any relationships and variations in composition in the four different variety of apple juices, the PCA method was used. In order to understand how the metabolites contribute to the varieties separation, the biplot need to be examined. Due to the low information content of PC3 and since a good separation in varieties is obtained from the first two PCs, the biplot reported in Figure 2.17

NMR characterization of Cannabis Sativa natural extract

Cannabis Sativa (also nown as "hemp") is a fast-growing, highly polymorphic and hybridized plant, belonging to the Cannabaceae family probably native to the Central Asia and the Indian subcontinent [START_REF] Li | An archaeological and historical account of Cannabis in China[END_REF][START_REF] Elsolhy | Phytochemistry of Cannabis Sativa L[END_REF][START_REF] Small | Classification of Cannabis Sativa L[END_REF]. It is one of the most ancient and versatile plants sources of textile fiber, bioactive extract and seed oil. In particular, in addition to the industrial application of hemp crops in the manufacture of textile fibers, Cannabis Sativa flower and seeds are commonly used in the production of extract characterized by high nutritional and/or therapeutic characteristics. Indeed, the seeds from which the oil is extracted, are rich in fatty acids with 3:1 ratio ω6/ω3 which give to the obtained oil a very good nutritional value [START_REF] Izzo | Chemical Analysis of Minor Bioactive Components and Cannabidiolic Acid in Commercial Hemp Seed Oil[END_REF]. Regarding the flowers of the plant, the female inflorescences of the Cannabis Sativa have been widely used in the traditional medicine of different populations thanks to the pharmacological activity of some molecules present in large quantities in these parts of the plant [START_REF] Mercuri | The long history of Cannabis and its cultivation by Romans in central Italy, shown by pollen records from Lago Albano and Lago di Nemi[END_REF]. This class of substances is composed by terpenophenolic 21-carbon atoms backbone molecules known by the name of phytocannabinoids of which almost 120 molecules have been identified and isolated to date (ElSolhy & Slade, 2017;[START_REF] Elsolhy | Phytochemistry of Cannabis Sativa L[END_REF]. The renewed and recent interest in Cannabis Sativa is due to the identification of these molecules and to the growing discovery of the different pharmacological activities that they possesses. Indeed, anti-inflammatory effects, inhibition of cell growth and tumor regression seem to be characteristics of Cannabis Sativa extracts which, although they were already known in ancient times, are now supported by numerous scientific works (Baker et al, 2003;[START_REF] Izzo | Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb[END_REF]. Among the phytocannabinoids, also called cannabinoids, the most representative are the well-nown psychotropic agent Δ 9trans-tetrahydrocannabinol (Δ 9 -THC), together with Cannabidiol (CBD), and its precursor Cannabidiolic acid (CBDA). Compared to THC, CBD show therapeutic benefits without euphoric or dysphoric effects, that represents an advantage for clinical applications (Baker et al, 2003;[START_REF] Izzo | Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb[END_REF]. CBD has become very popular over the years for its health benefits and nowadays is commercially available as a dietary supplement, lotion, and most importantly as a CBD oil. Indeed, the interest from the scientific community in the therapeutic potential of CBD oil is growing every day. The reason is simple: it has already been used in various scientific studies, for the treatment of numerous health problems and is now recognized as one of the main elements of the so-called "Therapeutic Cannabis" (Russo, 2018). Hemp crops with a low THC content have also been exploited as food, but they disappeared in the 1970s due to the association with the type of plants rich in THC [START_REF] Pisanti | Cannabis: A plurimillennial history of an evergreen[END_REF]. The reintroduction of the cultivation of some hemp cultivars with a THC content lower than 0.2% w/w, took place 20A00016)). This document fixes the content of THC at a maximum of 2 mg per kilo (0.002%) in hemp seeds, flour and derived foods and at a maximum of 5 mg per kilo (0.005%) in the oil obtained from hemp seeds. Currently the "Union method for the quantitative determination of the Δ 9 -tetrahydrocannabinol content in hemp THC and it provides a tolerance allowed equal to 0.03% in absolute value. However, this official method is quite laborious and the scientific community is always looking for advanced methodologies that allow to rapidly analyze natural mixtures without requiring manipulations or separations. The research presented in this work is placed in this scenario and aims to apply NMR methodologies to the study of natural extracts from seeds and inflorescences of Cannabis Sativa. Specifically, the work aims to perform a chemical profiling via NMR of the inflorescences extracts and seeds oil of Cannabis Sativa grown in Calabria region. It must be undelined that the study carried out presented in this section, is a preliminary study and it is part of a project that involved the LXNMR S.C.An. group in which I worked during my Ph.D., together with the company "Calabria Maceri e Servizi S.p.A." (Rende, CS, Italy) and the farm "Le Querce S.r.l" (Montalto Uffugo, CS, Italy). The aim of the aforementioned project was the characterization of metabolites present in the Cannabis Sativa extracts and at the same time optimize and validate an extraction method able to provide extracts rich in in both component, triacylglyceroles and medical cannabinoids, that can be employed in food and pharmaceutical industries for the production of enriched foods and supplements.

Samples preparation and experimental procedure

The seeds and flower used in this study came from Futura 75 cultivar which was harvested in september 2019. The storage and pre-treatment sample steps are mostly in accordance with the official "Union method for the quantitative determination of the Δ9-tetrahydrocannabinol content in hemp varieties" (Annex III of the Commission Delegated egulation (EU) No. 639/2014, 11 March 2014). In particular, all the collected hemp inflorescences were natural airdrying, manually separated from twigs and finely chopped. After this procedure, the samples were stored in the dark at 4.0°C until extraction procedure was performed. Seed samples were collected and, once dryed, they were ground into a powder and stored in the dark at 4.0°C until extraction procedure was performed. Dry inflorescence and seed oil obtained by using ethanol as extracting solvent were provided by "Calabria Maceri e Servizi S.p.A." (Rende, CS, Italy), while the crops were produced by the farm "Le Querce S.r.l" (Montalto Uffugo, CS, Italy).

Extracts from hemp flowers and seeds of Futura 75 were obtained by means of dynamic maceration with an ultrasound probe (Hielcher UP 100Hz, 100W pulse, 30 kHz frequency). A weighed amount of each sample, dried, chopped ad stored (2.00 g), was extracted with 50 mL of Ethanol at a room temperature for 20 min, under magnetic stirring. The solution was then paper filtered, evaporated under vacuum at 30°C and the residue was extracted with the same procedure one more time with other 50 mL of same solvent.

To prepare NMR tubes, after the evaporation under vacuum, the inflorescence extracts residue was dissolved in 1.20 ml of CDCl3 (CDCl3 -99.95 atom % D) and 600 µL of this solution were transferred to 5 mm o.d. NMR tube. The seeds oil was directly dissolved using 600 µL of CDCl3 in a 5 mm o.d. NMR tube. All the NMR spectra were recorded at room temperature on a 500 MHz Bruker Avance spectrometer (11.7 T) (Bruker, Fällanden, Switzerland) equipped with a 5 mm TBO probe and a standard variable-temperature unit BVT-3000, and on a 400 MHz Bruker Avance spectrometer (9.4 T) equipped with a TBI probe. Once obtained the spectra, all the data processing steps were carried out using TopSpin 3.6 software (Bruker BioSpin, Rheinstetten, Germany) (TopSpin, 2018).

Signal assignments

NMR-based metabolomics where applied in the characterization of hemp extract by using onedimensional (1D) and two-dimensional (2D) NMR experiments. In addition, in the case of hemp flowers extracts, Jij couplings between some pair of protons were measured thanks to the homonuclear 2D experiment 1 H-1 H J-Res. In the following sections the characterization of Futura 75 seed extracts and Futura 75 flower extracts are reported.

Hemp seeds oil

In Figure 2.18 is reported the 1D 1 H NMR spectrum of the hemp seeds oil obtained as described above.

Figure 2.18. 500 MHz 1 H NMR spectrum of hemp seeds oil dissolved in CDCl3 recorded at 298K. The spectrum was recorded using zg30 Bruker standard pulses sequence and for each experiment 128 FIDs were accumulated by using a spectral width of 10.00 ppm and a relaxation delay of 5 s. The NMR FID was Fourier-transformed, phased, baseline corrected and aligned using the TMS signal as reference.

From the proton 1D spectrum it was possible to recognize the classical profile of the polyunsaturated fatty acid esters (PUFAs) [START_REF] Sacchi | 1 H and 13 C NM of Virgin Olive Oil. An Overview[END_REF]. Indeed, in the 2D 1 H COSY it was possible to recognize all the correlation of the triacylglycerols containing both saturated and unsaturated fatty acids as shown in Figure 2.19 and, among them, it was possible to distinguish the signal of the terminal -CH3 of both 𝜔 6 and 𝜔 3 fatty acid which presence gives hemp seeds oil a very high nutritional power. As usual for all the assignments made, summarized in the Table 2.3, the informations obtained from the various recorded spectra have been used. shown together with the correspondent enlargement on the CBD and CBDA correlations taken as an example of the metabolic identification procedure adopted. In addition to the classical 1D and 2D experiments, in order to fully characterize the inflorescences extract, a J-Resolved experiment was performed. This kind of experiment allowed to separate on the F1 dimension the J-coupling contributions that can be easily measured. In order to show how this kind of experiment has been useful in the characterization of the extract, let's consider the case of the H9cis signal of CBD. In the 1D 1 H NMR spectrum, the signal of the proton H9cis is a multiplet from wich it is impossible to extract any useful information. On the contrary, by extrapolating the column in correspondence of the chemical shift of H9cis on the F2 dimension of the J-Resolved spectrum, as shown in the Figure 2.21, it is possible to obtain the 1D profile of the considered signal in which it is possible to measure all the coupling constants. Indeed, the resulting signal, classified as multiplet by analysing the only 1D 1 H NMR spectrum, actually is a dublet of quartet due to the interaction of the proton H9cis with the proton H9trans (dublet, 2 J9cis-trans=2.6 Hz) and with the protons H10 (quartet, 4 J9cis-10=0.9 Hz). The case just discussed is shown in the enlargement of the Figure 2.22 in which the obtained J-Resolved spectrum is reported.

All the assignation made are resumed in Table 2.4 together with al the information extracted from the 1D and 2D experiments. In addition to the cannabinoids, by comparison the protonic spectra of hemp seeds oil and hemp infuorescences extract, it was possible to recognize in this last spectrum also some signals that can be referred to the triacylclycerols constituent. Indeed, the use of ethanol as extracting solvent seems to be able to extract both component allowing to obtain extracts reach in bioactive compounds that can potentially be used in food and pharmaceudic industries for the production of enriched foods and supplements. All the signal assignment is reported in the Figure 2.23.

Conclusions

In this study NMR spectroscopy was applied in order to investigate the metabolic profile of Calabrian Cannabis Sativa extracts. In particular, hemp seeds oil and inforescences extract, obtained both through solute-solvent extraction ultrasound assisted using ethanol as extracting solvent, were studied. For the hemp seed oil the protonic characterization was performed and the triacylglyceroles profile was recognized by using 1 H COSY experiment. Regarding the inflorescences extract, it was possible to recognize three cannabinoids, CBD, CBDA and CBG together with the triacylglycerolic profile. The fact that any signal referred to THC was recognized indicates that the concentration of this substance is lower thant the detection limit of NMR spectroscopy [START_REF] Eisenmann | Untargeted NMR spectroscopic analysis of the metabolic variety of new apple cultivars[END_REF] and therefore also lower than the law limit.

Several studies have been conducted on these complex matrices [START_REF] Mazzoccanti | Cannabis through the loo ing glass: Chemoand enantio-selective separation of phytocannabinoids by enantioselective ultra high performance supercritical fluid chromatography[END_REF][START_REF] Nagy | Chemical Characterization of Leaves, Male and Female Flowers from Spontaneous Cannabis (Cannabis sativa L.) Growing in Hungary[END_REF], one of them, published very recently, involved the characterization of hydroalcoholic extracts of Cannabis Sativa using NMR spectroscopy [START_REF] Ingallina | Cannabis sativa L. Inflorescences from Monoecious Cultivars Grown in Central Italy: An Untargeted Chemical Characterization from Early Flowering to ipening[END_REF]. In this study different classes of compounds have been identified (six sugars, six organic acids, thirteen amino acids, choline and trigonelline) but no cannabininoids where detected. The presence of cannabidivarin (CBDV), cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN), (-)-∆9-tetrahydrocannabinol (THC) and cannabichromene (CBC) was verified only through UHPLC targeted analysis. This proves that, in order to obtain extracts rich in triacylglyceroles and medical cannabinoids, our extraction method is promising. Indeed the possibility to obtain an extract reach in both component by using ethanol as extracting solvent worth furter analysis and must be explored in order to confirm the evidence obtained in this preliminary study and try to validate a methodology able to extract both bioactive components that could represent a turning point in the production of functional food.

Metabolic composition and authenticity evaluation of Calabrian Bergamot Essential Oil (BEO)

Citrus bergamia, Risso, commonly known by the name of bergamot, is a fruit bearing tree belonging to the Rutaceae family, genus Citrus, which grows almost exclusively in ItalyIn particular, the pedoclimatic conditions of Calabrian strip coast between Scilla and Monasterace are exceptionally suitable for cultivating this kind of citrus fruit, as reported in the "Politiche Agricole, 2020" (Politiche agricole, 2020). Due to its composition in antioxidants, vitamins and biomolecules in general, bergamot possess healthly properties ranging from antioxidant and anti-inflammatory to anti-cholesterol properties [START_REF] Di Donna | Statin-Like Principles of Bergamot Fruit (Citrus Bergamia): Isolation of 3-Hydroxymethylglutaryl Flavonoid Glycosides[END_REF][START_REF] Di Donna | Hypocholesterolaemic Activity of 3-Hydroxy-3-Methyl-Glutaryl Flavanones Enriched Fraction from Bergamot Fruit (Citrus Bergamia): "In Vivo" Studies[END_REF][START_REF] Impellizzeri | The Anti-Inflammatory and Antioxidant Effects of Bergamot Juice Extract (BJe) in an Experimental Model of Inflammatory Bowel Disease[END_REF][START_REF] Gabriele | Citrus Bergamia Powder: Antioxidanti, Antimicrobial and Anti-Inflammatory Properties[END_REF][START_REF] Da Pozzo | Antioxidant and Antisenescence Effects of Bergamot Jouice[END_REF][START_REF] Ballistrieri | Evaluation of Lipid and Cholesterol-Lowering Effect of Bioflavonoids from Bergamot Extract[END_REF]. Moreover, bergamot juice possesses interesting nutraceutical potential to be exploited in the food industry and in particular in functional foods enriched in the polyphenolic fraction [START_REF] Pernice | Bergamot: A Source of Natural Antioxidants for Functionalized Fruit Juices[END_REF][START_REF] Dugo | The Composition of Bergamot Juice. In Citrus Bergamia Bergamot and Its Derivatives[END_REF][START_REF] Russo | Bergamot (Citrus Bergamia Risso) as a Source of Nutraceuticals: Limonoids and Flavonoids[END_REF][START_REF] Poli | Nutraceuticals and Functional Foods for the Control of Plasma Cholesterol Levels. An Intersociety Position[END_REF][START_REF] Manzoor | Flavonoids: Health Benefits and Their Potential Use in Food Systems. In Functional Food Products and Sustainable Health[END_REF][START_REF] Baron | Analytical Profile and Antioxidant and Anti-Inflammatory Activities of the Enriched Polyphenol Fractions Isolated from Bergamot Fruit and Leave[END_REF]. However, bergamot is cultivated mainly for the high-quality essential oil that is extracted by rasping and cold pressing the peel of the fruit. Indeed, due to its unique fragrance and freshness and thanks to its anti-seborrheic, stimulating and toning properties, Bergamot Essential Oil (BEO) is a raw substance with a high commercial content widely used as an essential component for the international perfume and cosmetics industry [START_REF] Bijaoui | Bergamot and Its Use in Perfumery. In Citrus Bergamia Bergamot and Its Derivatives[END_REF][START_REF] Dreger | Application of Essential Oils as Natural Cosmetic Preservatives[END_REF][START_REF] Valussi | Bergamot Oil: Botany, Production, Pharmacology[END_REF]. The current production of BEO in the Calabrian coast area represents more than 90% of the total world production and this product is considered to be of the highest quality in the international markets [START_REF] Crispo | Bergamot in the Calabrian Economy and the ole of Consorzio del Bergamotto[END_REF][START_REF] Navarra | Citrus Bergamia Essential Oil: from Basic esearch to Clinical Application[END_REF]. Three cultivars of bergamot (Castagnaro, Fantastico, and Femminello) are commercially grown in the pedoclimatic condition of Calabrian coast and then industrially processed, to extract both their essential oils and their juices [START_REF] Valussi | Bergamot Oil: Botany, Production, Pharmacology[END_REF]. Generally, although these cultivars have different characteristics, the industrial process underlying the essential oil uses an indiscriminate mix of the three cultivars. It should be noted that today BEO is extensively used also in food industry and in the pharmaceutical and medical fields for its efficiency and health benefits due to a large number of bioactive molecules that contains [START_REF] Pizzimenti | Antimicrobial Activity of Citrus Bergamia isso & Poiteau. In Citrus Bergamia Bergamot and Its Derivatives[END_REF]Radford, 2013;[START_REF] Navarra | Citrus Bergamia Essential Oil: from Basic esearch to Clinical Application[END_REF]. In several papers, including very recent ones, it has been described that BEO possesses a wide variety of activities such as melanogenic, antinociceptive, antiproliferative, sedative, anxiolytic, neuroprotective, antioxidant and antimicrobial activities [START_REF] Cirmi | Anti-Infective Potential of Citrus Bergamia isso et Poiteau (Bergamot) Derivatives: A Systematic eview[END_REF][START_REF] Rombolà | Bergamot Essential Oil Attenuates Anxiety-Like Behavior in ats[END_REF]Dosiky & Setzer, 2018;[START_REF] Shaaban | Novel Bergamot Oil Nanospanlastics Combined with PUVB Therapy as a Clinically Translatable Approach for Vitiligo Treatment[END_REF][START_REF] Lombardo | Mechanisms Underlying the Anti-Inflammatory Activity of Bergamot Essential Oil and Its Antinociceptive Effects[END_REF][START_REF] Arena | Potential Use of Citrus Essential Oils Against Acute espiratory Syndrome Caused by Coronavirus[END_REF][START_REF] Maugeri | Bergamottin and 5-Geranyloxy-7-methoxycumain Cooperate in the Cytotoxic Effect of Citrus Bergamia (Bergamot) Essential Oil in Human Neuroblastoma SH-SY5Y Cell Line[END_REF][START_REF] Valussi | Bergamot Oil: Botany, Production, Pharmacology[END_REF]. For example, clinical studies showed that bergamottin, a natural furocoumarin first identified in bergamot essential oil (hence the name), is able to significantly decrease the electrocardiographic changes that are typical of coronary arterial spasms, hence the antianginal and antiarrhythmic properties of BEO [START_REF] Navarra | Citrus Bergamia Essential Oil: from Basic esearch to Clinical Application[END_REF]. More recently, the same bergamottin and the 5-geranyloxy-7methoxycoumarin have been claimed to be relevant in the anti-proliferative effects exerted by bergamot essential oil in the SH-SY5Y human neuroblastoma cells [START_REF] Maugeri | Bergamottin and 5-Geranyloxy-7-methoxycumain Cooperate in the Cytotoxic Effect of Citrus Bergamia (Bergamot) Essential Oil in Human Neuroblastoma SH-SY5Y Cell Line[END_REF].

Furthermore, it has been established that the analgesic effect of BEO is linked to the high content of linalool while its significant cytotoxicity is closely connected to the association of limonene and linalyl acetate, suggesting a combined action of these monoterpenes in the death of cancer cells (Russo et al, 2013). In addition, in the food sector, BEO is used as a flavoring in various products such as tea, ice cream, soft drinks, and as a natural alternative to chemicals for food preservation (Radford, 2013;[START_REF] Avila-Sosa | Bergamot (Citrus Bergami) Oils[END_REF]. Therefore, because of the high commercial value and the importance that the BEO plays in various fields, a subpar production or downright adulteration of the product is quite common. Over the years, the chemical composition of BEO has been extensively studied and it is well known that BEO is composed of a volatile fraction (93-96%) and a non-volatile fraction (7-4%). The non-volatile fraction consists mainly in coumarins, psoralens and pigments, while the volatile substances are mainly monoterpene and sesquiterpene hydrocarbons and their oxygenated derivatives [START_REF] Navarra | Citrus Bergamia Essential Oil: from Basic esearch to Clinical Application[END_REF][START_REF] Avila-Sosa | Bergamot (Citrus Bergami) Oils[END_REF]Gioffrè et al, 2020;[START_REF] Valussi | Bergamot Oil: Botany, Production, Pharmacology[END_REF]. The composition of the volatile fraction is highly variable, and this variability depends on several parameters such as cultivar [START_REF] Russo | Bergamot Peel and Leaf Extracts by Supercritical Fluids Technology and Composition. In Citrus bergamia: Bergamot and its Derivatives[END_REF]Giuffré et al, 2020), harvesting [START_REF] Marzocchi | Effect of Harvesting Time on Volatile Compounds Composition of Bergamot (Citrus × bergamia) Essential Oil[END_REF]Gioffrè et al, 2020), geographic origin and pedoclimatic characteristics of the production area [START_REF] Statti | Chemical and biological diversity of Bergamot (Citrus bergamia) in relation to environmental factors[END_REF], agronomic practices and extraction system [START_REF] Lazarotto | Chemical Composition and Antibacterial Activity of Bergamot Peel Oil from Supercritical CO2 and Compressed Propane Extraction[END_REF]. All these variables combined with the limited production, availability and high market value of the BEO, makes it a favourite target of fraudulent manipulation [START_REF] Valussi | Bergamot Oil: Botany, Production, Pharmacology[END_REF]. Dilution by adding less valuable oils, addition of chemical additives and preparation of the oil by mixing pure substances are some of the most frequent frauds in the essential oil sector that represent a serious problem for regulatory agencies [START_REF] Bonaccorsi | Adulteration of Bergamot Oil" In Citrus Bergamia Bergamot and Its Derivatives[END_REF]. For all these reasons, the use of advanced methodologies, able to identify and quantify the compounds present in BEO, is essential to ensure its quality and genuineness. The analytical techniques commonly used to perform compositional studies and currently accepted for the release of the "Protected Designation of Origin (PDO)" of "Bergamotto di eggio Calabria -Essential oil", are Gas Chromatography (GC) and High-Performance Liquid Chromatography (HPLC) [START_REF] Bonaccorsi | Adulteration of Bergamot Oil" In Citrus Bergamia Bergamot and Its Derivatives[END_REF][START_REF] Crispo | Bergamot in the Calabrian Economy and the ole of Consorzio del Bergamotto[END_REF]. A valid alternative to traditional analytical methods is NMR spectroscopy which advantages have been shown in chapter 1. In recent years, NMR has also gained ground in the field of fraud detection, especially in cases where established methods of fraud detection fail, such as when multiple and sophisticated adulterants are used [START_REF] Sobolev | Use of NM Applications to Tac le Future Food Fraud Issues[END_REF]Pacholczyk-Sienicka et al, 2021;Solovyey et al, 2021). Even though different works are present in literature on the application of NMR for the determination of metabolic profile of some essential oil [START_REF] Skakovskii | Application of NM Spectroscopy for Analysis of the Composition of Pine Needle Essential Oil[END_REF][START_REF] Skakovskii | Characterization of Essential Oil of Agastache ugosa By NM Spectroscopy[END_REF]Cerceau et al, 2016;[START_REF] Freitas | Chemometric Analysis of NM and GC Dataset for Chemotype Characterization of Essential Oils from Different Species of Ocimunìm[END_REF], to the best of our knowledge, such analysis has never been performed on BEO, the only works in the literature being limited to the molecular characterization of single components or fractions of bergamot deriving from separative techniques [START_REF] Di Donna | Statin-Like Principles of Bergamot Fruit (Citrus Bergamia): Isolation of 3-Hydroxymethylglutaryl Flavonoid Glycosides[END_REF][START_REF] Formisano | Detailed Phytochemical Characterization of Bergamot Polyphenolic Fraction (BPF) by UPLC-DAD-MS and LC-NM[END_REF]. Thus, in this context, the current study was designed to explore the feasibility of using versatile high-resolution NMR spectroscopy to provide useful experimental informations on both chemical composition, including qualitative and quantitative information, and fraud detection of a BEO sample, without sample pretreatment and in a rapid and non-destructive way. To this end and with the intention of fine-tuning the methodology, we decided to consider only a single representative sample of certified BEO, using only aliquots of this sample in all the experiments conducted in the work. Therefore, for the first time, NMR spectroscopy was applied to a pure and certified BEO sample, coming from the Italian consortium "Consorzio di Tutela del Bergamotto di Reggio Calabria", with a dual purpose, namely to verify whether the technique was able to: a) identify, characterize and quantify the main components of this complex mixture; b) discriminate a sample of pure BEO from adulterated ones, obtained simulating the dilution adulteration by adding different quantity of a less valuable oil, i.e.

Orange Essential Oil (OEO), to the pure BEO, and by combining the 1 H NMR spectra, acquired on the adulterated samples and on the pure BEO, with chemometric analysis (PCA).

Samples preparation and experimental procedure

For this study, the samples of BEO were supplied from "Consorzio di Tutela del Bergamotto di eggio Calabria" whose essential oil has the "Protected Designation of Origin (PDO)" of "Bergamotto di eggio Calabria -Essential oil" mar . The "Consorzio " provided us the following information on the sample we used: the sample was obtained from a mixture of fruit peels originated from the fruits of the three cultivars, Castagnaro, Fantastico and Femminello, grown in the same location and harvested in the same period of the year, i.e. December 2019.

For the metabolites identification, a sample was prepared by directly inserting 50 μL of BEO into 5-mm o.d. NMR-tube, with 500 μL of deuterated chloroform, CDCl3 containing 0.03% of tetramethylsilane, TMS. The CDCl3 was purchased from Eurisotop and it was used as fieldfrequency lock signal. The TMS contained in the solvent was used as internal reference for chemical shifts. The signal assignation was performed using 1D 1 H and 13 C-{ 1 H} NMR experiments and 2D NMR ( 1 H COSY, 1 H-13 C HMQC and 1 H DOSY) experiments and taking advantage of the data available in the literature [START_REF] Kolehmainen | Complete Spectral Analysis of the 1H NM 16-Spin System of -Pinene[END_REF][START_REF] Skold | Studies on the Autoxidation and Sensitizing Capaciti of the Fragrance Chemical Linalool, Identifying a Linalool Hydroperoxide[END_REF][START_REF] Wishart | HMDB: the Human Metabolome Database[END_REF][START_REF] Chunyan | Isolation and Purification of Psoralen and Bergapten from Ficus Carica L. Leaves by High-Speed Countercurrent Chromatography[END_REF][START_REF] Wishart | HMDB: a nowledgebase for the human metabolome[END_REF][START_REF] Wishart | HMDB 3.0 -The Human Metabolome Database in 2013[END_REF][START_REF] Wishart | HMDB 4.0 -The Human Metabolome Database for 2018[END_REF]Liu et al, 2017). However, in order to verify a correct assignment of the peaks, samples of the single available metabolites were also prepared. The pure compounds (R)-limonene, terpinene, -pinene, linalool and linalyl acetate were purchased from Fluka and Acros respectively, and NMR samples have been prepared by dissolving ~ 25 mg of each compounds in 500 μL of CDCl3. For the quantitative analysis of the main compounds present in BEO, a sample of BEO in CDCl3 was prepared by carefully weighing all the components and using benzoic acid as an internal standard. Thus, a mixture of 24.7 mg of BEO, 2.9 mg of benzoic acid and 857 mg of CDCl3 was prepared directly into the NMR tube and a 1 H NMR spectrum recorded. For the adulteration study, as an adulterant agent a sample of commercial Orange Essential Oil (OEO), from herbalist firm "Erboristeria Magentina Arancio Amaro -Olio

Essenziale", has been used. Adulteration procedure was simulated by preparing three solutions containing different weight ratios of pure BEO and pure OEO: i) solution A containing 95% of BEO and 5% of OEO; ii) solution B containing 90% of BEO and 10% of OEO; iii) solution C 70% of BEO and 30% of OEO. For each solution 14 different NMR samples were prepared inserting ~ 50 μL into NM tubes with 500 μL of CDCl3, for a total of 42 samples. In addition, other 7 NMR samples containing only BEO were prepared in the same way. On these 49 samples 1 H NMR spectra were recorded and subsequently used for statistical analysis. A last adulterated solution was prepared: solution D 50 % of BEO and 50% of OEO. This solution was not used for statistical purposes but only to conduct a study from the diffusive point of view, together with solutions B, C and pure BEO. All the chemical compounds were used without further purification and no additional treatment was necessary for the preparation of NMR samples. NMR spectra were recorded at room temperature on a 500 MHz Bruker Avance spectrometer (11.7 T) (Bruker, Fällanden, Switzerland) equipped with a 5 mm TBO probe and a standard variable-temperature unit BVT-3000, and on a 400 MHz Bruker Avance spectrometer (9.4 T) equipped with a TBI probe. Once obtained the spectra, all the data processing steps were carried out using TopSpin 3.6 software (Bruker BioSpin, Rheinstetten, Germany) (TopSpin, 2018).

Signals attribution

NMR-based metabolomics generally makes use of the 1 H NMR spectra which contains, in principle, a large amount of chemical information, but which, in the case of very complex mixtures, are often very difficult to extract from these spectra. Indeed, the superposition of the spectral lines makes it difficult to recognize metabolites through the single inspection of the 1D spectrum. This is the case of the BEO which presents a 1 H and a 13 C-{ 1 H} NMR spectrum consisting of a tangle of superimposed lines from which it was not possible to extract any useful information, as can be seen from Figure 2.24. The reported 1D NMR FIDs were Fouriertransformed, phased, baseline corrected and aligned using the TMS signal as reference. In the case of 13 C-{ 1 H} NMR spectra, it was filtered with an exponential 1 Hz line broadening before Fourier-transformation. spectral width of 14.00 ppm and a relaxation delay of 3 s.; B) 13 C-{ 1 H} NMR spectrum (zgig Bruker pulse sequence), performed with proton broad-band decoupling collecting 8K free induction decays (FIDs) using a spectral width of 280.00 ppm and a relaxation delay of 4s.1D NMR FIDs were Fouriertransformed, phased, baseline corrected and aligned using the TMS signal as reference. 13 C-{ 1 H} NMR spectra were filtered with 1 Hz line broadening before Fourier-transformation.

Correlation study

To overcome these limitations inherent the 1D spectra, the identification of the BEO mixture constituents was performed by 2D NMR experiments and verifying the assignments through Connectivity information obtained from 2D spectra together with the use of literature data [START_REF] Kolehmainen | Complete Spectral Analysis of the 1H NM 16-Spin System of -Pinene[END_REF][START_REF] Skold | Studies on the Autoxidation and Sensitizing Capaciti of the Fragrance Chemical Linalool, Identifying a Linalool Hydroperoxide[END_REF][START_REF] Wishart | HMDB: the Human Metabolome Database[END_REF][START_REF] Chunyan | Isolation and Purification of Psoralen and Bergapten from Ficus Carica L. Leaves by High-Speed Countercurrent Chromatography[END_REF][START_REF] Wishart | HMDB: a nowledgebase for the human metabolome[END_REF][START_REF] Wishart | HMDB 3.0 -The Human Metabolome Database in 2013[END_REF][START_REF] Wishart | HMDB 4.0 -The Human Metabolome Database for 2018[END_REF]Liu et al, 2017), allowed to assign the resonances of eleven compounds present in the BEO. However, although conventional 1D and 2D experiments allowed us to identify these compounds, many signals remained unassigned in all regions of the spectra due to signals overlap, as the case of aliphatic region.

Diffusion study

In an attempt to increase the assignments achieved with 1D NMR and 2D correlation experiments, high-resolution 2D DOSY spectra were therefore recorded and analyzed. The DOSY experiment allows to separate the signals of different compounds in solution on the basis of their different self-diffusion coefficients (Di). In the DOSY plot, chemical shifts are in one dimension (F2) and molecular self-diffusion coefficients (Di) in the other one (F1). Considering that D is a molecular property, because it depends from molecular masses and shape, all peaks from one molecule will be aligned at the same value of D in the F1 dimension and thus, can be distinguished from the other components with different values of Di [START_REF] Sobolev | Proton High-Field NM Study of Tomato Juice[END_REF].

The diffusion study in the case of a multicomponent system such as an essential oil turns out to be quite complex, not only for the high number of substances present in it, but also for the similarity between the structures of the molecules present in the oil. Indeed, as states the Stoke-Einstein relation (equaztion 1.46), the diffusion coefficient is strictly dependent on the size of the diffusing particle that, in the case of the essential oil, are terpene structures with very similar molecular weight. As consequence, carrying out diffusion studies aimed at discriminating the various species is challenging. In order to test this possibility, this study started by analyzing the diffusion properties of a two-component mixture prepared dissolving (R)-Limonene in CDCl3. Limonene is one of the major component of the bergamot essential oil, it is a chiral volatile hydrocarbon classified as a cyclic monoterpene. It is colourless at room temperature and has a strong smell of lemons or turpentine depending on the enantiomer considered. It takes its name from the lemon due to the lemon peel which, like other citrus fruits, contains large quantities of this chemical compound and is largely responsible for the characteristic smell [START_REF] Dugo | The Composition of Bergamot Juice. In Citrus Bergamia Bergamot and Its Derivatives[END_REF][START_REF] Baron | Analytical Profile and Antioxidant and Anti-Inflammatory Activities of the Enriched Polyphenol Fractions Isolated from Bergamot Fruit and Leave[END_REF].

By studing this simple mixture, it has been possible to optimize the experimental protocol and all the necessary parameters which were then applied to the more complex mixtur of the BEO.

Indeed, the first step performed involved the pulse sequence selection. As showed in the section 1.3.2.2, the choice of a pulse sequence for DOSY depends on both the capabilities of the As already mentioned, the substances that make up the BEO are very similar and consequently diffusion coefficients are expected to be not very far from each other. For this reason, before studying the natural complex mixture, other samples were analyzed in order to understand how the possible variables influence the diffusion coefficient and also to evaluate the sensitivity of NMR to detect the possible differences. Indeed, once optimized all the needed parameter, it was tested the possibility to differentiate diffusion coefficient of Limonene on the basis of a) temperature; b) samples concentration and c) viscosity of the solvent.

In particular were studied samples of (R)-Limonene in deuterated chloroform at 6 M, 5.2 M, 3.9 M, 3.0 M, 2.2 M and 1.2 M at 306.9 K. On the sample 2.2 M, a diffusion study at different temperature has been performed, in particular 258.7 K, 282.8 K, 306.9 K and 320.4 K were investigated. Last, samples of (R)-Limonene in different solvents were studied in order to evaluate the effect of changes in viscosity on diffusion coefficient. DMSO, DMF, THF and acetone were used in the preparation of samples of (R)-limonene in concentration 2.2 M and on these samples DOSY experiments were recorded at 306.9 K. All the results obtained are shown in Figures 2.28 On the basis of the results shown, it is possible to draw some conclusions. The first deduction that can be made relates to the sensitivity of the technique: NMR in fact has been shown to be able to highlight changes in the diffusion coefficient of limonene following changes in the experimental conditions. Regarding the trends obtained, in the case of the samples in different concentration of limonene, the values obtained lie on a hyperbole-like curve and this is in agreement with the data reported in literature regarding binary solutions studied with other techniques different from NMR (Tyn & callus, 1975). Concerning the study at different temperatures, in this case the Stokes-Einstein equation (equation 1.46) can be considered in order to study the results obtained. Indeed, from the experimental point of view, the diffusion coefficient grow when the temperature increase and this is in line with the theory. However, in this case the relation between diffusion coefficient and temperature is not linear as the Stokes-Einstein equation states. This can be attributed to the fact that at different temperatures the studied sample also have different viscosity, a parameter which affects the diffusion coefficient as states the equation 1.46. Indeed, in the case of limonene samples dissolved in solvents with different viscosities, different diffusion coefficient values are obtained. Also in this case the relation seems not to be linear from the experimental point of view and this is probably due to the effect of other variables that can affect the studied system (different solute-solvent interaction, for example). All these tests were designed and performed to demonstrate that the technique used represents a good tool for the diffusion study of binary mixtures and allows to distinguish different conditions. A last test were performed in order to examine the possibility to carry out this kind of study on complex mixture: a model mixture were prepared and studied by using the most aboundant metabolite recognized in the BEO. The sample prepared consists in linalool (0.954 g), linalyl acetate (0.483 g), (R)-limonene (1.225 g), (S)-limonene (0.029 g), -terpinene (0.255 g) and -pinene (0.190 g). The amounts of each metabolite were weighed in order to have the relative amount of the various species equal to those reported in the literature for the BEO [START_REF] Dugo | Characterization Of Cold-Pressed And Processed Bergamot Oils By Using GC-FID, GC-MS, GC-C-IRMS, enantio-GC, MDGC, HPLC and HPLC-MS-IT-TOF[END_REF]. The DOSY obtained is reported in Figure 2.31. 2D DOSY 1 H NMR spectrum of BEO is reported in Figure 2.31 and, as example, in the box the extrapolation of the line corresponding to the diffusion coefficient of limonene is reported. As it can be seen, the signals of the methyl groups of limonene (H1'' and H1'), impossible to be assigned by using conventional experiments, are those that resonate at 1.65 ppm and 1.73 ppm respectively. Similarly, the signals at 1.28 ppm, 1.59 ppm and 1.67 ppm were assigned to the methyl groups of linalool, the signals at 1.53 ppm, 1.59 ppm, 1.67 ppm and 2.00 ppm to the methyl groups of linalyl acetate, the signals at 0.72 ppm and 1.23 ppm to the methyl groups of -pinene and, finally, the signals at 1.01 ppm and 1.66 ppm to the methyl groups of -terpinene. that about 90% of the BEO sample analysed has been characterized and quantified via NMR.

Moreover, the percentage obtained via NMR for each quantified metabolite is substantially in agreement with the range of percentages reported in the literature about the same compounds and obtained from other analytical procedures on BEO samples from three different seasons [START_REF] Dugo | Characterization Of Cold-Pressed And Processed Bergamot Oils By Using GC-FID, GC-MS, GC-C-IRMS, enantio-GC, MDGC, HPLC and HPLC-MS-IT-TOF[END_REF]. * The data reported in the column were obtained by analyzing the 1 H-13 C HMQC spectrum, wich allow to obtain information on the directly linked 1 H and 13 C nuclei, and confirmed by the 13 C-{ 1 H} NMR spectra acquired on pure compounds, while the quaternary carbons resonances were obtained only from 13 C-{ 1 H} NMR spectra acquired on pure compounds.

Authenticity evaluation

As largely proved in the previous sections, 1D and 2D NMR spectroscopy is a powerful tool for high-throughput characterization of metabolites in natural extract. However, an accurate identification of compounds in complex mixture remain a challenging task, especially in spectral regions characterized by high degree of signal overlap. The number of information present in each spectrum is so huge that it is unthinkable to perform an accurate comparison between two spectral profiles. To overcome these complications, the use of statistical tools is the only way in the authentication detection field. In order to highlight possible variations in the metabolites composition deriving from dilution adulteration with other essential oils, spectra recorded on mixtures containing BEO and OEO in different concentrations were compared with the pure BEO. In Figure 2.35, the 1 H-NMR spectra of the pure BEO and three adulterated sample containing 5%, 10% and 30% of OEO are shown, respectively. As it can be seen from the Figure, no differences are detected in the spectral profile of the four spectra, so that, for a systematic comparison of the samples, the use of multivariate analysis is essential. In order to evaluate the possibility to distinguish the various situations, three approach were adopted. First were tested the possibility to distinguish different degrees of adulteration through diffusion studies. This approach was followed by a quantitative study of the metabolites present in major quantities in the different adulterated samples, and finally the data obtained from the NMR study conducted on various samples were statistically treated to highlight the type of adulteration studied.

Diffusion study

The possibility to distinguish the different diffusion coefficients of limonene in the case of different solute concentrations or different solvent viscosities was reported in section 2.5.4.2.

The results obtained suggest that NMR spectroscopy has the potential to distinguish the diffusion coefficient of limonene if it is present in pure BEO rather than in an adulterated sample in which the conditions of concentration and viscosity could vary. In order to verify once again this assumption, three model mixture were prepared using the amount of most abuntant metabolites found during the quantitative study and using for the remaining 10% three different substances, one for each model mixture prepared. Since the literature shows that BEO is made up of organic species consisting of six to ten carbon atoms [START_REF] Dugo | Characterization Of Cold-Pressed And Processed Bergamot Oils By Using GC-FID, GC-MS, GC-C-IRMS, enantio-GC, MDGC, HPLC and HPLC-MS-IT-TOF[END_REF] As can be seen from the results in

B (90% in BEO) (1.12 ± 0.01) × 10 -5 C (70% in BEO) (1.11 ± 0.02) × 10 -5 D (50% in BEO) (1.32 ± 0.01) × 10 -5 Pure BEO Solution B Solution C Solution D 1,00E-05 1,05E-05 1,10E-05 1,15E-05 1,20E-05 1,25E-05 1,30E-05 1,35E-05 40% 50% 60% 70% 80% 90% 100% Diffusion Coefficient (cm 2 /s) % in BEO
As can be deduced from the results, adopting this methodology it is possible to discriminate an high degree of adulteration such in the case of solution D containing 50% of adulterating agent, on the contrary it is not possible to distinguish the adulterated solution containing BEO in concentrations of 70% and 90% from the pure BEO because the diffusion coefficients obtained for the three situations are very similar to each other. To note that the data presented refer to a preliminary study on adulteration detection using diffusion studies, indeed further tests should be performed and a validation step is needed in order to propose this method as an alternative to the classic techniques commonly accepted.

Quantitative study

The second methodology tested in the adulteration detection is the quantitative study of the most abountant metabolites. In particular the quantitative study was performed on solution A,B ad C, in order to evaluate if this approach is able to detect even small amount of adulterating agent. The results obtained were compared with the quantitative data previously obtained for the pure BEO and they are represented in Figure 2.38. From the quantitative study results an increase in concentration of limonene and a reduction in concentration of linalyl acetate, linalool, -pinene and -terpinene as the degree of adulteration increases. In addition, it is possible to distinguish the 70% sample in BEO from the pure BEO, distinction that was not possible by using the approach based on diffusion. However, even if the trend of the amount of each metabolite is quite clear, also in this case it is difficult to distinguish samples with a low degree of adulteration from samples of pure BEO. Indeed, it is still impossible to distinguish pure BEO from the oil adulterated with 5% and 10% OEO and, in order to achieve this kind of discrimination, multivariate analysis is mandatory.

Statistical approach

In this study, the Principal Component Analysis (PCA) has been employed in order to evaluate whether the 1 H NMR spectra can be used as a criterion to discriminate the samples according to the different percentage of adulteration, placing more attention on solutions with a low amount of adulterating agent. The exploratory PCA analysis was applied to the 49 1 H NMR spectra (7 of pure BEO and 14 for each degree of adulteration) and it was carried out using R software (R Core Team, 2019). Note that, in this study, the PCA has been employed in a descriptive way to simplify and condense the global information provided by the spectra describing the entire chemical composition into a few parametric descriptors. The unsupervised procedure was applied to the data matrix obtained bucketing the 1 H NMR spectra in 0.05 ppm section. The bucketing procedure was performed using TopSpin 3.6 software (Bruker BioSpin, Rheinstetten, Germany) (TopSpin, 2018). Manually, the list of the integration ranges has been defined on the first spectrum and the other spectra were bucketed automatically using the saved list. The obtained integrals were normalized and exported in an excel file in which the data matrix has been mean-centered and scaled (correlation method) [START_REF] Borgognone | Principal Component Analysis in Sensory Analysis: Covariance or Correlation Matrix?[END_REF].

The 3D score plot obtained is reported in Figure 2.39, where a cumulative percentage of explained data variance of 75.7% (39.9%, 23.5% and 12.3% for PC1, PC2 and PC3 respectively) summarizes the relationship between samples. A well-defined separation between the samples of the four analysed mixtures is clearly evident from the score plot of Figure 2.39. Indeed, the group consisting of the pure BEO samples (in blue) are well clustered and clearly distinguishable from the adulterated samples. In turn, the samples adulterated at 30% in OEO (in red), those at 10% (in pink) and those at 5% (in green), are well grouped within each percentage and, although those at 5% and at 10% are closely spaced, they are distinguishable and far enough from the cluster of pure BEO samples. Hence, according to the score plot, it can be assumed that, in addition to distinguish the pure BEO samples from those adulterated, it is also possible to differentiate among the degrees of adulteration. The main component that would seem to best discriminate the four cases considered is PC2. Specifically, the pure BEO samples are characterized by negative PC2 values while, as the degree of adulteration increases, the scores move towards increasingly positive values and at the maximum of the adulteration considered in this study (30% in OEO) all the samples have positive PC2 values.

In order to determine which chemical compounds are crucial for the discrimination, an accurate examination of the loadings has been performed. In the Figure 2.40 is reported the 2D graph obtained plotting scores and loading on the second and third components, the two that better contribute to the clustering. The biplot provides clear evidence to identify the variables responsible for the sample clustering observed in the score diagram. Indeed, the loadings show how both components considered are affected by the terpene fraction of the oils. Of this volatile fraction the loadings of linalool, linalyl acetate, -terpinene, limonene and -pinene dominate the PC2. Specifically, from the biplot analysis it emerges that the pure BEO samples are rich in linalool, linalyl acetate, -terpinene and -pinene, while, on the contrary, the adulterated samples appear to be rich in limonene. Therefore, limonene represents a discriminant in the volatile fraction. On the other hand, for the non-volatile fractions, coumarins and psoralens are characterized by negative values of both components PC2 and PC3.

They therefore appear to contribute to the discrimination between the various clusters and, in particular, represent markers of adulteration as they are more present in bergamot oil, as it can be seen from the biplot (Figure 2.40).

Conclusions

The use of NMR techniques in new fields of application such as the quantitative monitoring of different classes of chemical compounds without the need of derivatizing or manipulating the sample, has had a considerable development in recent years and nowadays it is recognized as one of the most powerful analytical techniques in the field of molecular investigation of complex mixtures, such as foods or other natural matrices. In this work, NMR spectroscopy was successful applied for the first time to a non-manipulated sample of certified bergamot essential oil (BEO) with a dual purpose: on one side, to perform the characterization and quantification of the main metabolites of this complex matrix, whereas, on the other side, to explore the possibility of investigating and discriminating against any fraud to which the BEO could be subject. The results reported by applying the high-resolution NMR methodologies to the BEO show the potentialities of this technique in obtaining a large set of information data, otherwise available only by combining different analytical techniques, suitable for the type of investigations we planned to. Indeed, by combining the experimental data extracted from the 1 H and 13 C one-dimensional spectra with the connectivity arising from the 1 H COSY and 1 H-13 C HMQC correlation spectra and using the diffusion coefficients derived from the 2D 1 H DOSY experiment, we were able to identify eleven low molecular weight compounds present 2.6.

Molecular Characterization of the Organic Fraction of Municipal Solid

Waste and Compositional Evolution during Oxidative Processes assessed by HR-MAS 13 C NMR Spectroscopy Waste management has become a relevant problem in modern society since the development of urban areas has led to a growth in waste production and consequently to a reduction in the places where all types of materials can be deposited (Minelgaite & Liovikiene, 2019). The reduction of the quantity of waste to be stored in landfills has become a priority problem and, consequently, different strategies and treatment methods suitable for this purpose are adopted [START_REF] Zorpas | Strategy Development in the Framewor of Waste Management[END_REF]Sankoh, 2020). First of all recycling, combined with avoidance, separate collection and reuse, contributes not only to waste reduction but also to energy saving and to the reduction of polluting and climate-altering emissions [START_REF] Hamoda | Air Pollutants Emissions fro Waste Treatment and Disposal Facilities[END_REF][START_REF] Das | Solid Waste Management: Scope and the Challenge of Sustainability[END_REF]. All these actions, and in particular separate collection, are very useful in reducing and recycling waste but are not sufficient to achieve complete abatement, hence the need to develop new waste disposal and treatment methods that can convert waste into new products to be used in different fields so that the potential of waste can be fully exploited [START_REF] All | A eview on Vermicomposting of Organi Wastes[END_REF][START_REF] Soudejani | Application of Zeolites in Organic Waste Composting: A eview[END_REF]. In order to choose the right technique, the first step is to classify waste according to their composition [START_REF] Diaz | Composting and ecycling Municipal Solid Waste[END_REF]. In this study, our attention is focused on the Organic Fraction of Municipal Solid Waste (OFMSW) which includes food residues or food preparations and assimilable fractions, and which constitutes more than 30% by weight of municipal solid waste [START_REF] Hailu | Municipal Solid Waste Source Identification, Characterization and Physical Composition Analysis, Case Study Wol ite Town, Ethiopia[END_REF]. This waste fraction is generally converted in biogas through anaerobic digestion [START_REF] Mata -Alvarez | Anaerobic Digestion of Organic Solid Wasts. An Overview of esearch Achievements and Perspectives[END_REF][START_REF] Khalid | The Anaerobic Digestion of Solid Organic Waste[END_REF][START_REF] Li | Soli-State Anaerobic Digestion for Methane Production from Organic Waste[END_REF][START_REF] Pramanik | The Anaerobic Digestion Process of Biogas Production from Food Waste: Prospects and Constraints[END_REF] or transformed into soil improvers through a biological process of composting for the use in agriculture [START_REF] Diaz | Composting and ecycling Municipal Solid Waste[END_REF][START_REF] Soumaré | Effect of a Municipal Solid Waste Compost and Mineral Fertilization on Plan Growth in two Tropical Agricultural Soils of Mali[END_REF][START_REF] Hargreaves | A eview of the Use of Composted Municipal Solid Waste in Agriculture[END_REF][START_REF] Kumar | Composting of Municipal Solid Waste[END_REF][START_REF] Cerda | Composting of Food Wastes: Status and Challenges[END_REF]. These practices have many disadvantages related both to the time required for bacteria to degrade the various organic substances and to the possible problems of sickening smell or environmental pollution due to the accumulation of this fraction. Henceforth, the need to develop new treatment methods that can be used as an alternative to the existing ones even economically and ecologically more advantageous. The chemical-physical process based on Fenton reagents, an oxidative method commonly used to treat a large variety of pollutants in water (phenols, formaldehyde, BTEX pesticides, rubber chemicals and so on) seems to be one of the promising methods in treatment of OFMSW [START_REF] Koppenol | The Centennial of the Fenton eaction[END_REF][START_REF] Oliveros | Industrial Waste Water Treatment: Large Scale Development of a Light-Enhanced Fenton eaction[END_REF][START_REF] Chamarro | Use of FENTON eagent to Improve Organic Chemical Biodegradability[END_REF][START_REF] Neyens | A eview of Classic FENTON's Peroxidation as an Advanced Oxidation Technique[END_REF][START_REF] Roccotelli | Organic Matter Characterization and Phytotoxic Potential Assessment of a Solid Anaerobic Digestate Following Chemical Stabilization by an Iron-Based Fenton eaction[END_REF]. Indeed, this treatment has been tested by us and other colleagues within the project called by the acronym ReSIFaC and it seems to be a fast composting methods by limiting the typical olfactory problems and allowing to obtain a good compost to be used as a soil conditioner. The characterization of the starting organic materials and understanding of all the transformations they undergo during the oxidation process is of considerable importance both for the evaluation of the method and for assessing whether the final compound obtained is stable to be used as soil improver. Among the instrumental techniques used to obtain this kind of information, solid-state nuclear magnetic resonance (SS-NMR) spectroscopy must certainly be included. In order to apply to the solid substances or solid mixtures, the NMR requires the use of special techniques to overcome many difficulties in the analysis of such samples as Cross-Polarization (CP) and Magic Angle Spinning (MAS) that reduce / eliminate the line broadening observed in the spectra of these anisotropic samples. The technique is known as CP-MAS NMR spectroscopy. 13 C CP-MAS NMR spectroscopic analyses were already used to characterize the families of chemical compounds present in several waste as reported in several papers [START_REF] Chefetz | Chemical and Biological Characterization of Organic Matter during Composting of Municipal Solid Waste[END_REF]Baldock, et al., 1997;[START_REF] Pichler | Changes in the Chemical Structure of Municipal Solid Waste during Composting as Studied by Solid-State Dipolar Dephasing and PSRE 13 C NMR ad Solid-State 15 N NM Spectroscopy[END_REF][START_REF] Pichler | Solid-State 13 C NMR Spectroscopic, Chemolytic and Biological Assessment of Pretreated Municipal Solid Waste[END_REF][START_REF] Castaldi | Study of the Organic Matter Evolution During Municipal Solid Waste Composting Aimed at Identifying Suitable Parameters for the Evaluation of Compost Maturity[END_REF][START_REF] Tambone | Assessing Amendment Properties of Digestate by Sudying the Organic Matter Composition and the Degree of Biological Stability During the Anaerobic Digestion of the Organic Fraction of MSW[END_REF][START_REF] Tambone | Organic Matter Characterization During the Anaerobic Digestion of Different Biomasses by Means of CPMAS 13 C NM Spectroscopy[END_REF]Tang & Hills, 2003).

Due to their nature, organic waste are in a state that is not simply solid but it is intermediate between the solid and liquid states. Indeed, the organic waste can be considered a heterogeneous systems endowed with liquid-like dynamics. For this kind of system, the High-Resolution Magic-Angle Spinning (HR-MAS) NMR spectroscopy has become an extremely versatile technique. Many studies of semisolid materials have been carried out using this technique, that combines the "high-resolution" NM for liquids and the "line-narrowing" for solids, in order to obtain NMR spectra of semisolids with a spectral resolution comparable to liquid-state NMR spectroscopy. There are numerous journal reviews and books dedicated to HR-MAS NMR spectroscopy, due to its large number of applications (cancer tissues, food science, cellular science, material science, etc.) [START_REF] Huhn | Metabolic Profiling of Normal and Hypertensive Rat Kidney Tissues by HRMAS-NM Spectroscopy[END_REF][START_REF] Beckonert | High-Resolution Magic-Angle-Spinning NMR Spectroscopy for Metabolic Profiling of Intact Tissues[END_REF][START_REF] Farooq | H -MAS NMR Spectroscopy: A Pratical Guide for Natural Samples[END_REF][START_REF] Corsaro | H -MAS and NM towards Foodomics[END_REF] but, to our knowledge, organic waste samples have not yet been studied with this spectroscopic technique, although their physical state makes them an ideal matrix. In this work we successfully applied, for the first time, HR-MAS NMR spectroscopy to the organic waste materials in order to investigate their chemical composition and to monitor the change of their organic composition during the oxidation process with Fenton's reagent to which they are subjected and which leads to the formation of compost. Specifically 13 C-{ 1 H} NMR spectra of several samples of organic waste have been used both for composition investigation and for monitoring different Fenton's treatments, in term of reaction conditions, carried out on portions of the same starting material. All HR-MAS NMR spectra were obtained on a Bruker Avance 500 MHz spectrometer, operating at a 13 C resonance frequency of 125.758 MHz, equipped with a BVT-3000 device for temperature control and an HR-MAS 5mm probe. All measurements were made at a spin rate of 4 kHz and at a room temperature, acquiring 32768 points with 5000 scans and using the inverse-gated pulse sequence (zgig) in order to avoid the Nuclear Overhauser Effect (nOe). Data were processed via Bruker TopSpin software: the time-domain spectra were apodised with exponential function, and then Fouriertransformed, phased and baseline-corrected automatically. In order to remove the background signal due to the Kel-F rotor cap, to each spectrum has been subtracted the 13 C spectrum obtained from a blank sample consisting in D2O and TMS. The chemical shifts were referred to the TMS signal (δ = 0.00 ppm).

Results and discussion

The most important advantage of HR-MAS technique compared to the solid-state NMR is the possibility to obtain well-resolved NMR spectra [START_REF] Wong | High-Resolution Magic-Angle-Spinning(HR-MAS) NMR Spectroscopy[END_REF]. Both 1 H and 13 C NMR spectra can be obtained with a spectral quality similar to the liquid state. However since the organic waste is a complex system, the 1 H HR-MAS NMR spectra are quite complex, due to the overlapping of the signals of the various compounds, and, therefore, for our investigations, the 13 C spectra have been used. A typical 13 C-{ 1 H} NMR spectrum of the organic waste obtained using the HR-MAS NMR technique is shown in Figure 2.42, where the carbon spectrum of sample 0 (in blue) is compared with an OFMSW spectrum obtained using the CP-MAS NMR technique (in black) [START_REF] Pichler | Changes in the Chemical Structure of Municipal Solid Waste during Composting as Studied by Solid-State Dipolar Dephasing and PSRE 13 C NMR ad Solid-State 15 N NM Spectroscopy[END_REF]. Although the two spectra show a similar distribution of the peaks, an improvement in resolution is clearly visible in the HR-MAS spectrum in which the signal lines are much narrower. Note that this region of the spectrum has the same profile as the typical CP-MAS starch spectrum as shown in the box of Figure 2.43 where the comparison of this region is reported (Tang & Hills, 2003). From 125 to 130 ppm it is the region of unsaturated carbon atoms signals, (Tang & Hills, 2003).

A detailed illustration of the overall chemical shift range of carbons is shown in Table 2.6 and these chemical shift ranges are used to evaluate the area of the signals. Indeed, unlike the CP-MAS spectrum in which it is possible to integrate only large signal ranges, in the spectrum of Table 2.6: Chemical shift regions of 13 C-{ 1 H} NMR spectra of OFMSW [START_REF] Chefetz | Chemical and Biological Characterization of Organic Matter during Composting of Municipal Solid Waste[END_REF]Baldock, et al., 1997;[START_REF] Pichler | Changes in the Chemical Structure of Municipal Solid Waste during Composting as Studied by Solid-State Dipolar Dephasing and PSRE 13 C NMR ad Solid-State 15 N NM Spectroscopy[END_REF][START_REF] Pichler | Solid-State 13 C NMR Spectroscopic, Chemolytic and Biological Assessment of Pretreated Municipal Solid Waste[END_REF][START_REF] Castaldi | Study of the Organic Matter Evolution During Municipal Solid Waste Composting Aimed at Identifying Suitable Parameters for the Evaluation of Compost Maturity[END_REF][START_REF] Tambone | Assessing Amendment Properties of Digestate by Sudying the Organic Matter Composition and the Degree of Biological Stability During the Anaerobic Digestion of the Organic Fraction of MSW[END_REF][START_REF] Tambone | Organic Matter Characterization During the Anaerobic Digestion of Different Biomasses by Means of CPMAS 13 C NM Spectroscopy[END_REF] For a semi-quantitative procedure, the global chemical shift range of carbon in the spectra was subdivided into sub-regions as shown in Table 2.6. Each signal was integrated using the TopSpin software and was expressed as a percentage respect to the total integration area. The integration errors were calculated using the root-mean-square (RMS) method, by which the noise RMS was calculated as:

RMS= √ ∑ 𝜆 𝑖 𝑛 𝑖 𝑛 (2.1)
where 𝜆 is the intensity of the i-point (in ppm) out of the total n-points. The RMS value, obtained in this way, represents the height of the noise. Subsequently, this value was used to estimate the error expressed as a percentage of the intensity of the signal and this percentage has been used

to estimate the error bar of the integral of each NMR peak [START_REF] Caricasole | Evolution of Organic Matter During Composting of Different Organic Wastes Asessed by CPMAS 13 C NM Spectroscopy[END_REF].

In Table 2.7 the 13 C NMR integrated area of the different carbon type of Sample 0 are reported. occurs at the time of about 8 hours against the 12 hours of the process A. Moreover, for Sample 5 it is possible to hypothesize a third stage between 9 and 12 hours: at this moment the trend previously observed in Stage I, i.e. an oxidation of the alcoholic groups in carbonyls with simultaneous loss of CO2, occurs. In Table 2.10 and Figure 2.46 are also reported the NMR data obtained for the Sample 6_8h in which the same concentrations of the Samples 5 are used but the reaction proceeds continuously for 8 hours. As can be seen from Table 2.10 and Figure 2.46, in Sample 6_8h there is a slight increase in the carbon atoms bound to the heteroatoms compared to the situation observed at 6 hours. A decrease in the aliphatic content at 8 hours is not observed, but a decrease in the unsaturation content is appreciable and suggests that this process (Stage II) begins with the oxidation of the double bonds and that only subsequently proceeds with the oxidation of the saturated chains as highlighted in Example 5_9h.

Conclusions

In this work was demonstrated that HR-MAS NMR spectroscopy is an innovative and nondestructive method able to characterize the chemical composition of the Organic Fraction of MSW and it is a good tool in the monitoring of the variations in terms of composition of the initial organic materials subjected to oxidation treatments. The better performance of the HR-MAS 13 C NMR approach with respect to the CP-MAS method, the most commonly used technique to analyse the organic fraction of waste, relies upon the possibility of obtaining high quality and much more resolved spectra whose analysis contributes to a better understanding of the chemical nature of these waste. In addition, this HR-MAS approach has also proved suitable for monitoring the oxidative processes based on the Fenton's reaction to which the initial waste matrix was subjected. Indeed, the results obtained highlight the qualitative/quantitative modifications of the different types of carbon atoms as a function of both concentrations of Fenton's reactants and duration of the oxidative process. To our knowledge, this case study represent the first example in which the HR-MAS approach has been successfully applied to the organic fractions of MSW. Finally, the advantages in terms of rapidity, simplicity, and diversity of obtainable information makes this technique very attractive for further studies on different matrix of OFMSW and also to monitor other degradation/transformation processes to which organic waste can be subjected.

Chapter 3.

Conformational studies of bioactive couples of enantiomers 3.1. Introduction In the previous chapter some studies carried out on natural matrices were presented with the aim to determine, by means of NMR spectroscopy, the metabolomic composition of the studied samples. The substances recognized in the metabolome of the investigated materials are bioactive molecules that may play important roles in the system biology. In particular, the behavior they exhibit in living systems is dependent on the kind of interactions they create with endogenous ligands and receptors (Boehr et al, 2009). These interactions are predetermined by the configuration and the conformations of the flexible metabolites involved, hence the importance to perform a conformational study of these bioactive flexible species in parallel with the characterization of the metabolome itself. From the theoretical point of view, during the years the conformational analysis using molecular modelling calculations has gained importance as a technique able to investigate structures and to determine conformational equilibria of flexible molecules in the field of drug design, screening processes and pharmaceutical formulation development [START_REF] Mavromoustakos | An Effort To Understand the Molecular Basis of Hypertension through the Study of Conformational Analysis of Losartan and Sarmesin Using a Combination of Nuclear Magnetic Resonance Spectroscopy and Theoretical Calculations[END_REF][START_REF] Fairlie | Conformational Selection of Inhibitors and Substrates by Proteolytic Enzymes: Implications for Drug Design and Polypeptide Processing[END_REF]. The possibility to obtain conformational information experimentally in liquid environment is even more interesting, since it is the state of the matter where molecules generally interact and play their biological role. In this field of research, NMR spectroscopy in weakly oriented media has found application. Indeed, in such media the solute reorientational motion is no longer isotropic and this leads to somewhat simple and fully informative high resolved NMR spectra where nonzero dipolar couplings are directly observed, allowing, thanks to their long-range nature, to determine the solute 3D structure and, based on robust suitable theoretical models, its in-solution conformational distribution (Emsley & Lindon, 1975;[START_REF] Canet | Nuclear Magnetic Resonance, Concepts and Methods[END_REF]Burnell & de Lange, 2003;Di Pietro et al, 2014;Di Pietro et al, 2017;Liu et al, 2017). As mentioned, the interactions that occur in living organisms are a function of the configuration of the species involved, as well as being influenced by the conformations they assume in solution if they are flexible. Indeed, most biological receptors are very sensitive to the differences between enantiomers as they are also made up of chiral molecules; they can therefore interact with the two species in a different way, inducing a consequent different reaction in the organism itself [START_REF] Bentley | The Nose as a Stereochemist. Enantiomers and Odor[END_REF].

Fortunately, in some cases this does not lead to problematic situations, but in more negative cases one of the two enantiomers can be dangerous for the health [START_REF] Strömland | Thaliodomide Embryopathy: Revisited 23 Years Later[END_REF].

Therefore, it is clear that chirality assumes a fundamental importance in the study of the properties of bioactive molecules and the possibility of distinguishing a pair of enantiomers is a crucial task.

From the experimental point of view, much effort has been dedicated to suggesting effective analytical methodologies able to distinguish a couple of enantiomers and determine the enantiomeric excesses. Chiroptical techniques (based on the optical rotatory power) as well as chiral gas chromatography (GC) or high-pressure liquid chromatography (HPLC) are generally employed as routine tools for robust and rapid determinations of the enantiomeric excesses. More sophisticated optical spectroscopy methods exist such as Raman optical activity (ROA) or vibrational circular dichroism (VCD) spectroscopy but they have to be combined with theoretical calculations so are not really considered for routine analyses (Lesot et al, 2020). Methods based on Nuclear Magnetic Resonance (NMR) spectroscopy have also been explored: the use of chiral derivatizing agents, chiral solvating agents and metal complexes, associated with the isotropic NMR technique, are methods commonly adopted to measure the enantiomeric excesses and to find the absolute configuration of optically active molecules. The methodology is based on the different chemical shift values presented by the derivatizing-enantiomer (R) and derivatizing-enantiomer (S) compounds. However, in some cases, the use of this procedure leads to a difference in chemical shift that is not sufficient to discriminate between the two enantiomers spectra [START_REF] Rinaldi | The Determination of Absolute Configuration Using Nuclear Magnetic Resonance Techniques[END_REF][START_REF] Fraser | Asymmetric Synthesis[END_REF][START_REF] Weisman | Asymmetric Synthesis[END_REF][START_REF] Yamaguchi | Asymmetric Synthesis[END_REF][START_REF] Morril | Methods in Stereochemical Analysis[END_REF][START_REF] Parker | NMR Determination of Enantiomeric Purity[END_REF]. A powerful approach able to discriminate enantiomers relies on NMR spectroscopy using chiral ordering agents as solvent, such as enantiopure liquid crystals. This method makes use, as solvents, of weakly orienting chiral phases (such as cholesteric non-aqueous-based lyotropic liquid crystals, commonly used for the conformational analysis) thus combining the advantages deriving from chiral agents with the prerogatives of the anisotropic phases [START_REF] Courtieu | Chiral Liquid Crystal NMR: a Tool for Enantiomeric Analysis[END_REF][START_REF] Aroulanda | Weakly Oriented Liquidcrystal NMR Solvents as a General Tool to Determine Relative Configurations[END_REF]Lesot et al, 2020;[START_REF] Aroulanda | Molecular Enantiodiscrimination by NMR Spectroscopy in Chiral Oriented Systems: Concept, Tools and Applications[END_REF]. The NMR spectra of samples in these phases are in fact extremely rich in information, as they are governed by interactions of isotropic but also anisotropic nature. In chiral crystalline liquid solvent a couple of enantiomers experiment different order and this is traduced in different anisotropic observables. This is due to the fact that the chirality of this phase associated with its anisotropy, is responsible for imparting a different statistical order to the two enantiomers within the solution. In fact, enantioselective interactions are established between the enantiomers and the mesophase itself and this entails a different average orientation of the two molecules with respect to the laboratory Z axis which is reflected in two matrices of the order parameters (𝑆 𝛼𝛽 𝑅 and 𝑆 𝛼𝛽 𝑆 ) different for the two species (Lesot et al, 2020):

𝑆 𝛼𝛽 𝑅,
From the spectral point of view, these difference in the interactions that occur between the R and S enantiomers and the solvent itself is often sufficient to distinguish two species through the observables NMR that are sensitive to the order of the solute. Therefore, all the observables that are affected by the order of the solution could be used to discriminate two enantiomers as shown in Figure 3.1 for the dipolar coupling and the chemical shift. While the differences in chemical shift of the two species are not always high enough to be appreciable, the differences in residual dipolar couplings (RDCs) are an effective tool for achieving this goal. The RDCs have extensively proved to be able to provide structural, conformational and orientational information on flexible molecules and during the years, the conformational analysis of several flexible non-steroidal anti-inflammatory drugs, belonging to the class of salicylates and α-arylpropionic acids (profens), have been performed by treating residual dipolar couplings with an appropriate theoretical model (Di Pietro et al, 2018). This

J ij n i iso R S R S |D ij R -D ij S | (n i aniso ) S (n i aniso ) R R R S S n i aniso R S R S J ij +2D ij A) B) C)
methodology can be applied to a pair of enantiomers in order to investigate their individual conformations and to understand the different solute-solvent interactions that the two enantiomers R and S form in a chiral solvent. With the purpose of undertaking this type of investigation, in the following sections are reported two conformational studies carried out on two couples of enantiomers dissolved in the weakly aligning solvent composed by the chiral phase of poly-γ-benzyl-L-glutamate (PBLG) and CDCl3. The first study presented in section

Assessing the Chirality-dependent Conformational Distribution of Small

Flexible Opposite Enantiomers of 4-F-α-methyl benzyl alcohol (4FMBOH)

The structural, configurational and conformational determination of flexible molecules in solution is one of the most unsolved problem in modern molecular analysis. It becomes even more challenging in the case of bioactive molecules because the spatial arrangement they adopt strongly affects the interactions with ligands and this influences their biological activity. In particular, flexible molecule largely used in the treatment of some health problems are chiral species. They are used sometimes as a racemic mixture while some others do need to be administrated as enantiopure due to extreme differences in the biological activity of the two enantiomers (as in the case of thalidomide, for example [START_REF] Strömland | Thaliodomide Embryopathy: Revisited 23 Years Later[END_REF]).

Hence the interest in understanding the conformational features of both molecules achieving a detailed conformational elucidation analysis in solution: the possibility to probe equilibria between more possible conformers for both enantiomers is crucial for understanding and rationalizing the biological activity of the studied species. Due to the importance of the topic, any molecular approach able to shed light on peculiar aspects of this intriguing field deserves to be applied and exploited. In this panorama, the use of chiral liquid crystalline phases coupled to NMR spectroscopy has historically contributed to provide a better understanding of stereochemistry, conformational equilibria and molecular configurations in a very significant way (Safarti et al, 2000;Di Pietro et al, 2018;Lesot et al, 2020). Indeed, two enantiomers dissolved in an isotropic phase are affected by the same interactions when the solution is subjected to the NMR strong magnetic field, therefore they generate two identical spectra. This is no longer true in the case of a chiral ordered phase. In fact for a couple of enantiomers, the chirality of the medium is essential for obtaining two different spectral profiles. This is due to the fact that the two enantiomers experience different order parameters in the chiral aligned solution and this results in two different sets of RDCs (Lesot et al, 2020).

In this section, a conformational study have been performed using as weakly aligning solvent the chiral phase obtained dissolving poly-γ-benzyl-L-glutamate (PBLG) in CDCl3 in order to detect the chirality signature on the conformational equilibrium of two species (Aroulanda et al, 2021). In particular, a simple molecule, composed by a single torsional angle, were studied approach (Pileio, 2005) described in 1.4, accurate descriptions of the probability distributions have been obtained for both enantiomers.

Samples preparation and experimental procedure

In order to perform a correct conformational study, the first step is to have access to a large number of experimental data, i.e. to extract as many observed RDCs (𝐷 𝑖𝑗 𝑜𝑏𝑠 ) as possible from the proton and 13 C spectra of the molecule dissolved in the liquid crystalline phase. As seen in chapter 1, from a spectrum of a sample in this phase, what is measured for a pair of nonequivalent nuclei i and j is the total coupling constant reported in equation 1.32 and 1.33. This implies that, to have access to the values of the 𝐷 𝑖𝑗 𝑜𝑏𝑠 , it is necessary to carry out NMR measurements in both isotropic media, to measure the 𝐽 𝑖𝑗 𝑖𝑠𝑜 , and in the liquid crystalline phase.

In addition, to obtain the two set of 𝐷 𝑖𝑗 𝑜𝑏𝑠 , one for each enantiomer, it is necessary to have both species and to prepare different anisotropic solutions. Indeed, it should be noted that the technique here presented does not allow to determine, from the signals, the absolute configuration of the molecules analysed but it allow only to detect the differences between the signals due to the two different enantiomers. The absolute configuration, and, therefore, the attribution of the signals relating to the R and S species, can be obtained in the case in which the spectral information of one of the two enantiomers, for which the absolute configuration is well established, is known. In the case of 4FMBOH, the single enantiomers were used and, therefore, the strategy adopted involved the extraction of the 𝐷 𝑖𝑗 𝑅 and the 𝐷 𝑖𝑗 𝑆 from the experiments conducted on samples composed by the pure enantiomers. Thus, the first experimental step involved the preparation of an isotropic solution of 4FMBOH. Since the two enantiomers dissolved in an isotropic phase are affected by the same interactions when the solution is subjected to the NMR strong magnetic field so that they generate two identical spectra, the isotropic sample can be obtained using one of the two or both the enantiomers without making any mistakes. Thus, the isotropic sample, studied for the extraction of 𝐽 𝑖𝑗 , was obtained by simple addition of 0.038 g of R-4FMBOH to 0.688 g CDCl3 into which the molecule was immediately dissolved. The preparation of the anisotropic sample, on the other hand, required a more elaborate procedure (Canet et al, 1995), in fact a first sample (R-FMBOHaniso) was obtained by dissolving 0.0487 g of R-4FMBOH in 0.7190 g of CDCl3 and 0.0874 g of PBLG was added to this solution, directly into the NMR tube. This led to the obtainment of a sample with a concentration of 10.2% wt in PBLG. This concentration is not included in the range reported in the literature in which the mesophase is stable but, since this range is also function of the degree of polymerization of the polymer used, in this case a concentration of 10.2% wt in PBLG is sufficient to obtain a sample with the desired anisotropic characteristics (Sarfati et al, 2000). This sample was heated several times up, centrifuged back and forth to give a very homogeneous birefringent phase. In order to verify that the solution was actually anisotropic, the 1D 2 H spectrum of the deuterated solvent was recorded. In the spectrum obtained, a singlet is visible if the phase is isotropic and a doublet if the phase is anisotropic. It is important that no isotropic domains are present in the sample and this is detectable by the absence of the peak that is located in the centre of the doublet due to the quadrupolar splitting. Following this procedure also the S-FMBOHaniso sample was prepared. The two samples were prepared trying to respect as much as possible the same weight concentrations of both the PBLG/CDCl3 solution and the solute dissolved in it in order to have in each sample a situation in which the solute experiments the same degree of order.

Since the magnitude of the dipolar couplings is dependent on the degree of order of the solute within the solution, preparing samples at a similar concentration is mandatory to obtain spectra of the pure enantiomers that are comparable. All the samples prepared and studied are reported in Table 3.1. The insert in Figure 3.3 shows the comparison of the signal corresponding to methyl group protons for the two enantiomers. It should be pointed out that, in anisotropic environment, this signal is not a doublet but a triplet of doublets, due to the coupling between the three equivalent protons of the methyl group and the coupling of these with the proton attached to the stereogenic carbon atom. This structure is not well resolved in the S-4FMBOHaniso spectrum so the measurement of the corresponding 𝑇 𝐻𝐻 𝑆 (𝑜𝑏𝑠) has been possible only through the homonuclear SERF experiment as shown in Figure 3.4 for the coupling 𝑇 𝐻8-𝐻8 𝑆 . R-4FMBOHaniso S-4FMBOHaniso In order to obtain more spectral information 2D 1 H-1 H J-resolved experiment was performed.

As an example, in Figure 3.5 is reported the J-resolved spectrum recorded on the R-4FMBOHaniso sample from which the projections shown in the boxes, corresponding to the columns centred on the chemical shifts of H2, H3 and H7 have been extracted. These projections have been analysed through the simulating program Arcana (Celebre et al, 1994) in order to provide the corresponding 𝑇 𝐻𝐻 𝑅 (𝑜𝑏𝑠) not directly measurable on the spectra by simple inspection.

2 T H8-H8 For the measurements of the 1 H-13 C short and long-range couplings, the heteronuclear selective refocusing 1 H-13 C NMR experiments has been used for all anisotropic samples. From all the experiments performed on the three samples (4FMBOHiso, R-4FMBOHaniso and S-4FMBOHaniso) a large data sets of of the remaining 𝐷 𝑖𝑗 (𝑜𝑏𝑠) was performed by a trial and error process during the fitting procedures that will be presented in the following section.

Conformational and orientational analysis

The sets of experimental 𝐷 𝑖𝑗 𝑅,𝑆 (𝑜𝑏𝑠) extracted from the analysis of the spectra have been exploited to investigate structure and conformational equilibrium of the two enantiomers. In particular, 26 𝐷 𝑖𝑗 𝑅 (𝑜𝑏𝑠) and 29 𝐷 𝑖𝑗 𝑆 (𝑜𝑏𝑠) are available for the conformational study and, among them, 6 for the R enantiomer and 8 for the S enantiomer, are relating to the interactions between the two rigid fragments in which the molecule can be divided, that is, between the alcohol fragment and the aromatic ring. From an operational point of view, the conformational analysis was performed using the AP-DPD approach, described in the section 1.4.4, using the dedicated software AnCon and using the experimental 𝐷 𝑖𝑗 𝑅,𝑆 (𝑜𝑏𝑠) as data input. To investigate the conformational distribution around the angle 𝜙 it is also need use as input data the geometries of the molecule and it is necessary to have an idea about the distribution 𝑃 𝑖𝑠𝑜 ({𝜙 }). The geometry of the two rigid sub-units of 4FMBOH has been fixed equal to the one found by performing, using GAUSSIAN 03 software (Gaussian 03, 2003), a geometry optimization by density functional theory (DFT) calculations at the B3LYP levels of theory and applying 6-31++G** function as basis set. Using the same theoretical approach, a relaxed scan have been carried out in order to build the theoretical probability distribution function for the torsional angle investigated 𝑃 𝑡ℎ𝑒𝑜 ({𝜙 }). Once obtained information regarding the "shape" of the curve, within the AP model the 𝑃 𝑖𝑠𝑜 ({𝜙 }) was modelled as a sum of two

Gaussian functions cantered at 0 ° and 180 ° as the following equation states:

𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑅,𝑆 ∝ ( |cos 𝜙 𝑅,𝑆 | -cos 𝜙 𝑅,𝑆 2 + 𝐴 𝜙 𝑅,𝑆 cos 𝜙 𝑅,𝑆 ) • 𝑒𝑥𝑝 [- 𝑠𝑖𝑛 2 (𝜙 𝑅,𝑆 -𝜙 𝑚𝑎𝑥 𝑅,𝑆 ) 2ℎ 𝜙 2 𝑅,𝑆 ] (3.3)
Where 𝜙 𝑚𝑎𝑥 𝑅,𝑆 is the first most probable value of the torsional angle (H7-C7-C4-C3) for each enantiomer R and S (the second is given by (180 ° + 𝜙 𝑚𝑎𝑥 𝑅,𝑆 ), 𝐴 𝜙 𝑅,𝑆 is the relative weight of the first Gaussian curve (the relative weight of the second is fixed equal to 1-𝐴 𝜙 𝑅,𝑆 but, in this particular case, 𝐴 𝜙 𝑅,𝑆 = 0.5 because the two Gaussians are equally populated) and ℎ 𝜙 𝑅,𝑆 is the width at half height of each Gaussian curve. In addition, according to the AP approach, it is necessary to describe the molecular fragment in terms of interaction tensors (see paragraph 1.2.2) and in our case 4 uniaxial components are needed 𝜀 2,0 𝑗 , two to describe the aromatic ring: 𝜀 2,0 𝐶1-𝐶4 and 𝜀 2,0 𝐶2-𝐶6 ; two to describe the alcohol subunit 𝜀 2,0 𝐶7-𝐶8 and 𝜀 2,0 𝐶8-𝐻7 .

Once defined the 𝑃 𝑖𝑠𝑜 ({𝜙 }) function, fixed the geometry and described the molecular fragment in terms of 𝜀 2,0 𝑗 , we tried to reproduce the experimental 𝐷 𝑖𝑗 𝑅,𝑆 (𝑜𝑏𝑠) by suitably iterating over the 4 orientational parameters 𝜀 2,0 𝑗 , over the conformational term 𝜙 𝑚𝑎𝑥 and parametrically varying ℎ 𝜙 up to achieve an acceptable RMS value. An acceptable reproducibility of the experimental values was obtained as shown in The first thing that can be noticed from these results is that the two enantiomers are characterized by different maxima in conformational probabilities, therefore two different curves are obtained. However, in both cases at the angles corresponding of maxima values of 𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑅,𝑆 hydrogen atom directly bonded to the stereogenic carbon almost in same plane of the aromatic ring so that the two bulky groups, therefore methyl and alcohol, are found one on each side of the plane with respect to the aromatic ring, an optimal situation to minimize steric repulsion. In addition, an interesting evidence that emerges from the comparison of the as antipyretic, anti-inflammatory and analgesic agents. These characteristics make NSAIDs largely applied for the treatment of pain, arthritic conditions, dysmenorrhea, pyrexia, and gout. The main mechanism of action of NSAIDs is the inhibition of the enzyme cyclooxygenase (COX) which allow to convert arachidonic acid into prostaglandins, species that cause vasodilation and play a role in the increase of the temperature set-point in the hypothalamus [START_REF] Vane | Mechanism of Action of Anti-Inflammatory Drugs[END_REF][START_REF] Emerson | Non-Steroida Anti-Inflammatory Drugs on Core Body Temperature During Exercise: A Systematic Review[END_REF]. These molecules show a large number of stereoisomers due to the presence of more torsional angles so it is clear that a deeper knowledge of the conformational features of NAIDs can help in understanding why and how they work, expanding knowledge on biological effects. Indeed, the exploration of the three-dimensional structure and the conformational equilibrium in solution of these drugs is particularly useful because it allows to acquire knowledge on the relationship between conformation and pharmacological activity, but, especially, it can be of valuable help for the rational drug design and screening processes as well as for the pharmaceutical formulation development. The NMR study in weakly ordered media, methodology in part used in this thesis, has been adopted during last years to carry out the conformational study of the most common families of NSAIDs: salicylates and 2-arylpropionic acids (or profens) (Di Pietro et al, 2018). Starting from the experimental RDCs measured in PBLG-based mesophases, the structure and the torsional distribution of these drugs molecules have been investigated with the AP-DPD theoretical approach, initially developed for highly oriented molecules (Pileio, 2005). Regarding the first class of molecules, the diflunisal (2′,4′-difluoro-4hydroxybiphenyl-3-carboxylic acid) and its non-fluorinated analogue phenylsalicylic acid, both species composed by a single bond rotation, have been useful as conceptual proofs to confirm the effectiveness and robustness of the AP-DPD approach when applied to lyotropic systems, since they are relatively simple cases to be treated from the conformational point of al, 2017;Di Pietro et al, 2018). However, in each of these studies, only the biologically active enantiomer was considered and it was never carried out a conformational study on both enantiomers of the same species. Indeed, for this class of compounds stereochemistry is a major determinant of both pharmacodynamics and pharmacokinetic properties. The (S)enantiomers of these molecules possess an higher pharmacological activity than the (R)enantiomers; further in some cases the (R)-(-) enantiomers were found to be converted in vivo into the corresponding (S)-(+)-enantiomers [START_REF] Nguyen | Chiral Drugs: An Overview[END_REF]. Since, in section 3.2 has been proved the effectiveness of the method based on the use NMR in weakly ordered media as a tool able to highlight chiral features of a very simple couple of enantiomers by studying their conformational behavior, in the following section the method has been applied on a couple of enantiomers belonging to the NSAIDs family. In particular, the main character of this section is the tiaprofenic acid, the 5-benzoyl--methyl-2-thiophene-acetic acid (Figure 3.11), that is a highly potent anti-inflammatory and nonnarcotic analgesic agent [START_REF] Plosker | Tiaprofenic Acid[END_REF]. This substance is commonly used for the treatment of pain, inflammation and it is associated to the treatment of different diseases [START_REF] Davies | Clinical Pharmacokinetics of Tiaprofenic Acid and its Enantiomers[END_REF][START_REF] Plosker | Tiaprofenic Acid[END_REF]. The tiaprofenic acid (TA), such as the other profens, contains a chiral center located at the C2 of the propionic moiety and, therefore, exists in two enantiomeric forms, R (-) and S (+). Both the enantiomers are bioactive and it does not show in vivo chiral inversion as some other profens, so that normally it is dispensed and administered as a racemic mixture [START_REF] Davies | Clinical Pharmacokinetics of Tiaprofenic Acid and its Enantiomers[END_REF]. The molecule consist of two cooperative torsions (𝜓 and 𝜃) and one independent torsion (𝜙); in the study presented in the following section, the attention was focused on the conformational behavior of the independent torsion (𝜙), involving the propionic portion and the thiophene ring, for both the enantiomers. On the samples prepared 1 H, 13 C and 13 C-{ 1 H } NMR spectra were acquired at T = 298 K on a 400 MHz Bruker Avance I spectrometer (9.4 T) (Bruker, Fällanden, Switzerland) equipped with a QXO probe. On these samples 2D correlation experiments ( 1 H COSY, 1 H-13 C HSQC and 1 H-13 C HMQC) were also performed for the correct attribution of the signals. For the extraction of all the coupling constants, in addition to the use of simple 1D NMR spectra, also 2D experiments such as homonuclear 1 H-1 H and heteronuclear 1 H-13 C J-resolved were performed together with the selective homonuclear and heteronuclear refocusing experiments, 1 H-1 H SERF and 1 H-13 C HetSERF. Once obtained the spectra, all the data processing steps were carried out using TopSpin 3.6 software (Bruker BioSpin, Rheinstetten, Germany) (TopSpin, 2018).

RDCs extraction

The study on TA acid begins with the isotropic sample in order to obtain the indirect coupling constants for the nuclei 1 H and 13 C, to be used in the calculation of the RDCs according the equations 1.32 and 1.33. Since the two enantiomers dissolved in an isotropic phase are affected by the same interactions when the solution is subjected to the NMR strong magnetic field, they generate two identical spectra from which a single set of 𝐽 𝑖𝑗 can be obtained. As a proof of this, Figure 3.12 shows the 13 C spectrum recorded on the isotropic sample RS-TAiso.

The enlargement in red shows the signal generated by the methyl group in position 3 whose carbon produces a quartet of doublets due to the coupling with the protons in position 3

(triplet) and with the proton in position 2 (doublet). From this signal it was possible to extract the two constants 1 JC3-H3 and 2 JC3-H2 which are the same for both enantiomers.

Sample Composition Concentration in PBLG

RS-TAiso 0.0116 g of RS-TA + 0.5556 g of CDCl3 -RS-TAaniso 0.0900 g of RS-TA + 0.5556g of CDCl3 + 0.1094 g of PBLG 14.5%wt Much more interesting is the extraction of the total coupling constants from the anisotropic sample prepared using the racemic mixture of TA. Indeed, as described in the section 3.1, the chiral liquid crystalline solvent PBLG/CDCl3 interacts in a different way with the two enantiomers and for each of them different values of 𝑇 𝑖𝑗 attributable one to the R enantiomer and the other to the S are expected are expected. Obviously, it is not possible to directly attribute the signals present in the spectra to the single enantiomers, however, it is possible to partially separate the couplings extracted from the spectra into two blocks, A and B, one for each enantiomer, without relating each block to a specific enantiomer. The broadness of the peaks due to the weakly ordered phase used, together with the existence of more spectral lines due to superposition of two spectral profile generated by the two enantiomers, did not allow to extract information from simple one-dimensional spectra and the use of 2D techniques was essential. In the Figure 3.13 is reported as an example the SERF spectrum selective on the coupling H2/H3 recorded on the RS-TAaniso sample. From the 2D map two different

1 J C3-H3 2 J C3-H2
quadruplet for the selected couplings can be extracted, and from them two 𝑇 𝐻2-𝐻3 values can be obtained, one for each enantiomer. There is no possibility of knowing which of the two constants corresponds to an enantiomer and which to the other, for this reason one has been attributed to a generic enantiomer A and the other to enantiomer B, as shown in the Figure. It must be emphasized that, once the first pair of constants have been assigned to species A and B, the subsequent attributions are not obvious. Indeed, sometimes it has not be possible to assign the Tij to one of the two species due to the lack of additional information deriving for example from known correlations. For example in the 2D HetSERF spectrum reported in Figure 3.14, is highlight the signal obtained by extrapolating the column corresponding to the C5 signal. From the extracted spectrum only one signal related to the interaction between C5 and H2 is detectable and the corresponding TC5-H2 cannot be assigned neither to the species A and neither to B. In this case the attribution was carried out during the conformational analysis step through a trial and error procedure. From all the experiments performed on the two samples (TAiso and RS-TAaniso) a large data . The determination of the remaining 𝐷 𝑖𝑗 (𝑜𝑏𝑠) was performed by a trial and error process during the fitting procedures that will be presented in the following section. From the operational point of view, the conformational analysis was performed using the AP-DPD approach, described in the section 1.4.4, using the dedicated software AnCon and using the experimental 𝐷 𝑖𝑗 𝐴,𝐵 (𝑜𝑏𝑠) as data input. To investigate the conformational distribution around the angle 𝜙 it is also need use as input data the geometries of the molecule and it is necessary to have an idea about the distribution 𝑃 𝑖𝑠𝑜 ({𝜙 }). The geometry of the two considered rigid sub-units of TA has been fixed equal to the one found by performing a geometry optimization by density functional theory (DFT) calculations at the B3LYP levels of theory and applying 6-31++G** function as basis set, using GAUSSIAN 03 software (Gaussian 03, 2003). Using the same theoretical approach, a relaxed scan have been carried

out in order to build the theoretical probability distribution function for the torsional angle investigated 𝑃 𝑡ℎ𝑒𝑜 ({𝜙 }). Once obtained information regarding the "shape" of the curve, within the AP model the 𝑃 𝑖𝑠𝑜 ({𝜙 }) was modelled. In particular, theoretical calculations predict 2 conformers of minimum energy for each enantiomer. The two conformers, unlike the other profens, are not related to each other by 180° due to the presence of sulfur atom in the aromatic ring which lowers the symmetry of the molecule. For these reasons, the 𝑃 𝑖𝑠𝑜 ({𝜙 }) was modelled as a sum of two Gaussian functions as the following equation states:

Regarding the other enantiomer, the torsional distribution 𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑆 is characterized by two differently populated maxima at 14° ± 7° (conformation IS) and at 202° ± 9° (conformation IIS) differently populated (𝐴 𝜙 𝑆 1 =0.7 and 𝐴 𝜙 𝑆 2

= 1 -0.7 = 0.3). Both curves are in reasonable agreement with the ones obtained through theoretical calculation. All the experimental data discussed are reported in the Table 3.4. As can be seen from Figure 3.16 and from the results reported in Table 3.4, the two enantiomers are characterized by different maxima in the conformational probabilities but the two curves are one mirror image of the other. This result is in perfect agreement with the result obtained from the study conducted on the two enantiomers of 4FMBOH (section 3.2).

For both enantiomers R-TA and S-TA, at the angles corresponding to maxima values of 𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑅,𝑆 the hydrogen atom directly bonded to the stereogenic carbon atom is almost in same plane of the aromatic ring so that the two bulky groups are one on each side of the plane. These experimental results reasonably agree both with the theoretical distribution obtained for the isolated molecules, and with the results obtained for other works involving the propionic acid of other NSAIDs (Di Pietro et al, 2018). As example, in Figure 3.16 is reported the comparison between the curves obtained for S-TA and S-Ketoprofen for the same torsional angle. As can be seen, the substantial difference between the two curves lies in the weight of the two conformers, in fact, the peculiarity of TA is the presence of the sulphur atom, which lowers its symmetry, when compared with the other profens studied, and this is probably the cause of the difference in population between the two conformational probability maxima.

Conclusions

The example reported in this section prove once again that NMR spectroscopy in weakly orienting PBLG phase, combined with the AP-DPD approach, is a valuable tool for investigating the structure and the conformational equilibrium of anti-inflammatory drugs. In the mechanism of action of the drug and the different biological activity of the two enantiomers, thus opening the way to new insight. In addition to the conformational results obtained for the species studied, this study proved the power of the adopted methodology.

Indeed, it was proven the possibility to use the chiral PBLG/CDCl3 liquid crystalline media combined with the theoretical AP-DPD approach, in order to let emerge the chiral features of enantiomer by observing their conformational equilibria in solution, the state in which they play their biological role. In addition, the proposed methodology appears to be able to lead to the determination of the configuration of a species whose stereochemistry is unknown. In our opinion, fundamental studies of this kind could pave the way for a deeper and improved description of chirality-affected molecular properties.

to obtain concentrated juices. Classic 1D and 2D correlation experiments together with literature data allowed to identify 23 low molecular weight compounds. 1 H NMR spectra of squeezed, pasteurized and concentrated juices were then processed with statistical tools (PCA) in order to distinguish between the samples arising from these three different industrial processes. Each process is clustered in one defined region of the score plot and, for each cluster, only slight differences between the different production phases are evident. This led to the conclusion that the thermal industrial process does not change the metabolic profile of the starting juice. The loading analysis allowed to identify the metabolites, responsible for the clustering and this shows that, during the concentration phases, i) the ethanol concentration decreases whereas the sugar content increases in the concentrated samples and ii) the clementine juice seems to be richer in the amino acids and other aliphatic compounds than orange juices.

The second example showed in the chapter is the NMR metabolic characterization of Calabrian apple juices coming from four different varieties. In particular, this work was aimed at the metabolic characterization spectroscopy of complex food matrices such as Calabrian apple juices from four different varieties by using NMR spectroscopy. Classic 1D and 2D pulse sequences were used together with literature data in order to identify 11 low molecular weight compounds present in the four varieties of apple juices studied. In addition, the combination of 1 H NMR spectra of the juices with multivariate data analysis (PCA) proved to be able to distinguish samples from the four varieties of apples. Indeed, in the score plot it is possible to highlight that Golden Delicious, Fuji and Royal Gala varieties have a more similar metabolic composition than the juices from the Pink Lady variety which are clustered. The analysis of the loadings showed that the sugars and malic acid contents are the compounds responsible for the separation between the four varieties.

The third study presented was the investigation of the metabolic profile of Calabrian Cannabis Sativa extracts by means of NMR spectroscopy. Hemp seeds oil and inflorescences extracts obtained through solute-solvent ultrasound assisted extraction using ethanol as extracting solvent, were studied by means of 1D and 2D NMR experiments. For the hemp seed oil, the 1 H characterization was performed and the triacylgliceroles (TAG) profile was recognized.

Regarding the inflorescences extract, it was possible to recognize three cannabinoids, CBD, CBDA and CBG together with the triacylglicerolic profile. This is an important result because

Cannabis Sativa is known to be rich in fatty acids with 3:1 ratio ω6/ω3 which represents a very good nutritional value and, the possibility to use an extraction technique able to provide extracts rich in both components deserves to be investigated more in deep with other studies. In addition, the fact that any signal referred to THC was recognized indicates that the concentration of this substance is lower than the detection limit of NMR spectroscopy and therefore also lower than the law limit. This preliminary study carried out was compared with the literature results and it allowed to conclude that our extraction method is promising for obtaining extracts rich in triacylgliceroles and medical cannabinoids that could represent a key turning point in the production of functional food.

The fourth study presented concerns the use of NMR spectroscopy to investigate for the firsttime the metabolic composition of a pure certified bergamot essential oil (BEO) sample without any manipulation or derivatization process. The main objectives of this study were, on one side, to perform the characterization and quantification of the main metabolites of this complex matrix, whereas, on the other side, to explore the possibility of investigating and discriminating against any adulteration to which the BEO could be subject. We were able to identify 11 low molecular weight compounds present in the studied sample by combining classical 1D and 2D correlation spectra with 2D 1 H DOSY experiments. Moreover, we were able to quantify about 90% of the sample components, getting average percentage values for the most abundant metabolites by employing benzoic acid as an internal standard and using the 1 H NMR spectrum.

Finally, as adulterations are not uncommon due to the high value of the BEO, we investigated the possibility of using three NMR approaches in order to detect possible frauds. The study was carried out by simulating a dilution adulteration by adding different quantities of orange essential oil. Diffusion studies, quantitative analysis and statistical study were used aiming to detect such manipulation. While the diffusion study allows to highlight the investigated fraud only in the case of high degrees of adulteration, the quantitative study seems to have a greater sensitivity in recognizing diluted oils. In any case, the technique that best allows to distinguish the various degree of adulteration and is able to provide more information is given by the combination of one-dimensional proton spectra with multivariate analysis tools.

The last example presented in this chapter is the application of HR-MAS NMR technique to the study of the organic fraction of the municipal solid waste (MSW). In particular, we demonstrated that HR-MAS NMR spectroscopy is an innovative and non-destructive method able to characterize the chemical composition of the Organic Fraction of MSW and it is an adequate tool in monitoring the variations in terms of composition of the initial organic materials subjected to oxidation treatments. The better performance of the HR-MAS 13 C NMR approach with respect to the CP-MAS method, the most commonly used technique to analyse the organic fraction of waste, relies upon the possibility of obtaining much more resolved spectra whose analysis contributes to a better understanding of the chemical nature of this propionic portion and the thiophene ring, for both enantiomers. The results obtained showed that the two enantiomers are characterized by a conformational curve composed by two maxima differently populated and the two curves obtained, one for each enantiomer, are mirrored image.

The presence of the sulfur atom in the structure, represents the major difference between the tiaprofenic acid and the structures of the other profens. The sulfur lowers the symmetry of the molecule and seems to be the cause of the different population of the two maxima of the conformational probability function. However, the two tiaprofenic acid enantiomers, as well as the other biologically active profens studied in literature, are characterized by the presence of the hydrogen atom directly bonded to the chiral carbon atom almost coplanar with the aromatic ring and this seems to be a peculiar characteristic of the NSAIDs (Non-Steroidal Anti-Inflammatory Drugs).

To conclude, the work carried out during this thesis project has faced several problems: a) the study of complex natural matrices using non-invasive NMR methodologies and, b) the exploration of the conformational behaviour in solution of chiral bioactive molecules. From one side, all the evidences emerged in the metabolomic studies presented allow us to conclude that NMR technique, combined with statistical analysis, is a powerful tool in different metabolomic applications. Indeed, it is able to discriminate among samples of different species, to detect metabolic variations during the thermal industrial process and degradation or oxidation processes, and to highlight frauds and sophistications. From the other side, in the case of the conformational studies, the example reported proved once again that NMR spectroscopy in weakly ordered solvent, combined with the AP-DPD approach, is a valuable tool for investigating the structure and the conformational equilibrium of anti-inflammatory drugs and in particular allow to study chiral features of species. Outcome demonstrated that NMR-based metabolomics has the potentiality to be applied in the characterization of complex natural extracts and in the industrial quality control field and, in addition to that, it is a powerful technique that can be applied in the study of complex flexible chiral species allowing to add new pieces for understanding the mechanism of action of the drug and the different biological activity of pairs of enantiomers, thus opening the way to new insight.

Nel secondo capitolo sono state presentate le applicazioni NMR nel campo della metabolomica esplorate durante il dottorato. Nel primo studio descritto è stata applicata la spettroscopia NMR per studiare il profilo metabolico dei succhi freschi di arancia e clementina Calabresi e per seguire le variazioni metaboliche che il succo fresco subisce durante il processo termico industriale che porta all'ottenimento di succo concentrato. I classici esperimenti 1D e di correlazione 2D insieme ai dati della letteratura hanno permesso di identificare 23 composti a basso peso molecolare. Gli spettri 1 H NMR di succhi spremuti, pastorizzati e concentrati sono stati quindi utilizzati per eseguire uno studio multivariato (PCA) per poter effettuare una discriminazione tra i campioni provenienti dai tre processi industriali. Dai risultati ottenuti è emerso che ogni processo è raggruppato in una regione ben definita dello score plot e che per ogni cluster sono evidenti solo lievi differenze nelle diverse fasi di produzione, cioè spremitura, pastorizzazione e concentrazione. Queste evidenze hanno permesso di concludere che il processo termico industriale non modifica il profilo metabolico del succo di partenza. L'analisi dei loadings ottenuti dal trattamento statistico ha permesso, inoltre, di identificare i metaboliti responsabili del clustering e questo mostra che, durante le fasi di concentrazione, l'etanolo diminuisce e il contenuto di zucchero aumenta nei campioni concentrati. In aggiunta a ciò, da questo tipo di analisi è emerso che il succo di clementine risulta essere più ricco in aminoacidi e altri composti alifatici rispetto al succo d'arancia.

Il secondo esempio presentato in questo capitolo è la caratterizzazione metabolica NMR di succhi di mela Calabresi. In particolare, questo lavoro è stato finalizzato alla caratterizzazione metabolica via NMR, di matrici alimentari complesse come i succhi di mela provenienti da quattro diverse varietà. Le classiche sequenze di impulsi 1D e 2D sono state utilizzate insieme ai dati riportati in letteratura per identificare 11 composti a basso peso molecolare presenti nelle quattro varietà di succhi di mela studiati. Inoltre, la combinazione degli spettri 1 H NMR ottenuti con l'analisi statistica dei dati (PCA) si è rivelata un mezzo in grado di distinguere i campioni dalle quattro varietà di mele. Infatti, nel grafico degli scores è possibile evidenziare che le varietà Golden Delicious, Fuji e Royal Gala hanno una composizione metabolica più simile rispetto ai succhi della varietà Pink Lady i quali appaiono ben clusterizzati. L'analisi dei loadings ha invece mostrato che gli zuccheri e l'acido malico sono i composti responsabili della separazione tra le quattro varietà e per tale ragione possono essere definiti dei composti marker.

Il terzo studio presentato riguarda l'indagine del profilo metabolico di estratti di Cannabis Sativa Calabrese tramite tecniche NMR mono-e bi-dimensionali. L'olio di semi di canapa e gli estratti di infiorescenze studiati sono stati ottenuti mediante estrazione solido-liquido, estrazione condotta utilizzando etanolo come solvente di estrazione e in presenza di ultrasuoni.

Sull'olio di semi di canapa è stata eseguita la caratterizzazione dello spettro 1 H in cui è stato riconosciuto il profilo tipico dei trigliceridi (TAG). Per quanto riguarda invece l'estratto di inflorescenze, in questl'ultimo è stato possibile riconoscere tre cannabinoidi, CBD, CBDA e CBG, insieme al profilo triglicerico. Questo è un risultato importante perché la Cannabis Sativa è nota per essere ricca di acidi grassi con rapporto ω6/ω3 pari a 3:1. Tale rapporto rappresenta un ottimo valore nutritivo e, la possibilità di utilizzare una tecnica di estrazione in grado di fornire estratti ricchi di entrambe le componenti merita di essere approfondita con ulteriori studi. Inoltre, il fatto che non sia stato riconosciuto alcun segnale riferito al THC, indica che la concentrazione di questa sostanza è inferiore al limite di rilevabilità della spettroscopia NMR e quindi anche inferiore al limite di legge. Questo studio preliminare effettuato è stato confrontato con i risultati presenti in letteratura e ha permesso di concludere che il nostro metodo di estrazione è promettente poiché permette di ottenere estratti ricchi in trigliceridi e cannabinoidi e ciò potrebbe rappresentare un punto di svolta nella produzione di alimenti funzionali.

La quarta applicazione presentata riguarda l'uso della spettroscopia NMR per studiare, per la prima volta, la composizione metabolica di un campione di olio essenziale di bergamotto puro e certificato (BEO) senza ricorrere ad alcun processo di manipolazione o derivatizzazione.

Nello specifico, gli obiettivi principali di questo studio erano, da un lato, eseguire la caratterizzazione e quantificazione dei principali metaboliti presenti in questa matrice complessa, mentre, dall'altro, esplorare la possibilità di indagare e discriminare eventuali frodi e sofisticazioni a cui il BEO potrebbe essere soggetto. Combinando gli spettri 1D e 2D classici con esperimenti DOSY 2D 1 H, è stato possibile identificare 11 composti a basso peso molecolare presenti nel campione studiato. Inoltre, è stato possibile quantificare circa il 90% del campione utilizzando l'acido benzoico come standard interno. Infine, poiché le adulterazioni non sono rare a causa dell'alto valore commerciale del BEO, è stata valutata la possibilità di utilizzare tre approcci NMR per rilevare possibili frodi. Tale studio è stato condotto simulando un'adulterazione da diluizione aggiungendo diverse quantità di olio essenziale di arancia al puro BEO. Studi di diffusione, analisi quantitativa e studio statistico sono stati utilizzati allo scopo di rilevare tale manipolazione. Mentre lo studio eseguito tramite diffusione consente di evidenziare la frode solo nel caso di elevati gradi di adulterazione, lo studio quantitativo sembra avere una maggiore sensibilità nel riconoscere gli oli diluiti. In ogni caso, la tecnica che meglio permette di distinguere i vari gradi di adulterazione ed è in grado di fornire maggiori informazioni risulta dalla combinazione di spettri protonici monodimensionali con gli strumenti di analisi statistica multivariata. L'ultimo esempio presentato in questo capitolo riguarda l'applicazione della tecnica HR-MAS NMR allo studio della frazione organica dei rifiuti solidi urbani. In particolare, è stato dimostrato che la spettroscopia HR-MAS NMR è un metodo innovativo e non distruttivo in grado di caratterizzare la composizione chimica della Frazione Organica di rifiuti solidi urbani ed è uno strumento adeguato a monitorare le variazioni in termini di composizione dei materiali organici iniziali sottoposti a trattamenti di ossidazione.

Le migliori prestazioni dell'approccio HR-MAS 13 C NMR rispetto al metodo CP-MAS, la tecnica più comunemente utilizzata per analizzare la frazione organica dei rifiuti, si basa sulla possibilità di ottenere spettri molto più risolti la cui analisi contribuisce a una migliore comprensione della natura chimica di questi rifiuti complessi. Inoltre, questo approccio HR-MAS si è dimostrato idoneo anche per il monitoraggio dei processi ossidativi basati sulla reazione di Fenton a cui è stata sottoposta la matrice iniziale. I risultati ottenuti, infatti, evidenziano le modificazioni qualitative e quantitative dei diversi tipi di carbonio in funzione sia delle concentrazioni dei reagenti di Fenton che della durata del processo ossidativo. A nostra conoscenza, questo caso di studio rappresenta il primo esempio in cui l'approccio HR-MAS è stato applicato con successo alle frazioni organiche di rifiuti solidi urbani. Infine, i vantaggi in termini di rapidità, semplicità e diversità delle informazioni ottenibili rendono questa tecnica molto interessante per ulteriori studi su diverse matrici di rifiuti e anche per monitorare altri processi di degradazione/trasformazione a cui possono essere sottoposti i rifiuti organici.

Nel terzo capitolo sono stati presentati i risultati ottenuti dall'applicazione della spettroscopia NMR nel secondo campo di ricerca indagato. In particolare, nel corso degli anni, le applicazioni

Titre:

  Application du potentiel analytique de la spectroscopie de Résonance Magnétique Nucléaire à l'étude de composés d'intérêt pharmaceutique et nutraceutique Mots clés : RMN, Métabolomique, PCA, Chiralité, Milieu Orienté Résumé : Dans cette thèse de doctorat, la spectroscopie RMN est appliquée dans deux domaines de recherche : dans l'étude métabolique des matrices alimentaires et extraits naturels, et dans l'étude structurale, orientationnelle et conformationnelle de petites molecules chirales flexibles d'intérêt pharmacologique. Dans le domaine métabolomique, le profil moléculaire des aliments constitue une «empreinte» du biosystème étudié et peut être utilisé pour son identification. Dans ce contexte, la spectroscopie RMN a été utilisée en combinant des séquences d'impulsions traditionnelles à des outils de chimiométrie dans le but de caractériser la composition métabolique des aliments et des extraits naturels, et de suivre l'évolution du profil métabolique lors des phases de traitement industriel. Puisque l'activité biologique d'un aliment est une fonction non triviale de celle de chaque métabolite individuel et qu'elle dépend à la fois de la configuration des centres stéréogènes, s'il s'agit d'une espèce chirale, et de la disposition spatiale des atomes au sein de la molécule, l'étude de la conformation et la configuration de ces espèces flexibles bioactives doivent être réalisées en parallèle de la caractérisation du métabolome lui-même. En particulier, une étude RMN dans des milieux chiraux faiblement orientés traditionnelles et plus sophistiquées à des modèles théoriques pour l'analyse des données.

  extracts. Hemp seeds oil and inflorescences extracts are studied by means of NMR experiments to characterize the complex mixtures and furnish information on these extracts in order to optimize the extraction technique that potentially could represent a turning point in the production of functional foods. The fourth example presented is the study of certified bergamot essential oil (BEO) sample. The main objectives of this study is the characterization and quantification of the main metabolites of this complex matrix, and the exploration of the possibility of discriminating fraud and sophistications to which this high value extract can be subjected. The second chapter ends with the application of HR-MAS NMR (High Resolution -Magic Angle Spinning Nuclear Magnetic Resonance spectroscopy) in metabolomics science.

  Dans le troisième chapitre, l'attention est portée sur l'étude conformationnelle espèces flexibles. En particulier, étant donné que l'interaction de chaque molécule bioactive avec des ligands et des récepteurs endogènes est dépendante de la configuration et des conformations de l'espèce flexible impliquée, l'attention c'est portée sur la possibilité de distinguer une paire d'énantiomères en fonction de leurs courbes conformationnelles expérimentales. L'étude RMN en milieux chiraux faiblement orientés a ensuite été appliquée à un premier couple d'énantiomères caractérisé par la présence d'un seul angle de torsion. Les d'alcools (RS)-4fluoro-α-méthylbenzylique ((RS)-4FMBOH) ont été étudiés afin d'explorer leurs équilibres conformationnels moléculaires et de faire émerger des différences dans la comparaison de leurs courbes de distribution de probabilité conformationnelle et les paramètres d'ordre indicatifs de la deux énantiomères. La chiralité devenant un problème crucial lorsqu'il s'agit d'espèces bioactives, la deuxième étude présentée dans ce dernier chapitre porte sur le cas de l'acide tiaprofénique. Cet acid est une espèce structurellement plus complexe dont un seul énantiomère est biologiquement actif en tant que médicament anti-inflammatoire. L'objectif principal de l'étude présentée est de comprendre si notre méthodologie en mesure de fournir des informations sur les équilibres conformationnelles en solution d'espèces bioactives chirales flexibles de structure non triviale, permettant ainsi d'accroître les connaissances fondamentales dans la compréhension des mécanismes d'action des médicaments.

  , 𝑡) = -𝛾 𝑖 𝐺 𝑦 (𝑡)𝑦𝐼 ̂𝑖𝑧 for gradient 𝐺 𝑦 along y axis (1.38) 𝐻 ̂𝑖𝑔𝑟𝑎𝑑 (𝒓, 𝑡) = -𝛾 𝑖 𝐺 𝑧 (𝑡)𝑧𝐼 ̂𝑖𝑧 for gradient 𝐺 𝑧 along z axis (1.39)

Figure 1 . 1 .

 11 Figure 1.1. The flow of information in system biology from the genome to the transcriptome, to the proteome and finally to the metabolome.

Figure 1 . 2 .

 12 Figure 1.2. Number of publications per year based on metabolomics studies. The data were obtained from Web Of Science (https://www.webofknowledge.com/ ). The orange data indicates the number of papers published up to 1 st October 2021.

Figure 1 . 3 .

 13 Figure 1.3. Scheme of metabolomics strategies.

  or the metabolites recognition are performed in order to achieve biological interpretations or generate new insights. A schematic summary of the steps that make up the metabolomics workflow is shown in the Figure1.4.

Figure 1 . 4 .

 14 Figure 1.4. Workflow of a metabolomic study and different steps involved.

Figure 1 . 5 .

 15 Figure 1.5. Pulse sequences for: A) a classical 1D NMR experiment (zg Bruker standard pulses sequence); B) 1D NMR experiments carried out observing a nucleus on the channel I and decoupling on S during both the preparation and the detection steps (zgpg Bruker standard pulses sequence); C) 1D NMR experiments carried out observing a nucleus on the channel I and decoupling on S during the detection step (zgig Bruker standard pulses sequence).

  SpectroscopY (COSY) is based on the transfer polarization by a mixing pulse between directly J-coupled spins and represents the most simple and commonly used 2D

Figure 1 . 6 .

 16 Figure 1.6. Pulse sequence for the classical COSY-90 experiment (cosyqf Bruker standard pulses sequence).

Figure

  Figure 1.7.Pulse sequences commonly used for the heteronuclear correlation spectroscopy: the 1 H-13 C HMQC pulse sequences is reported in the box A) (hmqcqf Bruker standard pulses sequence), 1 H-13 C HMBC is reported in B) (hmbclpndqf Bruker standard pulses sequence) and 1 H-13 C HSQC pulse sequences is reported in C) (hsqcph Bruker standard pulses sequence).

  and this made J-Res NMR spectroscopy a popular method for a wide range of NMR-based metabolomics studies[START_REF] Emwas | NMR Spectroscopy for Metabolomics Research[END_REF].

Figure 1 . 8 .

 18 Figure 1.8. Pulse sequences used for the J-Resolved NMR spectroscopy: the simple J-Res pulse sequence reported in the box A), while in B) is represented the experiment recorded for X-nuclei ( 13 C)

  phenomenon is to use a quantity called flux. The flux (𝐽 𝐴 𝑧 ) of a mass A along the z

Figure 1 .

 1 Figure 1.10. Spin-Echo (SE) pulse sequence (zggpse Bruker standard pulses sequence).

  magnetization, causing a reduction in the intensity of the signal. Different experiments like this are executed changing the gradient pulse magnitude G I , they are Fourier transformed and different monodimensional spectra are obtained. The acquired 1D spectra are subjected to the Laplace transform and the 2D DOSY experiment is obtained in which the dimension F2 shows the conventional chemical displacements while the diffusion coefficient is reported on F1 (Figure 1.11) (Yuan et al., 2017).

Figure 1 .

 1 Figure 1.11. Schematic flow for obtaining a 2D DOSY spectrum

Figure 1 .

 1 Figure 1.12. Representation of the curve reported in the equation (1.47). The intensity of the signals I I are plotted against the applied gradient strenght G I .

Figure 1 .

 1 Figure 1.13. STimulated-Echo (STE) pulse sequence. (stegp1s Bruker standard pulses sequence).

Figure 1 .

 1 Figure 1.14. Longitudinal Eddy current Delay (LED) pulse sequence. (ledgp2s Bruker standard pulses sequence).

  qNMR) represents an application of NMR spectroscopy able to provide an highly accurate and reproducible measurement of metabolites concentration. This is possible because in NMR spectroscopy the signal integral is directly proportional to the number of each nuclei and it is dependent to the amount of the substances present in the sample. This is true if the studied spectra are acquired under fully relaxed conditions and no polarization transfer occurs. Among the possible nuclei and pulse sequences, simple 1D 1 H NMR spectra, acquired using the pulse sequence reported in Figure1.5A, are the most used in qNMR study. The first quantitative proton NMR study was reported in 1963 by Jungnickel and Forbes simultaneously

Figure 1 .

 1 Figure 1.15. Workflow of a qNMR study and different steps involved.

  REference To access In vivo Concentrations) was published in 1999 by

  Figure 1.16.

Figure 1 .

 1 Figure 1.16. Workflow of the data preprocessing for the statistical study and different steps involved.

Figure 1 . 18 .

 118 Figure 1.18. Structure of the chiral homopolypeptide poly-γ-benzyl-L-glutamate (PBLG).

Figure 1 . 19 .

 119 Figure 1.19. Molecular organization in the cholesteric phase obtained by dissolving the PBLG in a suitable organic co-solvent.

Figure 1 . 20 .

 120 Figure 1.20. Representation of the uniaxial nematic chiral liquid crystalline phase generate by the liquid crystalline phase PBLG/co-solvent when the system is immerged in the NMR magnetic field 𝐵 0 ⃗⃗⃗⃗ .

Figure 1 .

 1 Figure 1.21.Pulses sequence used the SERF experiment is reported on the left. On the right, an example of the SERF spectrum obtained using this sequence. The offset of the selective pulse was set at Ha on the proton channel and the offsets of the refocusing selective pulse at Hb.

Figure 1 . 22 .

 122 Figure 1.22. Pulses sequence used for the HetSERF experiment is reported on the left. On the right, an example of the HetSERF spectrum obtained using this sequence. The offset of the selective pulse was set at Hb on the proton channel.

  the case of direct dipolar couplings, 𝐷 𝐶𝐻 1 , it is quite easy to recognize the correct value. Indeed, since 𝐽 𝐶𝐻 1 are known to be positive and in PBLG phase 𝐷 𝐶𝐻 1 are of similar magnitude or even 180 soft (H b )

1. 4 . 3

 43 Relationship between experimental NMR data and structure, order and conformation of flexible moleculesIn the case of a flexible molecule, the RDCs, obtained following the methodology just described, are in reality "average" values of the observables. This is due to the fact that the orientational motion of the molecule and the internal molecular motions are "faster" than the NMR technique and, consequently, the experimental information obtained is averaged on the aforementioned motions. To consider the dependence of the NMR observables on the properties of the conformers and the corresponding orientational parameters, it is possible to express the average dipolar coupling constant 𝐷 𝑖𝑗 𝑜𝑏𝑠 approximately as(Di Pietro et al, 2014; Di Pietro et al, 2015; Di Pietro et al, 2017):

Figure 1 . 23 .

 123 Figure 1.23. Flowchart showing the strategy adopted by the AnCon package.

Figure 2 . 1 .

 21 Figure 2.1. 500 MHz NMR spectra of Calabrian squeezed fresh orange juice recorded at 298K.A) . 1 H NMR spectrum, recorded using a pre-saturation sequence zgpr (Bruker standard pulses sequence) in order to suppress the residual water signal, and for each experiment 256 FIDs were acquired using a spectral width of 14.00 ppm and a relaxation delay of 2 s; B) 13 C-{ 1 H} NMR spectrum, acquired with proton broad-band decoupling by collecting 8000 free induction decays (FIDs), by using a spectral width of 200.00 ppm and a relaxation delay of 4s.

Figure 2 . 2 .

 22 Figure 2.2. Structure of citric acid and atomic numbering adopted.

  the signals investigated in the proton spectrum. As can be seen from the 1D experiment shown in Figure2.3b, it is possible to recognize the two previously hypothesized doublets, which resonate at 2.79 ppm and 2.93 ppm, and it is possible to extract the geminal coupling constant 2 JH3a-H3b = 2 JH5a-H5b = 15.6 Hz. Both the values of chemical shifts and the values of the coupling constant are in agreement with the data reported in the literature[START_REF] Gall | Discrimination between Orange Juice and Pulp Wash by 1H Nuclear Magnetic esonance Spectroscopy: Identification of Mar er Compounds[END_REF][START_REF] Alves Filho | 1H NM Spectroscopy and Chemometrics Evaluation of Non-Thermal Processing of Orange Juice[END_REF][START_REF] Ludwig | Birmingham Metabolite Library: a Publicly Accessible Database of 1-D 1H and 2-D 1H J-resolved NMR Spectra of Authentic Metabolite Standards (BML-NM )[END_REF].

Figure 2 . 3 .

 23 Figure 2.3. A) 500 MHz 1 H COSY NMR spectrum recorded on squeezed orange juice in D2O (298K).The 1 H COSY experiments were acquired employing pre-saturation sequence for water signal suppression and using a SW of 9 ppm on both dimensions, 2K data points, 4 scans and 512 increments. In red is reported the enlargement of the region 2.80 -3.15 ppm on both dimensions; B) region ranging from 2.80 to 3.15 ppm of the 500 MHz 1 H NMR spectrum recorded on squeezed orange juice in D2O (298K).

Figure 2 .

 2 Figure 2.4. 500 MHz 1 H-13 C HMQC NMR spectrum recorded on squeezed orange juice in D2O (298K).The 1 H-13 C HMQC spectra were recorded employing pre-saturation sequence for water signal suppression and using a SW of 9 ppm ( 1 H) and 200 ppm ( 13 C), 2K datapoints, 256 scans and 16 experiments. In red is reported the enlargement of the region (F1: 39.5-46.5 ppm; F2: 2.50-3.25ppm).

Figure 2 . 5 .

 25 Figure 2.5. Expanded 1 H NMR spectrum and signal assignment of Calabrian squeezed fresh orange juice: A) Amino acid region; B) Sugar region and C) Aromatic region.

Figure 2 . 6 .

 26 Figure 2.6. Comparison of 500MHz 1 H NMR spectra of squeezed (in blue), pasteurized (in red) and concentrated (in green) orange juice coming from the process A (298K).

Figure 2 . 7 :

 27 Figure 2.7: Comparison between orange squeezed juice 1 H NMR spectrum (in blue) and orange and clementine squeezed juice (in red). Signals of phlorin are marked by asterisks.

Figure 2 .

 2 Figure 2.8 shows the PC1 vs PC2 score plot, in which a clear separation between the samples

Figure 2 . 8 :

 28 Figure 2.8: Principal Component Analysis (PCA) of Orange (A and B) and Orange and Clementine (C) juices. The scores plot showing the first two PCs (PC1 and PC2) with their respective variation. R 2 X(PC1) = 68.2%, R 2 X(PC2) = 14.0%.

Figure 2 .

 2 Figure2.9 shows the PC1 vs PC3 score diagram. All concentrated juices are located at positive scores of PC3 and all the squeezed juices are positioned at negative scores of PC3, while there is a clear variability as regards the data coming from the pasteurization process. Indeed, the pasteurization process seems a non-reproducible step because the pasteurized clementine and orange juices (sample C) are located at positive PC3 values, near to the concentrated cluster and far from the squeezed one, while the corresponding orange juices are at negative values of PC3 and overlapped to the squeezed cluster. These data can be explained by assuming that the

Figure 2 . 9 :

 29 Figure 2.9: Principal Component Analysis (PCA) of Orange (A and B) and Orange and Clementine (C) juices. The score plot showing the two PCs: PC1 and PC3, with their respective variation. R 2 X(PC1) = 68.2%, R 2 X(PC3) = 7.8%.

Figure 2 .

 2 Figure 2.10: Loading plots of PCA carried out on NMR spectra of Orange and Orange and Clementine juices: A) showing how variables influenced the two PCs: PC1 and PC2; B) showing how variables influenced the two PCs: PC1 and PC3.

  of Figure 2.10A provides clear evidence to identify the variables responsible for the sample clustering observed in the score diagram reported in Figure 2.8. Indeed, loadings highlight that PC1 is dominated by amino acid, phlorin and sugars. Particularly, amino acid, phlorin and α-glucose are negatively correlated with sucrose; while PC2 is influenced by citric acid, DMP and sugar (sucrose and sugars are negatively correlated with α-glucose, DMP and citric acid). The loadings of Figure 2.10 B shows that ethanol and aliphatic compounds contribute negatively with sugars to PC3. These data indicate that the clementine and orange juice (sample C) is richer in amino acids and phlorine than orange juice alone. The markers that differentiate them are the sucrose and the citric acid whose concentration is different for the two samples A and B analysed. This, most likely, is due to the different origin and quality, in terms of cultivar and ripening, of the starting fruits. As expected, during the concentration

Figure 2 . 11 :

 211 Figure 2.11: Dendrogram of cluster analysis of juice samples. Capital letters "S", "P" or "C" stand for Squeezed, Pasteurized or Concentrated samples, while small letters "o" and "c" represent orange or clementine juices.

  5 g of the supernatant at the right pH value in 5 mm o.d. NMR tube together with about 0.03 g of the solution of TMSP-d4 (100mM) and NaN3 (2mM) in D2O. TMSP-d4 (i.e. 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt) was used as internal standard for the chemical shifts calibration. The aqueous solution of NaN3 was added to minimize the alteration in the composition of the juice derived from microbial activity, both during the time of sample preparation and the acquisition of NMR spectra, and D2O was used as the field frequencies lock signal. All chemicals and reagents were of commercial origin: 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (98 atom % D)

Figure 2 .

 2 Figure2.12. 500 MHz NMR spectra of Calabrian Golden Delicious juice recorded at 298K. A) 1 H NMR spectrum, recorded using the noesypr1d sequence (Bruker standard pulses sequence) in order to suppress the residual water signal, and for each experiment 256 FIDs were acquired using a spectral width of 11 ppm and a relaxation delay of 10 s; B) 13 C-{ 1 H} NMR spectrum, performed with proton broad-band decoupling collecting 5000 free induction decays (FIDs) using a spectral width of 266.80 ppm and a relaxation delay of 10s.

Figure 2 .

 2 Figure 2.13. 500 MHz 1 H COSY NMR spectrum recorded on Calabrian Golden Delicious juice in D2O (298K) on the left. The 1 H COSY experiments were acquired employing pre-saturation sequence for water signal suppression and using a SW of 11 ppm on both dimensions, 2K data points, 28 scans and 512 increments. In the red box is reported the enlargement of the region containing the correlations of the -glucose and sucrose anomeric groups (F1:5.0-6.0 ppm; F2:3.0-4.5 ppm). On the right are reported the structures and the atom numbering of -glucose and sucrose.

Figure 2 .

 2 Figure 2.14. 500 MHz 1 H-13 C HMQC NMR spectrum recorded on Calabrian Golden Delicious juice in D2O (298K). The 1 H-13 C HMQC experiments were acquired employing pre-saturation sequence for water signal suppression and using a SW of 11 ppm ( 1 H) and 265.0 ppm ( 13 C), 2K datapoints, 256 scans and 32 experiments. In the red box is reported the enlargement of the region containing the correlations of the -glucose and sucrose anomeric groups (F1: 100.0-90.0 ppm; F2: 4.8-5.8 ppm).

Figure 2 .

 2 15, then the metabolites recognized for the Golden Delicious variety are recognizable also in the other apple varieties, which show no other signals than those already identified, except different relative intensity of the signals and small variations in chemical shift.

Figure 2 . 15 .

 215 Figure 2.15. Comparison of 500MHz 1 H NMR spectra of Golden Delicious (in blue), Royal Gala (in red), Pink Lady (in green) and Fuji (in orange) juice recorded at 298K.

Figure 2 .

 2 Figure 2.16. Principal Component Analysis (PCA) of Golden Delicious, Pink Lady, Fuji and Royal Gala juices. The score plot showing the first three PCs: PC1, PC2 and PC3 with their respective variation. R 2 X(PC1) = 52.3%, R 2 X(PC2) = 22.2%. R 2 X(PC3) =7.1%.

  is a 2D plot where only the PC1 and PC2 are considered. From the biplot emerged that Pink Lady variety is characterized by a major sucrose and malic acid content respect to other ones, while Golden Delicious and Fuji are richer in α-glucose, β-glucose, α-D-fructofuranose and β-Dfructofuranose. Royal Gala seems to have an intermediate composition between Pink Lady variety and Golden Delicious and Fuji varieties even if it is closer to the last two. Minor compounds such as ethanol, quinic acid, chlorogenic acid, L-rhamitol and citramalic acid, have null values of PC2, i.e. the dimension in which the apple varieties are better separated. This means that these 5 metabolites do not affect the separation and can be neglected as marker of variety separation.

Figure 2 .

 2 Figure2.17. Biplot of the first two PCs, PC1 and PC2, taking into account the loadings and the scores obtained from the PCA carried out on NMR spectra of the Golden Delicious, Pink Lady, Fuji and Royal Gala juices.

  only several years later, i.e. in 2009, by means of an appropriate Regulation published by the European Union (European Commission (2009) Regulation (EC) No 1107/2009). Nowadays, in many countries, Cannabis Sativa cultivations and cannabis medicines have been legalized under certain conditions due to the immense prospects in various medicinal applications. The Italian legislation on Cannabis Sativa cultivation is somewhat ambiguous about the legal and illegal uses and cultivation of the plant. The law 242/2016 "Dispositions for the promotion of cultivation and supply chain of agro-industrial Hemp" is the most recent regulation in that direction and the reference text governing industrial hemp production in Italy only for fiber or other industrial uses, different from pharmaceutics, starting from certified seeds (Legge 2 Dicembre 2016, n.242. Disposizioni per la Promozione della Coltivazione e della Filiera Agroindustriale della Canapa (16G00258)). However, on 4 November 2019, Italian Ministry of Health approved and ratified "Definition of maximum levels of tetrahydrocannabinol (THC) in food" (GU n.11, 15-1-2020) (Decreto 4 novembre 2019 Definizione di livelli massimi di tetraidrocannabinolo (THC) negli alimenti. (

  varieties", described by the annex III of the Commission Delegated egulation (EU) 2017/1155 (the last update on 15 February 2017), is the only official procedure that member states must use for the quantitative determination of THC by gas chromatography (GC) after extraction with a suitable solvent (Annex III, Commission Delegated Regulation (EU) 2017/1155 of 15 February 2017 Amending Delegated Regulation (EU) No 639/2014). It describes in detail sampling, samples dimensions, drying and storage, techniques and reagents for extraction and determination of

Figure 2 . 19 :

 219 Figure 2.19: Triacylglycerols structures recognized in hemp seeds oil dissolved in CDCl3 (left); on the right is reported an enlargement (F1: 0.5 -6.0 ppm, F2: 0.5 -6.0 ppm) of the 500 MHz 1 H COSY NMR spectrum acquired on hemp seeds oil sample (298K) using a SW of 11.00 ppm on both dimensions, 2K data points, 16 scans and 256 increments. A sine filter was applied on both dimensions, F1 and F2, of COSY experiment before Fourier-transformations.

Figure 2 .

 2 Figure 2.21.A) The structures of CBD and CBDA are reported together with the atoms numbering adopted. B) 500 MHz 1 H COSY spectrum of inflorescence extracts sample dissolved in CDCl3 recorded at 298K. The spectrum was acquired using a SW of 11.00 ppm on both dimensions, 2K data points, 16 scans and 256 increments. A sine filter was applied on both dimensions, F1 and F2, of COSY experiment before Fourier-transformations. In the enlargement is reported the region F1:1.5-5.0ppm, F2:3.8-5.2ppm containing the correlations of protons H9 and H10 of CBD and CBDA. C) 500 MHz 1 H-13 C HMQC NMR spectrum of inflorescence extracts sample dissolved in CDCl3 (298K) using a SW of 10.00 ppm ( 1 H) and 200.00 ppm ( 13 C), 2K datapoints, 512 scans and 40 experiments. A qsine filter was applied on both dimensions, F1 and F2, on HMQC experiment before Fourier-transformations. In the enlargement is reported the region F1:107.0-114.5 ppm, F2:4.1-5.0 ppm containing the correlations of protons H9 of CBD and CBDA with the respective carbon atom.

Figure 2 .

 2 Figure 2.22. 500 MHz 1 H-1 H J-resolved spectrum of inflorescence extracts sample dissolved in CDCl3 recorded at 298K. The spectrum was acquired using a SW of 11.00 ppm on F2 and a SWH of 100 Hz on F1, 2K data points, 48 scans and 256 increments. In the enlargement is reported the signal obtained by extrapolating the column at the chemical shift of the proton H9cis of CBD.

  3 JH1-H6 = 13.0Hz; 3 JH1-H2 = 3.5Hz; 4 JH1-H5 = 2.5Hz) 5.57 (m) 2.05 -2.09 (m) 2.22 (m) 1.78-1.84 (ddd, 3 JH5-H6 = 5.3Hz; 3 JH5-H4a = 1.3Hz; 3 JH5-H4b = 0.6Hz) 2.40 (dd, 3 JH6-H1 =13.0Hz, 3 JH6-H5 = 5.0Hz) 1.79 (d, 3 JH7-H2 = 0.5Hz) 4.64 (dq, 2 J9cis-trans = 2.6Hz, 4 J9trans-10 = 1.5Hz) 4.39 (dq, 2 J9cis-trans = 2.6Hz, 4 J9cis-10 = 0.9Hz) 1.70 (dd, 4 J10-9cis = 0.9Hz, 4 J10-9trans = 1.5Hz)

  3 JH5-H6 = 5.3Hz, 3 JH5-H4a = 1.3Hz; 3 JH5-H4b = 0.6Hz) 2.40 (dd, 3 JH6-H1 =13.0Hz; 3 JH6-H5 = 5.0Hz) 1.79 (d, 4 JH7-H2 = 0.5Hz) 4.51 (dq, 2 J9cis-trans = 3.0Hz ; 4 J9trans-10 = 1.8 Hz) 4.39 (dm, 2 J9cis-trans = 3.0Hz) 1.70 (m)

Figure 2 .

 2 Figure 2.23. 500 MHz 1 H NMR spectrum of inflorescence extracts sample dissolved in CDCl3 showing signals assignmentS: A) 0,7-2,2 ppm; B) 2,2-3,1 ppm; C) 3,7-4,8 ppm and D) 4,8-6,5 ppm.

Figure 2 .

 2 Figure 2.24. 500 MHz NMR spectra of pure Bergamot Essential Oil (BEO) recorded at 298 K. A) 1 H NMR spectrum recorded using zg Bruker standard pulses sequence, collecting 128 FIDs by using a

  have in common a cyclic α,β-unsaturated fragment that originates a characteristic proton spectrum similar in multiplicity but with signals that resonate at different frequencies for each single compound.The COSY correlation spectrum was essential in assigning the Hb (H4 for the citropten and GMC; H5 for the bergapten and bergamottin) proton peaks of the cycle to each individual component. Indeed, as can be seen from the 1D spectrum of Figure2, the Hb proton of the fragment generates a doublet of multiplets for each molecule which, although easily recognizable since they have the same multiplicity but different chemical shifts and intensities, are difficult to assign to each individual molecule. On the contrary, in the COSY spectrum, the correlation of the signals generated by the Hb proton with those generated by the Ha proton (H3 for the citropten and GMC; H6 for the bergapten and bergamottin) allowed to reconstruct the other correlations and to correctly assign the four signals to the single molecules as shown in Figure 2.25. These assignments have been confirmed by the data present in literatureregarding the 1 H NMR spectra of each single species[START_REF] Chunyan | Isolation and Purification of Psoralen and Bergapten from Ficus Carica L. Leaves by High-Speed Countercurrent Chromatography[END_REF] and by the analysis of 1 H-13 C HMQC NMR spectrum (Figure2.26) which was essential for the 13 C assignments.

Figure 2 .

 2 Figure 2.26. 500 MHz 1 H-13 C HMQC NMR spectrum acquired on pure Bergamot Essential Oil (BEO) sample (298K) using a SW of 18.00 ppm ( 1 H) and 280.00 ppm ( 13 C), 2K datapoints, 512 scans and 32 experiments. A qsine filter was applied on both dimensions, F1 and F2, on HMQC experiment before Fourier-transformations.

Figure 2 .

 2 Figure 2.27. 400 MHz 2D 1 H DOSY NMR spectrum recorded at 306.9 K on the (R)-Limonene/CDCl3 sample.

Figure 2 . 28 .

 228 Figure 2.28. Diffusion coefficients obtained for samples of (R)-Limonene at different concentration. On the right, the diffusion coefficient is plotted against concentration of limonene.

Figure 2 . 29 .Figure 2 . 30 .

 229230 Figure2.29. Diffusion coefficients obtained at different temperature for samples of (R)-Limonene in chloroform (2.2 M). On the right, the diffusion coefficient of the limonene is plotted against temperature at which the experiment was carried out.

Figure 2 .

 2 Figure 2.31. 400 MHz 2D 1 H DOSY NMR spectrum recorded at 306.9 K on the model mixture sample.

Figure 2 . 2

 22 Figure 2.32. 400 MHz 2D 1 H DOSY spectrum of BEO recorded at 306.9 K (left); limonene structure, numbering and signal attribution (right).

Figure 2 .

 2 Figure 2.33. Expanded 500 MHz 1 H NMR spectrum and signal assignment of BEO: A) 0.3 -3.2 ppm; B) 3.5 -6.5 ppm and C) 6.2 -10.5 ppm.

Figure 2 .

 2 Figure 2.34. 500 MHz 1 H NMR spectrum and expanded signals used for the quantitative study. The H(C) and H(BB') in the benzoic acid box are the standard signals integrated for the study. In the bottom the deconvolution of the linalool and linalyl acetate peaks is shown.

Figure 2 .

 2 Figure 2.35. Comparison of 500 MHz 1 H NMR spectra of pure BEO (in blue), adulterated BEO (5%OEO) (in red), adulterated BEO (10%OEO) (in green) and adulterated BEO (30%OEO) (in violet).

  , the three substances choosed for the model mixtures were n-hexane, cyclohexane and n-decane. The composition of each model mixture and the results obtained for the diffusion coefficient of limonene are shown in Figure 2.36.

Figure 2 .

 2 Figure 2.36. Composition of the model mixtures prepared (left) and diffusion coefficient obtained for the limonene by means of the 400 MHz 2D 1 H DOSY NMR spectrum recorded at 306.9K (right).

Figure 2 .

 2 36, it is possible to distinguish the diffusion coefficient of limonene in the three case. The values are not very far from each other but they are still different. At this point the solution B (90% of BEO and 10% of OEO), solution C (70 % of BEO and 30% of OEO) and solution D (50% of BEO and 50% of OEO) were studied from the diffusion point of view and the results obtained were compared to the one found for pure BEO. All the data obtained are reported in Figure2.37.

Figure 2 . 37 .

 237 Figure 2.37. Diffusion coefficients obtained for the limonene by means of the 400 MHz 2D 1 H DOSY NMR spectrum recorded at 306.9K on the pure BEO sample and the adulterated solutions. On the left the data obtained are summarized in a Table and on the right the diffusion coefficients obtained are plotted against the concentration in BEO of the studied samples.

Figure 2 .

 2 Figure 2.38. Histograms representing the quantitative results obtained for each metabolite in the different samples: pure BEO and adulterated ones.

Figure 2 .

 2 Figure 2.39. Principal Component Analysis (PCA) of pure BEO (100BEO) and adulterated BEO with 5, 10 and 30% of OEO (95BEO:5OEO, 90BEO:10OEO and 70BEO:30OEO respectively). The score plot shows the three PCs: PC1, PC2 and PC3, with their respective variation. R2X(PC1) = 39.9%, R2X(PC2) = 23.5% and R2X(PC3) = 12.3%.

Figure 2 .

 2 Figure 2.40. Biplot of PCA carried out on NMR spectra of pure and adulterated BEO samples showing how variables affected the two PCs: PC2 and PC3.

Figure 2 .

 2 Figure 2.41. Experimental steps, oxidative reaction conditions, and NMR samples collected for each process the initial sample of the organic fraction of municipal solid waste (OFMSW) was subjected to.

Figure 2 .

 2 Figure 2.42. HR-MAS 13 C-{ 1 H} NMR spectrum of Sample 0 (in blue) compared with CP-MAS 13 C NMR spectrum of OFMSW sample reported in the literature (in black)(Pichler, et al, 200).

Figure 2 .

 2 Figure 2.43 shows the NMR HR-MAS 13 C-{ 1 H} spectrum of sample 0 in which the different regions are indicates. The region from 12 to 40 ppm is the classic region of aliphatic carbons signals, probably due to the presence of lipids and fatty acids in the OFMSW mixture that we used. The region from 58 to 100 ppm is due to the carbon atom signals linked to heteroatoms.

  i.e. signals due to unsaturation in fatty acids and/or to aromatic species; finally the region from 169 to 183 ppm is related to the signals of carbonyl carbons (acids, esters, ketones, amides and aldehydes).

Figure 2 .

 2 Figure 2.43. HR-MAS 13 C NMR spectrum of Sample 0 and signal assignment. In the box, the comparison with the CP-MAS spectrum of starch in the same region is shown(Tang & Hills, 2003).

Figure 2 .

 2 Figure 2.43 the good resolution allows a better integration on the individual signals. In particular, the area of each signal of the spectrum was integrated and the quantitative information were grouped according to the nature of the atoms (aliphatic carbon atoms: Caliph; carbon atoms linked to heteroatoms: CX; unsaturated carbon atoms: Cunsat and carbonyl carbon atoms: Ccarb).

Figure 3 . 1 .

 31 Figure 3.1. In Figure are shown the signals relating to the interactions between a pair of nuclei i and j in the three phases: A) achiral isotropic phase, where only the indirect coupling and the isotropic chemical shifts are present, B) achiral anisotropic phase, which allows us to observe the anisotropic chemical shift interactions and direct coupling, in addition to the interactions of isotropic origin, and C) the chiral anisotropic phase in which the anisotropic interactions that appear to be different for the two enantiomers are visible.

3. 2

 2 is referred to the simplest case consisting of the R and S enantiomers of the 4-fluoro-αmethylbenzyl alcohol (4FMBOH) molecule, a single rotor species. Then the work performed on the two enantiomers of the more complex molecule of 2-(5-benzoylthiophen-2yl)propanoic acid (tiaprofenic acid), with a larger number of internal torsions, is presented in section 3.3.

  and the two enantiomers of the chiral flexible species (RS)-1-(4-fluorophenyl)ethan-1-ol (IUPAC name), also called (RS)-4-F--methyl benzyl alcohol, ((RS)-4FMBOH; Figure3.2) have been investigated using liquid crystal NMR technique. The analysis of NMR spectra of this orientationally ordered systems provide important and rich of information anisotropic observables, corresponding to the partially averaged components of several interactions Among them, in this study the observed Residual Dipolar Couplings, RDCs (𝐷 𝑖𝑗 𝑜𝑏𝑠 ) between the magnetically active nuclei present in the two enantiomers have been used in order to have access to the structural, conformational and orientational information. By combining these experimental parameters, extracted by using different one-and two-dimensional NMR experiments, with the theoretical Additive Potential-Direct Probability Description (AP-DPD)

Figure 3 . 2 .

 32 Figure 3.2. Structure, atomic labelling and torsional angle of (RS)-4FMBOH.

3. 2 . 2 .

 22 RDCs extractionIt is known very well that there is no difference in the spectral profile between two species R and S in an non chiral isotropic environment, while the different solute-solvent interactions that each molecule experiences in the chiral liquid crystalline solvent PBLG/CDCl3 originate spectra with different profile and affected by values of 𝑇 𝑖𝑗 𝑅 (𝑜𝑏𝑠) ≠ 𝑇 𝑖𝑗 𝑆 (𝑜𝑏𝑠). These differences are evident in Figure3.3, where the 1 H anisotropic spectra of R-4FMBOHaniso and S-4FMBOHaniso are compared.

Figure 3 . 3 .

 33 Figure 3.3. 1D 1 H spectra recorded at 298 K for the samples R-aniso (in red) and S-aniso (in blue). In the enlargement the signals of the methyl group (H8) of the two enantiomers are reported.

Figure 3 . 4 .

 34 Figure 3.4. 2D SERF spectrum recorded at 298 on S-4FMBOHaniso sample where the offset of the selective pulse was set at n8. The total coupling between the three equivalent protons of the methyl group is easily read on the 2D map by measuring the distance between the triplet spots as shown by the red sign.

Figure 3 . 5 .

 35 Figure 3.5. 2D 1 H-1 H J-resolved spectra recorded at 298 K on S-4FMBOHaniso sample. Enlarged maps show projections corresponding to the columns centered on the chemical shifts of H7, H2, H3.

Figure 3 .Figure 3 . 6 .

 336 Figure 3.6 displays the HetSERF spectrum recorded on R-4FMBOHaniso sample selecting H8 frequency on the proton channel. As it can be seen, this selective spectrum allow to extract easily the long-range coupling 𝑇 𝐶3-𝐻8 𝑅

Figure 3 .

 3 7 (RMS = 0.94 Hz for R isomer and RMS = 0.84 Hz for S isomer). The curves obtained for the two enantiomers 𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑅 and 𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑆 were sampled between 0 ° and 360 ° in steps of 2° and are shown in Figure 3.8. The torsional distribution 𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑅 is characterized by two equally populated maxima at 149° ± 7° (conformation IR) and at 329° ± 7° (conformation IIR). Regarding the other enantiomer, the torsional distribution 𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑆 is characterized by two equally populated maxima at 35° ± 5° (conformation IS) and at 215° ± 5° (conformation IIS). Both curves are in reasonable agreement with the ones obtained through theoretical calculation.

Figure 3 . 7 .

 37 Figure 3.7. Comparison of experimental and calculated RDCs for both the enantiomers R-4FMBOH in orange and S-4FMBOH in blue.

Figure 3 . 8 .

 38 Figure 3.8. Comparison of experimental (solid line) and calculated (dot line) probability distribution function (𝑃 𝑖𝑠𝑜 ({𝜙 }) and 𝑃 𝑡ℎ𝑒𝑜 ({𝜙 }) obtained for both the enantiomers R-4FMBOH (in orange) and S-4FMBOH (in blue).

  view (Di Pietro et al, 2014). More complicated is the treatment of conformational distribution of profen derivatives. The molecules of (S)-naproxen ((S)-2-(6-methoxynaphthalen-2yl)propanoic acid) and (R)-flurbiprofen ((R)-2-(2-fluoro-4-biphenyl)propanoic acid) were chosen as illustrative of two-rotor molecules with noncoupled rotations (Di Pietro et al, 2015), while two other molecules (S)-ibuprofen ((S)-2-(4-(2-methylpropyl)phenyl)propanoic acid) and (S)-ketoprofen ((S)-2-(3-(benzoylphenyl)propionic acid) were chosen as examples of even more complex conformational situations with cooperative torsional motions (Di Pietro et

Figure 3 .

 3 Figure 3.11. Structure, atomic labelling and torsional angle of (RS)-TA.

Figure 3 .

 3 Figure 3.12. 1D 13 C spectrum recorded at 298 K for the sample RS-TAiso. In the red box is reported the enlargement of the C3 signal.

Figure 3 .

 3 Figure 3.13. 2D SERF spectrum recorded at 298 on RS-TAaniso sample where the offset of the excitation selective pulse was set at n2 and the offsets of the refocusing selective pulse at n2 and n3. The total coupling between the selected protons is easily read by extracting the column and by measuring the distance between the line of the resulting two quadruplets as shown in the enlargement in the red box.

Figure 3 .

 3 Figure 3.14. 2D 13 C-1 H HETSERF spectrum of RS-TAaniso sample. The offset of the selective pulse was set at n2 on the proton channel. Enlarged map shows the spectrum obtained by extrapolating the column corresponding to the C5 signal and the TC5-H2 can be easily measured.

Figure 3 .

 3 Figure 3.15. Comparison of experimental (solid line) and calculated (dot line) probability distribution function (𝑷 𝒊𝒔𝒐 ({𝝓 }) and 𝑷 𝒕𝒉𝒆𝒐 ({𝝓 }) obtained for both the enantiomers R-4FMBOH (in orange) and S-4FMBOH (in blue).

Figure 3 .

 3 Figure 3.16. Comparison of experimental probability distribution function (𝑷 𝒊𝒔𝒐 ({𝝓 }) obtained for the S-TA (in red) and S-Ketoprofene (in green).

  particular, from the study of the TA enantiomers emerged that the two enantiomer are characterized by a conformational curve composed by two maxima differently populated and the two curves obtained, one for each enantiomer, are one the mirror image of the other. The same result was obtained from the theoretical study and from the experimental study carried out on the two enantiomers of a simpler species: 4FMBOH presented in section 3.2. The presence of the sulfur atom represents the major difference between the structure of the TA and the structures of the other profens as in the case of ketoprofen for example. The sulfur lowers the symmetry of the molecule and seem to be the cause of the different population of the two maxima of the conformational probability function. Nevertheless, the two TA enantiomers, as well as the other biologically active profens studied, are characterized by the presence of the hydrogen atom directly bonded to the chiral carbon almost in same plane of the aromatic ring and this seems to be a peculiar characteristics of the NSAIDs. It must be noted that the work presented in this section is a preliminary study. In fact, only one torsional angle has been investigated for the two species but, as pointed out in the introduction, two other torsional angles contribute to determining the large number of stereoisomers that tiaprofenic acid can present. Considering the two enantiomers in their entirety by studying all the torsions from a conformational point of view, allows to add a new piece for understanding

  NMR in fasi chirali debolmente orientanti si sono ampiamente dimostrate in grado di fornire RDC che, se trattate con un opportuno modello teorico, possono portare all'ottenimento di informazioni strutturali, conformazionali e orientazionali di molecole flessibili. Questa metodologia è stata applicata in questo capitolo ad una coppia di enantiomeri al fine di indagare le loro conformazioni individuali e per comprendere le diverse interazioni soluto-solvente che le due specie, R e S, formano in un ambiente chirale. In particolare, il primo studio presentato in questo capitolo è riferito ad un caso molto semplice, costituito dagli enantiomeri R e S della molecola di alcol 4-Fluoro-α-metilbenzilico (4FMBOH), una specie che presenta un solo angolo torsionale. L'esempio riportato in questa sezione ha dimostrato il potere unico dell'NMR in fasi liquido cristalline nell'esplorazione degli equilibri conformazionali molecolari al fine di far emergere, per la prima volta secondo le nostre conoscenze, differenze nelle curve di distribuzione torsionale e nei parametri dell'ordine orientazionale degli enantiomeri R e S disciolti in un solvente debolmente ordinante chirale non racemico. In particolare, i risultati ottenuti hanno mostrato che i due enantiomeri sono caratterizzati da una curva conformazionale composta da due massimi ugualmente popolati e le due curve ottenute, una per ciascun enantiomero, sono l'una l'immagine speculare dell'altra. Inoltre, il modello teorico AP-DPD applicato, noto per essere un solido approccio nel trattamento dei dati sperimentali per molecole sia altamente che debolmente orientate, sembra essere appropriato per trattare anche le caratteristiche chirali degli enantiomeri studiando il loro comportamento conformazionale.Poiché la chiralità diventa un problema cruciale quando si tratta di specie bioattive, come ad esempio nel caso di farmaci, il secondo studio condotto ha riguardato una specie più complessa di cui un solo enantiomero è attivo come farmaco antinfiammatorio: l'acido tiaprofenico (TA).La molecola studiata è composta da 2 torsioni cooperative e 1 torsione indipendente e, nello studio presentato, l'attenzione si è concentrata sul comportamento conformazionale della sola torsione indipendente che coinvolge la porzione propionica e l'anello tiofenico. I risultati ottenuti dallo studio condotto su entrambi gli enantiomeri, hanno mostrato che le due specie sono caratterizzate da una curva conformazionale composta da due massimi diversamente popolati e le due curve ottenute, una per ciascun enantiomero, sono l'una l'immagine speculare dell'altra. La presenza dell'atomo di zolfo nella struttura dell'acido tiaprofenico, rappresenta la maggiore differenza tra l'TA e le strutture degli altri profeni. Infatti, lo zolfo abbassa la simmetria della molecola e ciò sembra essere la causa della diversa popolazione dei due massimi nella funzione di probabilità conformazionale. Tuttavia, i due enantiomeri di TA, così come gli altri profeni biologicamente attivi studiati in letteratura, sono caratterizzati dalla presenza dell'atomo di idrogeno direttamente legato al carbonio chirale quasi nello stesso piano dell'anello aromatico e questa sembra essere una caratteristica peculiare dei FANS.Per concludere, il lavoro svolto durante questo progetto di tesi ha affrontato diversi problemi: a) lo studio di matrici naturali complesse utilizzando metodologie NMR non invasive e b) l'esplorazione del comportamento conformazionale in soluzione di molecole bioattive chirali. Da un lato, tutte le evidenze emerse negli studi metabolomici presentati ci consentono di concludere che la tecnica NMR, combinata con l'analisi statistica, è un potente strumento in diverse applicazioni metabolomiche. Infatti, è in grado di discriminare tra campioni di specie diverse, di rilevare variazioni metaboliche durante il processo termico industriale e processi di degradazione o ossidazione, ed evidenziare frodi e sofisticazioni. D'altra parte, nel caso degli studi conformazionali, gli esempi riportati hanno dimostrato ancora una volta che la spettroscopia NMR in fase debolmente orientante, combinata con l'approccio AP-DPD, è un valido strumento per indagare la struttura e l'equilibrio conformazionale di farmaci antiinfiammatori e in particolare consentono di studiare le caratteristiche chirali delle specie. I risultati hanno dimostrato che lo studio metabolomico via NMR ha la potenzialità di essere applicato nella caratterizzazione di estratti naturali complessi e nel campo del controllo diConclusions généralesL'objectif de ce projet doctoral, mené conjointement entre l'Université de Calabre et l'Université Paris Saclay, était d'explorer différentes applications de la spectroscopie RMN pour l'analyse métabolomique de matrices naturelles et dans le domaine pharmacologique. Aujourd'hui, la spectroscopie RMN est couramment utilisée dans divers secteurs et parmi ces applications, le domaine de la métabolomique occupe certainement une place importante. Les métabolites présents dans un système biologique donné sont le résultat de processus internes déterminés génétiquement mais sont également fortement influencés par des facteurs environnementaux externes. Pour ces raisons, l'étude de toutes ces petites molécules, substrats, intermédiaires et produits du métabolisme cellulaire, fournit des informations importantes sur l'organisme étudié (mécanismes cellulaires, mutations, vieillissement, alimentation, stress, pathologies, origine géographique). De plus, chaque métabolite est une molécule bioactive capable d'interagir avec des ligands et des récepteurs endogènes. Ces interactions sont prédéterminées par la configuration et les conformations des espèces flexibles impliquées, ce qui justifie l'importance d'effectuer des études conformationnelles et des discriminations configurationnelles des espèces flexibles bioactives en parallèle avec la caractérisation du métabolome lui-même. Dans le premier chapitre de cette thèse de doctorat, les bases théoriques de la spectroscopie RMN ont été exposées et son application dans les deux domaines de recherche explorés au cours du projet a été illustrée, à savoir la caractérisation métabolomique de matrices ou d'extraits naturels et l'étude orientationnelle, structurale et conformationnelle de petites molécules chirales flexibles biologiquement actives. En particulier, après la description du projet et de ses principaux objectifs, un bref aperçu des principes de base de la spectroscopie RMN a été fourni dans le premier chapitre. Le chapitre s'est poursuivi en dessinant une description générale de la métabolomique et des approches scientifiques couramment utilisées, puis les principales plateformes d'analyse et les outils de traitement nécessaires pour effectuer l'analyse des données ont été présentés. Le chapitre se termine par la description de la méthodologie RMN utilisée pour obtenir des informations conformationnelles et configurationnelles relatives à un soluté flexible dissous dans des milieux chiraux partiellement orientés en utilisant des couplages dipolaires expérimentaux (RDCs). Plus précisément, un aperçu des outils de base de la spectroscopie RMN en milieu partiellement orienté a été fourni, les séquences d'impulsions capables de fournir les RDCs nécessaires à ce type d'étude et le modèle théorique adopté pour obtenir les informations structurales, orientationnelles et conformationnelles à partir des observables expérimentales ont été présentées. Dans le deuxième chapitre, les applications de la RMN dans le domaine de la métabolomique explorées au cours de la thèse ont été présentées. Une première étude décrit l'utilisation de la spectroscopie RMN pour étudier le profil métabolique de jus d'orange et de clémentine de Calabre fais ainsi que pour suivre les changements métaboliques que subit un jus frais au cours du processus thermique industriel qui conduit à l'obtention du jus concentré. Les expériences classiques de corrélation 1D et 2D ainsi que les données de la littérature ont permis d'identifier 23 composés de faible poids moléculaire. Les spectres RMN 1 H de jus pressés, pasteurisés et concentrés ont ensuite été utilisés pour réaliser une étude multivariée (ACP) afin de distinguer les échantillons issus de trois procédés industriels. Des résultats obtenus, il vient que chaque processus est regroupé dans une région bien définie du graphique de score et, pour chaque groupe, seules de légères différences sont évidentes entre les différentes phases de production, à savoir le pressage, la pasteurisation et la concentration, il a été conclu que le procédé thermique industriel ne modifie pas le profil métabolique du jus de départ. L'analyse des loadings issues du traitement statistique a permis d'identifier les métabolites responsables du clustering et cela montre que, lors des phases de concentration, le teneur en éthanol diminue tandis que la teneur en sucre augmente dans les échantillons concentrés. De plus, ce type d'analyse a révélé que le jus de clémentine est plus riche en acides aminés et autres composés aliphatiques que le jus d'orange. Le deuxième exemple présenté dans ce chapitre est la caractérisation métabolique par RMN des jus de pomme de Calabre. En particulier, ce travail visait la caractérisation métabolique par RMN, de matrices alimentaires complexes telles que des jus de pomme de quatre variétés différentes. Les séquences d'impulsions classiques 1D et 2D ont été utilisées avec les données reportées dans la littérature pour identifier 11 composés de faible poids moléculaire présents dans les quatre variétés de jus de pomme étudiées. De plus, la combinaison des spectres RMN 1 H obtenus avec l'analyse statistique des données (PCA) s'est révélée être un moyen de distinguer les échantillons des quatre variétés de pommes. En fait, dans le graphique de scores, il est possible de mettre en évidence que les variétés Golden Delicious, Fuji et Royal Gala ont des compositions métaboliques plus similaires entre elles que les jus de la variété Pink Lady qui apparaissent bien regroupés et différenciés. L'analyse des loadings a plutôt montré que les sucres et l'acide malique sont les composés responsables de la séparation entre les quatre variétés: pour cette raison, ces molecules peuvent être définies comme des composés marqueurs pertinents. La troisième étude présentée concerne l'étude du profil métabolique des extraits de Calabrese Cannabis Sativa à l'aide de techniques de RMN mono et bidimensionnelles. L'huile de graines de chanvre et les extraits d'inflorescence étudiés ont été obtenus par extraction solide-liquide, extraction réalisée à l'aide d'éthanol comme solvant d'extraction et sous d'ultrasons. La caractérisation du spectre 1 H de l'huile de graines de chanvre a permis de reconnaitre le profil typique des triglycérides (TAG). Quant à l'extrait de fleurs, il a été possible de reconnaître trois cannabinoïdes, CBD, CBDA et CBG, ainsi que le profil triglycérique. C'est un résultat important car le Cannabis Sativa est connu pour être riche en acides gras avec un ratio ω6/ω3 de 3:1. Ce rapport représente une excellente valeur nutritionnelle et la possibilité d'utiliser une technique d'extraction capable de fournir des extraits riches en ces deux composants mérite d'être explorée au cours d'études complémentaires. De plus, le fait qu'aucun signal attribué au THC n'ait été reconnu indique que la concentration de cette substance est inférieure à la limite de détection de la spectroscopie RMN et donc également inférieure à la limite légale. Cette étude préliminaire a été comparée aux résultats présents dans la littérature et nous a permis de conclure que notre méthode d'extraction est prometteuse puisqu'elle permet d'obtenir des extraits riches en triglycérides et en cannabinoïdes : ce résultat pourrait représenter un tournant clés dans la production d'aliments fonctionnels. La quatrième application présentée concerne l'utilisation de la spectroscopie RMN pour étudier, pour la première fois, la composition métabolique d'un échantillon d'huile essentielle de bergamote (BEO) pure et certifiée sans recourir à aucun procédé de manipulation ou de dérivatisation. Plus précisément, les principaux objectifs de cette étude étaient, d'une part, d'effectuer la caractérisation et la quantification des principaux métabolites présents dans cette matrice complexe, et, d'autre part, d'explorer la possibilité d'enquêter et de discriminer toute fraude et sophistication à dont le BEO aurait pu faire l'objet. En combinant les spectres classiques 1D et 2D avec les expériences DOSY 2D 1 H, il a été possible d'identifier 11 composés de bas poids moléculaire présents dans l'échantillon étudié. De plus, environ 90 % de l'échantillon a pu être quantifié en utilisant l'acide benzoïque comme étalon interne. Enfin, comme les adultérations ne sont pas rares en raison de la valeur élevée du BEO, la faisabilité d'utiliser trois approches RMN pour détecter d'éventuelles fraudes a été évaluée. Cette étude a été menée en simulant l'adultération par dilution en ajoutant différentes quantités d'huile essentielle d'orange à de l'huile BEO pur. Des études de diffusion, une analyse quantitative et une étude statistique ont été utilisées pour étudier cette modification. Alors que l'étude de diffusion ne permet pas de mettre en évidence la fraude sauf en cas de fort degré d'adultération, l'étude quantitative semble avoir une plus grande sensibilité dans la reconnaissance des huiles diluées. Dans tous les cas, la technique qui permet le mieux de distinguer les différents degrés d'adultération et est en mesure de fournir le plus d'informations résulte de la combinaison de spectres RMN 1 H monodimensionnels avec des outils d'analyse statistique multivariée. Le dernier exemple présenté dans ce chapitre concerne l'application de la technique RMN HR-MAS à l'étude de la fraction organique des déchets solides municipaux. En particulier, il a été montré que la spectroscopie RMN HR-MAS est une méthode innovante et non-destructive capable de caractériser la composition chimique de la fraction organique des déchets solides municipaux et est un outil adéquat pour suivre les variations en termes de composition des matières organiques initiales soumises à des traitements d'oxydation. La valeur ajoutée de l'approche HR-MAS 13 C RMN par rapport à la méthode CP-MAS, technique la plus couramment utilisée pour analyser la fraction organique des déchets, repose sur la possibilité d'obtenir des spectres bien plus résolus dont l'analyse contribue à une meilleure compréhension de la nature chimique de ces déchets complexes. Par ailleurs, cette approche HR-MAS s'est également révélée adaptée au suivi des processus oxydatifs fondés sur la réaction de Fenton auxquels la matrice initiale a été soumise. Les résultats obtenus mettent en effet en évidence les modifications qualitatives et quantitatives des différents types de atomes de carbone qui composent les molécules dans l'échantillon, étudié en fonction à la fois des concentrations des réactifs de Fenton et de la durée du processus oxydatif. À notre connaissance, cette étude de cas représente le premier exemple dans lequel l'approche HR-MAS a été appliquée avec succès aux fractions organiques des déchets solides municipaux. Enfin, les avantages en termes de rapidité, de simplicité et de diversité des informations obtenables rendent cette technique très intéressante pour des études ultérieures sur différentes matrices de déchets et également pour le suivi d'autres processus de dégradation/transformation auxquels peuvent être soumis les déchets organiques. Le troisième chapitre a permis de reporter les résultats obtenus à partir de l'application de la spectroscopie RMN dans le second domaine de recherche étudié. En particulier, au fil des années, les applications RMN dans les phases chirales faiblement orientantes se sont révélées largement capables de fournir des RDCs qui, traités avec un modèle théorique approprié, conduisent à l'obtention d'informations structurales, conformationnelles et orientationnelles de molécules flexibles. Cette méthodologie a été appliquée dans ce chapitre à une paire d'énantiomères afin d'étudier leurs conformations individuelles et de comprendre les différentes interactions soluté-solvant que les deux énantiomères forment dans un environnement chiral. En particulier, la première étude présentée dans ce chapitre fait référence à un cas très simple, constitué des énantiomères de la molécule d'alcool 4-fluoro-α-méthylbenzylique (4FMBOH), une molécule qui ne possède qu'un seul degré de liberté torsionnel. L'exemple rapporté dans cette section a démontré le pouvoir unique de la RMN dans les phases cristal-liquides fin d'explorer des équilibres conformationnels moléculaires et de mettre en évidence, pour la adultérations. D'autre part, dans le cas des études conformationnelles, les exemples reportés ont montré une fois de plus que la spectroscopie RMN dans les cristaux liquides faiblement orientants, combinée à l'approche AP-DPD, est un outil pertinent pour étudier la structure et l'équilibre conformationnel des médicaments anti-inflammatoires ; ils permettent notamment d'étudier les caractéristiques chirales de l'espèce. Les résultats ont démontré que l'étude métabolomique par RMN a le potentiel d'être appliquée dans la caractérisation d'extraits naturels complexes et dans le domaine du contrôle-qualité industriel. De plus, il s'agit d'une technique puissante qui peut être appliquée dans l'étude d'espèces complexes, chirales et flexibles permettant d'ajouter de nouvelles informations pour comprendre le mécanisme d'action des médicaments et les différentes activités biologiques de couples d'énantiomères, ouvrant ainsi la voie à de nouvelles connaissances.

Figure A3 :

 A3 Figure A3: 1D 1 H NMR spectrum (Bruker pulse sequence: zg) recorded on -terpinene in CDCl3 (9.4 T).

Figure A4 :

 A4 Figure A4: 13 C-{ 1 H} NMR spectrum (Bruker pulse sequence: zgdc) recorded on -terpinene in CDCl3 (11,7 T).

Figure A5 :

 A5 Figure A5: 1D 1 H NMR spectrum (Bruker pulse sequence: zg) recorded on linalool in CDCl3 (9.4 T).

Figure A6 :

 A6 Figure A6: 13 C-{ 1 H} NMR spectrum (Bruker pulse sequence: zgdc) recorded on linalool in CDCl3 (11,7 T).

Figure A7 :

 A7 Figure A7: 1D 1 H NMR spectrum (Bruker pulse sequence: zg) recorded on limonene in CDCl3 (9.4 T).

Figure A8 :

 A8 Figure A8: 13 C-{ 1 H} NMR spectrum (Bruker pulse sequence: zgdc) recorded limonene in CDCl3 (11,7 T).

Figure A9 :

 A9 Figure A9: 1D 1 H NMR spectrum (Bruker pulse sequence: zgdc) recorded on linalyl acetate in CDCl3 (9,4 T).

  In questo contesto, un ruolo particolarmente importante è rappresentato dall'applicazione della spettroscopia NMR per lo studio di matrici complesse quali alimenti e matrici/estratti naturali in genere. Di rilevante interesse è anche il ricorso a tale tecnica per lo studio di quelli che vengono definiti composti nutraceutici, ovvero sostanze che oltre ad avere un semplice valore nutrizionale, determinano effetti positivi sul benessere e sulla salute delle persone. Nello specifico da un lato risulta essere di fondamentale interesse individuare le specie nutraceutiche e riconoscerne la presenza

all'interno di un determinato alimento, dall'altro risulta essere molto importante effettuare la caratterizzazione, la determinazione strutturale e comprendere il comportamento conformazionale in soluzione delle molecole biologicamente attive con lo scopo di stabilire le relazioni tra configurazione/conformazione e bioattività. Nell'ambito delle scienze alimentari, la caratterizzazione di cibi è un argomento molto impegnativo. Per perseguire tale obbiettivo l'approccio più adatto risulta essere la metabolomica

(Kell & Oliver, 2016)

, disciplina scientifica in grado di identificare e quantificare i diversi metaboliti che determinano e caratterizzano il bio-sistema studiato. Essa si basa sull'identificazione globale di un elevato numero di metaboliti presenti in una matrice complessa quale un alimento; questo consente di caratterizzare il profilo metabolico e permette di identificare quali metaboliti o pattern di metaboliti possono essere utili nella discriminazione tra differenti gruppi di studio. Il profilo metabolico ottenuto costituisce una "impronta digitale" per il biosistema studiato e può essere utilizzato per la sua univoca identificazione. Infatti, l'identificazione di metaboliti o pattern di metaboliti è in grado di diversificare un determinato alimento e di discriminarne le caratteristiche geografiche, cultivar o di lavorazione, rispetto ad altri. Le tecniche più comuni impiegate per determinare il profilo metabolico dei prodotti alimentari sono la Spettrometria di Massa e la Spettroscopia di Risonanza Magnetica Nucleare (NMR) coadiuvate da strumenti di analisi statistica multivariata.

  valutare la possibilità di utilizzare questa tecnica come strumento di controllo della qualità nei processi industriali. Il secondo esempio presentato riguarda l'applicazione della spettroscopia NMR nella caratterizzazione del profilo metabolico di succhi di mela calabrese provenienti da diverse cultivar al fine di valutare se è possibile distinguere i diversi tipi di frutti sulla base del loro spettro protonico. Il capitolo prosegue con uno studio preliminare effettuato sugli estratti di Cannabis Sativa. In particolare, gli estratti di olio di semi di canapa e di infiorescenze vengono studiati mediante esperimenti NMR in modo da caratterizzare tali miscele complesse e fornire informazioni su questi estratti in modo da ottimizzare la tecnica di estrazione che potenzialmente potrebbe rappresentare una svolta nella produzione di alimenti funzionali. Il quarto esempio presentato è lo studio del campione

). Vengono quindi presentate le sequenze di impulsi utilizzate per l'estrazione delle RDC necessarie insieme al modello teorico adottato che consente di ottenere le informazioni strutturali, orientative e conformazionali dalle osservabili sperimentali.

Il secondo capitolo si concentra sulle applicazioni NMR nel campo della metabolomica esplorate durante questo dottorato e, in particolare, il capitolo inizia con la caratterizzazione del profilo metabolico dei succhi di arancia e clementina Calabresi. L'obiettivo principale di questo studio è valutare la possibilità di seguire eventuali variazioni metaboliche che il succo appena spremuto subisce durante il processo termico comunemente utilizzato allo scopo di ottenere succhi concentrati e certificato di olio essenziale di bergamotto (BEO). Gli obiettivi principali di questo studio sono stati la caratterizzazione e la quantificazione dei principali metaboliti presenti nella matrice complessa e lo studio della possibilità di discriminare frodi e sofisticazioni a cui questo estratto di alto valore può essere soggetto. Il secondo capitolo si conclude con l'applicazione della tecnica HR-MAS NMR nel campo metabolomico. In particolare, la tecnica viene utilizzata come metodo innovativo e non distruttivo in grado di caratterizzare la composizione chimica della frazione organica dei rifiuti solidi urbani e verificare se rappresenta un valido strumento nel monitoraggio delle variazioni in termini di composizione del materiale organico sottoposto a trattamenti di ossidazione. Nel terzo capitolo l'attenzione è spostata sullo studio conformazionale di specie flessibili. In particolare, poiché l'interazione di ogni molecola bioattiva con ligandi e recettori endogeni è dipendente dalla configurazione e dalle conformazioni della specie flessibile coinvolta, in questa sezione è stata concentrata l'attenzione sulla possibilità di distinguere una coppia di enantiomeri in base alle loro curve di probabilità conformazionale sperimentale. Lo studio NMR in mezzi chirali debolmente ordinanti è stato quindi applicato a una prima coppia di enantiomeri caratterizzati dalla presenza di un solo angolo torsionale. Le specie (RS)-4-fluoro-α-metilbenzil alcol ((RS)-4FMBOH) sono state studiate per esplorare i loro equilibri conformazionali molecolari e lasciare emergere differenze tra le curve di distribuzione di probabilità conformazionale e tra i parametri di ordine orientativo relativi ai due enantiomeri. Poiché la chiralità diventa un problema cruciale quando si tratta di specie bioattive, il secondo studio presentato in quest'ultimo capitolo è relativo al caso dell'acido tiaprofenico. L'acido tiaprofenico è una specie strutturalmente più complessa di cui un solo enantiomero è biologicamente attivo come farmaco antinfiammatorio. L'obiettivo principale dello studio presentato è stato dunque capire se la metodologia da noi adottata fosse in grado di fornire informazioni conformazionali in soluzione di specie bioattive chirali

Introduction générale

  Au fil des ans, la spectroscopie RMN a acquis une place de choix dans tous les secteurs des sciences et technologies ; il s'agit aujourd'hui d'un outil inégalé dans l'étude d'un grand nombre de systèmes chimiques, physiques et biologiques. Dans ce contexte, l'application de la spectroscopie RMN joue un rȏle particularment important pour l'étude de matrices complexes telles que les aliments et les extraits naturels. C'est aussi une technique d'un grand intérêt pour l'étude de molécules nutraceutiques, c'est-à-dire des substances qui, en plus d'avoir une simple valeur nutritive, ont des effets positifs sur le bien-être et la santé des être vivants. Concrètement, d'une part, il est d'un intérêt fondamental d'identifier les espèces nutraceutiques et de reconnaître leur présence au sein d'un aliment donné; d'autre part, il est très important de réaliser la

caractérisation, la détermination structurale et de comprendre le comportement conformationnel en solution des molécules biologiquement actives afin d'établir les relations entre configuration/conformation et bioactivité (structure-activité). Dans le contexte des sciences alimentaires, la caractérisation des aliments est un sujet très difficile. À cette fin, l'approche metabolomique semble la plus adaptée (Kell & Oliver, 2016). Il s'agit d'une discipline scientifique capable d'identifier et de quantifier les différents métabolites qui constituent et caractérisent le biosystème étudié. Elle repose sur l'identification globale d'un grand nombre de métabolites présents dans une matrice complexe telle qu'un aliment; cela permet de caractériser le profil métabolique et permet d'identifier les métabolites ou modèles de métabolites utiles dans la discrimination entre différents groupes d'étude. Le profil métabolique obtenu constitue une "empreinte digitale" du biosystème étudié et peut être utilisé pour son identification unique. Tout cela est rendu possible grâce à l'analyse des données spectroscopiques à l'aide d'outils d'analyse statistique multivariée et, plus précisément, la technique de RMN s'est avérée adaptée, et dans certains cas indispensable

  intramoléculaires qui déterminent les conformations que peut prendre la molécule dans le milieu réactionnel donné ou au sein des systèmes biologiques. Cela est dû au fait que, se référant au modèle "clé -serrure", une certaine enzyme est capable de distinguer à la fois une paire d'énantiomères et les différentes conformations qu'une molécule peut adopter et donc chaque conformère accessible thermiquement joue un rôle important dans les mécanismes de reconnaissance moléculaire(Boehr et al, 2019). On en déduit ainsi des informations sur les équilibres conformationnels des espèces bioactives jouant un rôle fondamental dans la réalisation de certaines actions biologiques. La connaissance de ces équilibres est indispensable, d'une part, pour comprendre le mécanisme d'action de l'espèce étudiée et d'autre part, pour la conception rationnelle de composés analogues capable de déclencher la même réponse être décrits en combinant l'utilisation de séquences d'impulsions traditionnelles ou plus sophistiquées avec des outils chimiométriques et des modèles théoriques pour l'analyse des données. En particulier, la spectroscopie RMN a été appliquée à l'étude métabolique de matrices alimentaires dans le but d'utiliser les informations obtenues dans la caractérisation et la traçabilité d'un produit alimentaire, dans le suivi du profil métabolique au cours des phases de transformation et pour l'étude structurelle, d'orientation et conformationnelle de petits énantiomères flexibles d'intérêt pharmacologique. Le premier chapitre de cette thèse présente une description du projet, ses principaux objectifs et illustre les aspects théoriques relatifs aux sujets abordés. Plus précisément, ce chapitre commence par un bref aperçu des principes de base de la spectroscopie RMN, puis fournit une En particulier, des extraits d'huile de graines de chanvre et d'inflorescences sont étudiés par des expériences RMN pour caractériser de tels mélanges complexes et fournir des informations sur ces extraits afin d'optimiser la technique d'extraction qui pourrait potentiellement représenter une percée dans la production d'aliments fonctionnels. Le quatrième exemple présenté est l'étude d'échantillons d'huile essentielle de bergamote certifiée (BEO). Les principaux objectifs de cette étude sont la caractérisation et la quantification des principaux métabolites présents dans la matrice complexe et d'explorer la possibilité de discriminer la fraude et l'adultération auxquelles cet extrait de haute valeur peut faire l'objet. Le deuxième chapitre se termine par l'application de la technique RMN HR-MAS (spectroscopie RMN haute résolution en rotation à l'angle Magique) dans le domaine

	puisque les mouvements de torsion internes interconvertissent les conformères possibles plus
	rapidement que ceux de l'expérience RMN, il est nécessaire de recourir à des modèles
	théoriques afin d'interpréter et de convertir correctement les RDCs et d'avoir accès aux
	conformations structurales, orientationnelles et conformationnelles de l'espèce examinée (Di
	Pietro et al, 2018).

particulier, sa bioactivité globale est une fonction non-triviale de celle de chaque métabolite individuel. L'activité d'un métabolite donné dépend à la fois de la configuration de des centres stéréogènes, s'ils sont présents au sein de sa structure, et de la disposition spatiale des atomes au sein de la molécule. Cette disposition peut favoriser ou non l'établissement d'une série d'interactions biologique. Les techniques à utiliser pour effectuer une analyse structurale et conformationnelle des espèces en solution sont très peu nombreuses et dans ce contexte la spectroscopie par résonance magnétique nucléaire représente un outil analytique puissant. En particulier, les progrès des méthodes RMN, fondées sur le développement de séquences d'impulsions plus ou moins sophistiquées, permettent l'étude de tels composés aussi bien en solution que dans des solvents anisotropes. L'utilisation de phases partiellement orientantes dans lesquelles la molécule cible est dissoute et est affectée par les interactions solute-solvent en s'orientant de manière appropriée par rapport au champ magnétique de la RMN est particulièrement pertinente

(Canet, 1996; Burnell & de Lange, 2003)

. L'anisotropie du solvent cristal liquide, permet d'obtenir des informations relatives à des interactions dont l'existence est strictement liée à la disposition spatiale des noyaux observés. En effet, les spectres RMN d'échantillons dans ces phases sont extrêmement riches en informations car régis par des interactions de nature anisotrope. Dans ce contexte, les couplages dipolaires résiduels (RDC) qui dépendent notamment des distances entre noyaux magnétiquement actifs et des orientations des vecteurs internucléaires par rapport au champ magnétique Zeeman prennent une importance particulière et, à ce titre, représentent une sonde sensibles aux interactions à longue distance, de portions de la molécule spatialement distantes les unes des autres. Dans le cas des molécules flexibles, C'est dans ce contexte que se développe ce projet doctoral. Plus précisément, l'objectif général de la recherche menée est l'application de la spectroscopie RMN dans les domaines de recherche qui viennent d'description générale de la métabolomique. Les différentes approches scientifiques couramment adoptées en métabolomique sont présentées ainsi que les principales plateformes analytiques utilisées dans ce type d'études, en se concentrant sur la spectroscopie RMN et les séquences d'impulsions couramment utilisées. Les outils de traitement spectral et de traitement des données sont ensuite présentés ainsi qu'une brève introduction à l'outil chimiométrique utilisé dans cette thèse, à savoir l'Analyse en Composantes Principales (PCA). Le premier chapitre se termine par la présentation de la méthodologie RMN utilisée pour obtenir des informations conformationnelles et configurationnelles liées à des solutés flexibles dissous dans des milieux chiraux partiellement orientés à l'aide de couplages dipolaires expérimentaux (RDCs). En particulier, un aperçu des outils RMN de base en milieu partiellement orienté est fourni en se concentrant sur la phase cristalline liquide lyotrope utilisée dans ce projet (PBLG / CDCl3). Les séquences d'impulsions capables de fournir les RDCs nécessaires sont ensuite présentées ainsi que le modèle théorique adopté qui permet d'obtenir les informations structurelles, d'orientation et de conformation à partir des observables expérimentales. Le deuxième chapitre porte sur les applications de la RMN dans le domaine de la métabolomique explorées au cours de cette thèse. En particulier, le chapitre débute par la caractérisation du profil métabolique de jus d'oranges et de clémentines de Calabre. L'objectif principal de cette étude est d'évaluer la possibilité de suivre les éventuelles variations métaboliques que subit le jus frais au cours du procédé thermique industriel afin d'obtenir des jus concentrés et d'évaluer la possibilité d'utiliser cette technique comme outil de contrôle qualité dans les procédés industriels. Le deuxième exemple présenté concerne l'application de la spectroscopie RMN dans la caractérisation du profil métabolique des jus de pomme de Calabre de différents cultivateurs afin d'évaluer la possibilité de distinguer différents types de fruits sur la base de leur spectre RMN proton. Le chapitre se poursuit par une étude préliminaire réalisée sur des extraits de Cannabis Sativa. métabolomique. En particulier, la technique est utilisée comme une méthode innovante et non destructive capable de caractériser la composition chimique de la fraction organique des déchets solides municipaux et de vérifier si elle représente un outil viable e pour surveiller les variations en termes de composition de la matière organique soumise à des traitements d'oxydation.

Table 2 .

 2 1. 1 H and 13 C chemical shifts of compounds in Calabrian squeezed fresh orange juice.

	(s=singlet; d=doublet; m=multiplet; o=overlapping signals).
	Compound	Group	1 H  (ppm)	13 C  (ppm)
	Valine	4 (CH3)	0.98 (d, J= 7.1 Hz)	17.43
		5 (CH3)	1.03 (d, J = 7.00 Hz)

Table 2 .

 2 2. 1 H and 13 C chemical shifts and multiplicity of compounds in apple juice of Golden Delicious variety (s=singlet; d=doublet; m=multiplet).

	Compound	Group	1 H δ (ppm)	δ 13 C (ppm)
	Ethanol
		(CH3)	1.17 (t, J=7.1 Hz)
	L-rhamnitol
		6 (CH3)	1.27 (t, J=6.4 Hz)
	Citramalic acid
		5 (CH3)	1.42 (s)
	Quinic acid
		3a (CH2)	1.89 (m)
		3b (CH2)	2.12 (m)
		7a (CH2)	1.89 (m)
		7b (CH2)	2.12 (m)

Table 2 .

 2 3: 1 H chemical shifts of compounds in hemp seeds oil (s=singlet; d=doublet; m=multiplet; o=overlapping signals).

	Compound	Group	1 H  (ppm)
		HA -CH2COOH	2.27-2.37 (m)
		HB	1.55-1.67 (m)
		-CH2CH2COOH
		HC	1.23-1.39 (m)
		-(CH2)n -
		HD	1.98-2.11 (m)
	Triacylglycerols	-CH2CH=CH-
		HE
		-CH=CH-	5.28-5.42 (m)
		HF	2.76 (t, J=6.5Hz)
		-CH=CHCH2CH=CH-	2.80 (t, J=5.5Hz)
		H G ω3	0.97 (t, J=7.5Hz)
		CH=CHCH2CH3
		H G ω6	t, 0.88 (t, J=7.3Hz)
		-CH2CH2CH2CH3
		H 𝐺	t, 0.89 (t, J=7.3Hz)
		-CH2CH2CH2CH3
		HH, L
		ROCH2CH(OR)CH2OR

Table 2 .

 2 4: 1 H chemical shifts of cannabinoids present in hemp inflorescences extract (s=singlet; d=doublet; m=multiplet).

	Compound	Group
		5''	0.86-0.88 (m)
		3''	1.27-1.32 (m)
		4''	1.27-1.32 (m)
		2''	1.52-1.61 (m)
		1"	2.43 (m)
		5'	6.16 (m)
		3'	6.26 (m)
		1	3.86 (ddt,
		2
		4a
		4b
		5
		6
		7
		9trans
		9cis
		10

1 H  (ppm)

13

C  (ppm)

Table 2 .

 2 5: 1 H and 13 C chemical shifts of compounds in BEO, concentration of the main metabolites and their diffusion coefficients. (s=singlet; d=doublet; m=multiplet).

	Compound (IUPAC Name)	Amount (%)	Diffusion coefficient (cm 2 /s)	Group	1 H  (ppm)	1 H -1 H COSY	13 C  (ppm)*
				1' a (CH2)	4.55 (m)	2.46 -H3 b 2.30 -H3 a
				1' b (CH2)	4.62 (m)	2.46 -H3 b 2.30 -H3 a	106.10-C3
				1 '' (CH3)	0.72 (s)
				1 ''' (CH3)	1.23 (s)		23.62-C6
				2 (-C-)			152.03-C2
				3 a (CH2)	2.30 (m)	2.46 -H3 b 1.82 -H4 b
	-pinene	4.9 ± 0.8	1.04×10 -5	3 b (CH2)	2.46 (m)	2.30 -H3 a 4.55 -H1 'a 4.62 -H1 'b 1.82 -H4 b	21.85-C3
						2.46 -H7 b
				7 a (CH2)	2.30 (m)	1.47 -H1
						1.93 -H5	26.13-C7
				7 b (CH2)	2.46 (m)	2.30 -H7 a
				4 a (CH2)	1.65 (m)	1.82 -H4 b
				4 b (CH2)	1.82 (m)	1.65 -H4 a 2.30 -H3 a	23.82-C4
						2.46 -H3 b
				5 (CH)	1.93 (m)	2.30 -H7 a	27.00-C5
				1 (CH)	1.47 (m)	2.30 -H7 a	51.83-C1
				6 (-C-)			40.4-C6
				1(-C-)			131.15-C1
	-terpinene			6(CH2)	2.60 (m)	5.43 -H5 1.66 -H7	27.60-C6
				5(CH)	5.43(m)	2.60 -H6	118.94-C5
				4(-C-)			140.56-C4
		11.6 ± 0.8 1.01×10 -5	3(CH2)	2.60 (m)	5.43 -H2	31.71-C3
				2(CH)	5.43 (m)	2.60 -H3	116.06-C2
				2'(CH)	2.20 (m)	1.01 -H1 ' 1.01 -H3 '	34.62-C2'
				1 ' (CH3) 3 ' (CH3)	1.01 (d, J=7.2 Hz)	2.20 -H2 '	21.34-C1'-C3'

  Table and on the right the diffusion coefficients obtained are plotted against the concentration in BEO of the studied samples.

		Model	Model	Model
	mixture II	mixture III	mixture IV
	Linalool	10.2%	10.6%	10.6%
	Linalyl acetate	32.8%	32.4%	31.7%		Limonene diffusion coefficient (cm 2 /s)
	Limonene	30.5%	30.4%	30.7%	Model mixture II	(1.35 ± 0.01) × 10 -5
	-terpinene	11.4%	11.7%	11.8%	Model mixture III	(1.51 ± 0.02) × 10 -5
	-pinene	4.9%	4.8%	4.9%	Model mixture IV	(1.56 ± 0.02) × 10 -5
	n-decane	10.2%
	n-hexane		10.1%
	Cyclohexane			10.3%
	Solutions	Diffusion Coefficient (cm 2 /s)
	Pure BEO	(1.15 ± 0.02) × 10 -5

Table 2 . 7 :

 27 Values of integrals expressed as percentages, and their errors, calculate for the four group of carbons (aliphatic, heteroatoms-linked, unsaturated and carbonylic) for the Samples 0.2.6.3. Oxidative treatment monitoringIn order to understand if the HR-MAS method is able to appreciate the possible chemical differences among the initial organic substrate and the products after the oxidative process, three samples, Sample 1, Sample 2 and Sample 3 were analyzed. The three samples are related to oxidative processes in which the initial OFMSW matrix was treated with the same amount of FeSO4 but with different quantities of H2O2. It is worth noting that the carbon spectra of the Sample 1, Sample 2 and Sample 3, have the same spectral profile of Sample 0 and not substantial variations in the chemical shifts values were observed in the spectra of the four samples. The integration process on the various types of carbon has been repeated for these samples and the results, reported in Table2.8 and illustrated in Figure2.44, were compared with the corresponding values obtained for Sample 0.

Table 2 . 8 :

 28 Values of integrals expressed as percentages, and their errors, calculate for the four group of carbons (aliphatic, heteroatoms-linked, unsaturated and carbonylic) for the Samples 0, 1, 2 and 3. Relative distribution in the assigned chemical shift regions of the four group of carbons (aliphatic, heteroatoms-linked, unsaturated and carbonylic) for the Samples 0, 1, 2 and 3.

	Figure 2.44:

Table 3 . 1 .

 31 Details about the preparation of the different samples of 4FMBOH studied.

	Sample	Composition	Concentration in PBLG
	4FMBOHiso	0.0380 g of R-4FMBOH + 0.6880 g of CDCl3	-
	R-4FMBOHaniso	0.0487 g of R-4FMBOH + 0.7190 g of CDCl3 + 0.0874 g of PBLG	10.2%wt
	S-4FMBOHaniso	0.0582 g of S-4FMBOH + 0.8767 g of CDCl3 + 0.1049 g of PBLG	10.1%wt
	Both enantiomers (99% pure), PBLG (150000 Da < MW < 350000 Da), and CDCl3 (99.96
	%D), were purchased from Aldrich and used for sample preparation without further
	purification. On the samples prepared 1 H, 13 C and 13 C-{ 1 H } NMR spectra were acquired at
	T=298 K on a 500 MHz Bruker Avance spectrometer (11.7 T) (Bruker, Fällanden,
	Switzerland) equipped with a 5 mm TBO probe and a standard variable-temperature unit
	BVT-3000. On these samples 2D correlation experiments were performed ( 1 H COSY and 1 H-
	13 C HMQC) for the correct attribution of the signals. From the extraction of all the coupling
	constants, in addition to the use of simple 1D NMR spectra, also 2D experiments such as
	homonuclear 1 H and heteronuclear 1 H-13 C J-resolved were performed together with the
	selective homonuclear and heteronuclear refocusing experiments, SERF and HetSERF. Once

  .32 and 1.33. A key point needs to be underlined: only the absolute values of the two coupling constants can be measured from spectra leading to an uncertainty on the signs and the magnitudes of RDCs. In the case of magnetically non-equivalent nuclei, since, by applying (1.32) four possible values of 𝐷 𝑖𝑗 (𝑜𝑏𝑠) are obtained if we consider all the possible combinations of 𝐽 𝑖𝑗 𝑖𝑠𝑜 and 𝑇 𝑖𝑗 . While for magnetically equivalent nuclei two values of 𝐷 𝑖𝑗 (𝑜𝑏𝑠) are obtained by considering the two possible signs of 𝑇 𝑖𝑗 . The sign of 𝐽 𝑖𝑗 𝑖𝑠𝑜 , in the case of 4FMBOH, has been assigned on the basis of data reported in the literature referring to similar molecules (Organic Chemistry Data Collection, 2021) and considering that the 1 JCH are known to be positive leading to only two possible values for 𝐷 𝑖𝑗 (𝑜𝑏𝑠)

	𝐽 𝑖𝑗 𝑖𝑠𝑜 , 𝑇 𝑖𝑗 𝑅 (𝑜𝑏𝑠) and 𝑇 𝑖𝑗 𝑆 (𝑜𝑏𝑠) have been measured. The 𝑅,𝑆 (𝑜𝑏𝑠) were calculated from the experimental 𝐽 𝑖𝑗 two sets of 𝐷 𝑖𝑗 𝑖𝑠𝑜 and 𝑇 𝑖𝑗 𝑅,𝑆 (𝑜𝑏𝑠) couplings using equations 1C3 + C5 4 TC3H8 R

1

. The determination

Table 3 . 2 .

 32 Experimental dipolar couplings 𝐷 𝑖𝑗 𝑜𝑏𝑠 determined by the NMR analysis of R and S 4FMBOH dissolved in PBLG/CDCl3 (the estimated experimental error affecting the data is ±0.5) and calculated dipolar couplings 𝐷 𝑖𝑗 𝑐𝑎𝑙𝑐 determined by AP-DPD approach (the estimated experimental error affecting the data is ±0.2), are reported in the Table together with all the output parameter obtained by performing the conformational study (i.e. potential, structural and orientational parameters).

			(R) FMBOH	(S) FMBOH
	i-j	𝑫 𝒊𝒋 𝒐𝒃𝒔 (Hz)	𝑫 𝒊𝒋 (Hz)	𝑫 𝒊𝒋 𝒐𝒃𝒔

Table 3 . 3 .

 33 Details about the preparation of the different samples of TA studied.

  From the spectra only the absolute values of the two coupling constants can be measured and this lead to an uncertainty on the signs and the magnitudes of RDCs. In the case of magnetically non-equivalent nuclei, since, by applying (1.32) four possible values of 𝐷 𝑖𝑗 (𝑜𝑏𝑠) are obtained if we consider all the possible combinations of 𝐽 𝑖𝑗 𝑖𝑠𝑜 and 𝑇 𝑖𝑗 . While for magnetically equivalent nuclei two values of 𝐷 𝑖𝑗 (𝑜𝑏𝑠) are obtained by considering the two possible signs of 𝑇 𝑖𝑗 . The sign of 𝐽 𝑖𝑗 𝑖𝑠𝑜 , in the case of TA, has been assigned on the basis of data reported in the literature referring to similar molecules (Organic Chemistry Data Collection, 2021) and considering that the 1 JCH are known to be positive leading to only two possible values for 𝐷 𝑖𝑗 (𝑜𝑏𝑠)

	sets of 𝐽 𝑖𝑗 𝑖𝑠𝑜 , 𝑇 𝑖𝑗 𝐴 (𝑜𝑏𝑠) and 𝑇 𝑖𝑗 𝐵 (𝑜𝑏𝑠) have been measured (21 𝑇 𝑖𝑗 (𝑜𝑏𝑠) for each species) . The
	two sets of 𝐷 𝑖𝑗 𝐴,𝐵 (𝑜𝑏𝑠) were calculated from the experimental 𝐽 𝑖𝑗 𝑖𝑠𝑜 and 𝑇 𝑖𝑗 𝐴,𝐵 (𝑜𝑏𝑠) couplings
	using equations 1.32 and 1.33. 1

  𝑜𝑏𝑠) are unknown. The determination of the right value of 𝐷 𝑖𝑗 (𝑜𝑏𝑠) was then performed by a trial and error process during the following conformational study. Indeed, after the RDCs have been calculated, the sets of experimental 𝐷 𝑖𝑗 𝐴,𝐵 (𝑜𝑏𝑠) have been exploited to investigate structure and conformational equilibrium of the two enantiomers. In particular, 15 𝐷 𝑖𝑗 𝐴 (𝑜𝑏𝑠)and 15 𝐷 𝑖𝑗 𝐵 (𝑜𝑏𝑠), together with 15 dipolar couplings not attributed to a specific species, are available for the conformational study of the investigated species. Among them, 5 RDCs are relating to the interactions between the thiophene rigid fragment and propionic portion. Since no interfragment RDCs have been measured for the two cooperative torsions (𝜓 and 𝜃) , only the conformational equilibrium around the angle 𝜙 has been investigated in this work.

	3.3.3. Conformational analysis
	The residual dipolar couplings 𝐷 𝑖𝑗 𝐴,𝐵 (𝑜𝑏𝑠) were calculated using equations 1.32 and 1.33 from
	the experimental 𝐽 𝑖𝑗 𝑖𝑠𝑜 and 𝑇 𝑖𝑗 𝐴,𝐵 (𝑜𝑏𝑠) couplings obtained from the spectral analysis previously
	discussed. Since the sign of 𝐽 𝑖𝑗 𝑖𝑠𝑜 can be assigned on the basis of data present in the literature
	referring to similar molecules (Organic Chemistry Data Collection, 2021), two different
	values of 𝐷 𝑖𝑗 (𝑜𝑏𝑠) were calculated for each coupling by considering that the signs of
	𝑇 𝑖𝑗 𝐴,𝐵 (	T C5-H2 ?

Table 3 .

 3 4. Experimental dipolar couplings 𝑫 𝒊𝒋 𝒐𝒃𝒔 determined by the NMR analysis of R-TA and S-TA dissolved in PBLG/CDCl3 (the estimated experimental error affecting the data is ±0.5) and calculated dipolar couplings 𝑫 𝒊𝒋 determined by AP-DPD approach (the estimated experimental error affecting the data is ±0.2), are reported in the Table together with all the output parameter obtained by performing the conformational study (i.e. potential, structural and orientational parameters).

			S-TA	R-TA
		i-j	𝑫 𝒊𝒋 𝒐𝒃𝒔 (𝑯𝒛)	𝑫 𝒊𝒋 (𝑯𝒛)	𝑫 𝒊𝒋 𝒐𝒃𝒔 (Hz)	𝑫 𝒊𝒋 (Hz)
	Propionic group
	C1-H2	-6.2	-6.2	5.4	8.2
	C2-H2	36.3	36.4	16.1	15.9
	C1-H3	3.4	1.9	0.2	-0.7
	C2H3	2.9	1.5	-1.3	-2.6
	C3-H3	-4,7	-5.3	10.0	10.0
	H2-H3	6.6	6.2	-7.8	-8.3
	H3-H3	-7.6	-7.8	-15.0	14.4
	Thiophenic group
	C5-H5	8.2	8.0	8.2	8.5
	C7-H5	1.5	0.5	1.5	0.6
	C6-H6	-52.8	-53.2	-52.8	-52.9
	C5-H6	-4.5	-5.1	-4.5	-4.1
	Interfragment
	C4-H2	4.7	5.5	-1.5	-1.8
	C6-H2	-1.4	-0.2	-1.4	-0.5
	C5-H3	-1.4	-0.2	-1.4	-0.9
	C1-H5	-1.7	-1.5	-1.7	-0.9
	C2-H5	-3.4	-2.6	-3.4	-2.7
	C3-H5	-1.5	0.0	-1.5	-1.6
	C4-H5	-3.9	-2.7	-4.7	-3.3
	C4-H3	-0.1	-0.9	-0.1	-1.1
	H2-H5	-15.4	-14.3	-10.0	-9.3
	H3-H5	4.0	2.9	-6.2	-5.1
	Parameter 𝜙 𝑚𝑎𝑥 1 (degree)	Optimized value 14 ±7	Optimized value 158±9
	ℎ 𝜙 1	(degree)	12(Parametrical value)	18 (Parametrical value)
	𝐴 𝜙 1 𝜙 𝑚𝑎𝑥 2 (degree)	0.7 202±9	0.3 346 ±7
	ℎ 𝜙 2	(degree)	18(Parametrical value)	12(Parametrical value)
	𝐴 𝜙 2	0.3		0.7
	RMS (Hz)	0.95	0.92

2.4.2 Signal assignments

OEO -Orange Essential Oil OFMSW -Organic Fraction of Municipal Solid Waste P -Pasteurized PAS STE -STimulated Echo TA-Tiaprofenic Acid (

The data were obtained from Web

The combination of 1 H-NMR spectra of squeezed, pasteurized and concentrated juices with Principal Component Analysis (PCA), permitted to successfully distinguished between the samples coming from the three industrial processes, indeed each process is clustered in one defined region. For each cluster, the PCA score plot shows that the thermal industrial process does not change the metabolic profile of the starting juice, highlighting only slight differences between the different production phases, which are more evident in orange juice mixed with clementines. Thanks to the loading analysis it was possible to identify the variable, i.e. the metabolites, responsible for the clustering. This shows, during the concentration phases, that for all the samples, the ethanol decreases and the sugar content increases in the concentrated samples. Therefore, the loading plot confirms that during the thermal process, the metabolic profile remains unchanged; there is no degradation or appearance of new metabolites, but the concentration of volatile compounds such as amino acids and other aliphatic components is reduced. Therefore, we can conclude that NMR technique, combined with statistical analysis tools, is able to discriminate among juice samples of different origin/species and to detect eventually metabolic variations during the thermal industrial process of concentrated juices.

Multivariate Analysis

The selection of the variable, needed for the PCA study, was achieved by choosing the NMR regions of characteristic compounds that most contribute to fruit juices: sugar and acid regions.

In particular, the integration of the proton spectrum was made considering chemical shift intervals of 0.05 ppm belonging to the following spectral windows: from 1.1 to 1.5 ppm that is the region of the ethanol, L-rhamnitol and citramalic acid methyl protons signals; from 1.9 to 2.25 ppm that is the region of quinic and chlorogenic acid aliphatic ring signals; from 2.6 to 3.0 ppm which includes the doublets of doublets related to the methylenic and methynic protons of malic acid; and finally from 3.15 to 4.25 ppm which is the sugar region. As can be seen, some regions have been excluded from the bucketing process: the interval from 4.30 to 5.10 ppm was omitted because of the residual water signal, and the interval from 5.6 to 10 ppm in which, due to the too low S/N, the signals are too low intensity to be significantly distinguishable from the background noise. A dataset of 20 samples (5 for each variety) and 51 variables has been obtained. The 3D score plot obtained is reported in Figure 2.16, where a cumulative percentage of explained data variance of 81.6% (52.3%, 22.2% and 7.1% for PC1, PC2 and PC3 respectively) summarizes the relationship between samples. The score plot shows a clear separation between the four varieties of apples. Specifically, the Pink Lady variety has negative values of PC2 while Royal Gala, Golden Delicious and Fuji have positive value of this component. This trend highlights that samples belonging to the Golden Delicious, Fuji and Royal Gala varieties have a more similar metabolic composition than the juices from the Pink Lady variety.

Hemp inflorescences extracts

In the case of inflorescences extract, the obtained spectra are more complex than the one obtained from the seeds oil. Indeed, as can be seen from the Figure 2.20, the superposition of the spectral lines makes it difficult to recognize metabolites through the single inspection of the 1D spectra. FIDs were accumulated by using a spectral width of 13.00 ppm and a relaxation delay of 5 s. B) 13 C-{ 1 H} NMR spectrum (zgig Bruker pulse sequence), performed with proton broad-band decoupling collecting 8K free induction decays (FIDs) using a spectral width of 200.00 ppm and a relaxation delay of 4s.1D NMR FIDs were Fourier-transformed, phased, baseline corrected and aligned using the TMS signal as reference. 13 C-{ 1 H} NMR spectra were filtered with 1 Hz line broadening before Fouriertransformation.

To overcome the limitations that are characteristic of the 1D spectra, the identification of hemp flowers constituents was performed by 2D NMR experiment and, in particular, 1 H COSY and 1 H-13 C HMQC were recorded. Thanks to these experiments, and by reference to literature data (Choi et al, 2004;Choi et al, 2004;[START_REF] Marchetti | Use of 13 C-qNMR Spectroscopy for the Analysis of Non-Psychoactive Cannabinoids in Fibre-Type Cannabis Sativa L. (Hemp)[END_REF], three cannabinoids were recognized in the spectra: CBD, CBDA, CBG. In addition, due to the absence of the THC signals and correlations, it was possible to assert that the amount of the psycotropic cannabinoid in the studied sample is lower than the limit of detection of NMR, i.e. 5-10 μM [START_REF] Eisenmann | Untargeted NMR spectroscopic analysis of the metabolic variety of new apple cultivars[END_REF], then it is lower that the law limits. In Figure 2.21 1 H COSY and 1 H-13 C HMQC are A B the spectra recorded on some single components or by literature reports [START_REF] Kolehmainen | Complete Spectral Analysis of the 1H NM 16-Spin System of -Pinene[END_REF][START_REF] Skold | Studies on the Autoxidation and Sensitizing Capaciti of the Fragrance Chemical Linalool, Identifying a Linalool Hydroperoxide[END_REF][START_REF] Wishart | HMDB: the Human Metabolome Database[END_REF][START_REF] Chunyan | Isolation and Purification of Psoralen and Bergapten from Ficus Carica L. Leaves by High-Speed Countercurrent Chromatography[END_REF][START_REF] Wishart | HMDB: a nowledgebase for the human metabolome[END_REF][START_REF] Wishart | HMDB 3.0 -The Human Metabolome Database in 2013[END_REF][START_REF] Wishart | HMDB 4.0 -The Human Metabolome Database for 2018[END_REF]Liu et al, 2017). 1 H-13 C HMQC spectra were recorded using a SW of 18.00 ppm ( 1 H) and 280.00 ppm ( 13 C), 2K datapoints, 512 scans and 32 experiments.

For example, the use of correlation experiments was fundamental in recognizing the individual signals of the four compounds that make up the majority of the non-volatile fraction, namely the two psoralens bergapten and bergamottin, and the two coumarins citropten and 5geranyloxy-7-methoxycumarin. Coumarins and furocoumarins (psoralen) have shown various biological effects: on the one hand they are active against diseases such as osteoporosis [START_REF] Chen | Psoralen Inhibited Apoptosis of Osteoporotic Osteoblasts by Modulating IRE1-ASK1-JNK Pathway[END_REF], since they possess antioxidant, anti-inflammatory [START_REF] Witaicenis | Antioxidant and intestinal Anti-Inflammatory Effects of Plant-Derive Coumarin Derivatives[END_REF] and anticancer properties [START_REF] Toshihiro | Chemical Aspects of Coumarin Compounds for the Prevention of Hepatocellular Carcinomas[END_REF]; on the other hand, however, especially furocoumarins in high concentrations, can induce skin disorders such as contact dermatitis and other allergic reactions, and in the worst cases they show phototoxicity (Kejlovà et al, 2007). available instrumentation and the nature of the system under study. When very small diffusion coefficients are involved, large gradient amplitudes are required to obtain adequate signal attenuation. As disturbances to the local field increase with the amplitude of the gradient pulse, the LED feature is often required. However, when only modest gradients are required and an efficient, well designed probe with active shielding is available, the STE sequence should be considered [START_REF] Russo | Bergamot (Citrus Bergamia Risso) as a Source of Nutraceuticals: Limonoids and Flavonoids[END_REF]. In the case of essential oils both sequences can be used, but in our case, the best results were obtained by selecting the LED sequence (ledgp2s Bruker standard pulses sequence). Next experimental step was the optimization of the experimental procedure and the acquisition parameters have been choosen in order to have the best attenuation of signals. All the values obtained from the optimization procedure, and used in all the diffusion studies presented in this thesis, are the following: the gradient amplitude (G I ) was changed from 0.8 G•cm -1 to 19.1 G•cm -1 using a 16-steps linear ramp. The diffusion time and the gradient pulse duration were set to = 0.2 s and = 0.0025 s, respectively.

In order to be able to precisely assess the self-diffusion coefficients values, temperature and gradient calibrations were carefully performed using respectively Bruker NMR standard samples (4% methanol in methanol-d4 and 80% ethylene glycol in DMSO-d6) and a homemade aqueous reference sample prepared with a very small quantity of hydrated sulfate copper (II) for limiting the sample longitudinal relaxation delay in the gradient calibration procedure.

Regarding the DOSY processing, the FIDs were apodized using an exponential line broadening of 1Hz prior to be Fourier-transformed. The diffusion coefficient was obtained plotting the intensities of the spectra against the gradient strength and by fitting the obtained curves by the equation (1.47). TopSpin 3.6 software (TopSpin, 2018) allowed the reconstruction of the DOSY spectrum with chemical shifts on the F2 axis and diffusion coefficients on the F1 axis. Each signal on the 2D map corresponds to the calculated diffusion coefficient, and the width of each signal on F1 dimension is correlated with the fitting error. Diffusion data were also dealt and controlled with Dynamic Center software (Dinamic Center) and best-fit agreement procedure using Excel solver macro.

Using this methodology the DOSY recorded on the (R)-Limonene/CDCl3 sample was obtained and the diffusion coefficient of Limonene was found to be (1.71 ± 0.01) × 10 -11 m 2 /s (Figure 2.27). Note that each diffusion experiment presented in this thesis has been executed in triplicate in order to estimate the error on each measurement by calculating the average deviation of the measures.

qNMR

Once the peaks of the main compounds present in the BEO were assigned, as described in the previous section, using the 1D 1 H NMR spectrum containing benzoic acid as internal standard and the equation (1.48), it was possible to quantify the most abundant metabolites, namely limonene, linalool, linalyl acetate, -pinene and -terpinene, and two other compounds, present in small quantities but whose signals are easily recognizable and integrable, namely geranial and neral. It should be noted that for these last two molecules, the quantification was carried out by integrating the signal of doublet corresponding to the aldehyde proton which resonates at 9.99 ppm for the geranial and at 9.89 ppm for the neral. These two signals are isolated in an area of the spectrum where there are no signals from other species and therefore they are easily recognizable and integrable. For the quantitative study preliminary pulses calibration and determination of spin-lattice relaxation times (T1) was required and performed by using the standard inversion recovery experiment (10 increments from 0.05 ms to 14 s). The 1 H qNMR spectra were recorded using a relaxation delay of 30 s and the parameters previously declared for the qualitative study. It is worth noting that, in order to obtain reliable quantitative values, data were collected in triplicate; i.e., on the sample containing the internal standard, three spectra were recorded and each of them processed three times. In Figure 2.34, the metabolites and standard signals integrated, for the quantitative study, are shown. The signals taken into consideration for benzoic acid are those corresponding to the protons H (C) and H (BB') and integrated together to avoid distortions in the integration since, as it can be seen in Figure 2.34, their resonance frequencies are very close. Due to the high complexity of the spectrum and, then, to the resulting overlap of different peaks, the signals considered for the integration process of the various metabolites were subjected to a preliminary deconvolution process, in order to obtain an accurate integration and quantification. As an example, Figure 2.34 shows the deconvolution process of the signals corresponding to linalool and linalyl acetate which are partially overlapped. The signals considered for these two metabolites are the doublet of doublets corresponding to the H2 proton of the double bond which resonate at 5.91 ppm for linalool and at 5.97 ppm for linalyl acetate. As it is evident in the Figure, the deconvolution process allows both signals to be well separated and therefore accurately integrated. The obtained quantitative information for the aforementioned metabolites, expressed as average percentage values, are reported in Table 2.5, together with the diffusion coefficients and the assignments of the peaks of the main compounds recognized in the BEO (the peaks of 1 H spectrum, are also reported in Figure 2.33). From the data given in the Table it can be inferred in the investigated sample. Moreover, by employing benzoic acid as an internal standard and using the 1 H NMR spectrum, we were able to quantify about the 90% of the sample, getting average percentage values for the most abundant metabolites, namely limonene, linalool, linalyl acetate, -pinene and -terpinene, comparable with the corresponding values obtained via other analytical procedures on BEO samples coming from three different seasons [START_REF] Dugo | Characterization Of Cold-Pressed And Processed Bergamot Oils By Using GC-FID, GC-MS, GC-C-IRMS, enantio-GC, MDGC, HPLC and HPLC-MS-IT-TOF[END_REF].

Finally, as adulterations are not uncommon due to the high price of the BEO and regulators should monitor market products, we investigated the possibility of using three NMR approaches in order to detect possible frauds, by simulating a dilution adulteration by adding different quantities of orange essential oil. Diffusion studies, quantitative analysis and statistical study were used with the aim of detect such manipulation. While the diffusion study allows to highlight the investigated fraud only in the case of high degrees of adulteration, the quantitative study seems to have a greater sensitivity in recognizing diluted oils. In any case, the technique that best allows to distinguish the various degree of adulteration and is able to fournish more information is given by the combination of one-dimensional proton spectra with multivariate analysis tools.The results obtained by combining 1 H NMR spectra, collected on the adulterated samples and on the pure BEO, with PCA proved that it possible to distinguish the adulterated samples from the pure BEO and to differentiate among the various percentages of adulteration and this approach also allows to recognize wich compounds can be defined as marker of adulteration. Thus, the combined use of experimental NMR spectra with multivariate data analysis appears to be a valid and the most powerful tool for investigating the quality and authenticity of BEO.

Samples preparation and experimental procedure

The samples were supplied from another unity of the Project and the experimental details of this process are outside this work, then only the highlights are reported below. The waste used for the Fenton treatment were obtained through a sampling procedure lasted 12 days. In each of these days waste were taken from a group of four different families, each day a different one.

The waste collected on each sampling day (about 5 kg), were mixed and milled at a millimeter size by means of a steel blender. The resulting minced was stored at -20 °C for 12 days. After this time, all the daily rates were defrosted and mixed together; the material obtained was divided into bags of 200 grams, re-frozen and then used as the starting matrix for the oxidative treatment. The oxidation reaction [START_REF] Koppenol | The Centennial of the Fenton eaction[END_REF] involves a thermal treatment of the organic matrix, followed by the use of hydrogen peroxide solution (H2O2) containing ferrous ions (typically Fe (II) sulphate, FeSO4). The samples used for the NMR experiments were collected during the phases of oxidation procedure and are differentiated according to the different percentages of H2O2/FeSO4 used. A first sample, called Sample 0, was collected from the batch of organic waste subjected to heat treatment (110 ° C for 48 hours). Three samples, called Sample 1, Sample 2, Sample 3, were collected treating waste for 12 hours with the same amount of FeSO4, 0.05%wt, but different concentration of H2O2, i.e. 0.002%wt, 0.0015%wt, and 0.0006%wt respectively. In order to understand the transformations that the waste undergoes during the oxidation process and to investigate the best experimental conditions, samples were collected at regular interval during two different process (process A and process B). For the process A, carried out by treating the waste with a solution of 0.002%wt of H2O2 and 0.01%wt of FeSO4, four samples were collected withdrawing substance from the reaction flask at regular 3-hour intervals, i.e. Sample 4_3h, Sample 4_6h, Sample 4_9h and Sample 4_12h. Other four samples were collected for the process B, carried out by adopting the condition of Sample 3 (0.0006%wt of H2O2 and 0.05%wt FeSO4), i.e. Sample5_3h, Sample 5_6h, Sample 5_9h and Sample 5_12h, always at 3 hour intervals. Moreover, another sample was collected for process B after a complete process of 8 hour, Sample 6_8h. In Figure 2.41 the experimental steps, reaction conditions and the NMR samples collected are reported.

Each sample analyzed shows different contents of C containing groups and these fluctuations cannot be attributed to the error in the evaluation of the integral area, but, evidently, they derive from the different oxidative processes. The greatest variations occur for the aliphatic zone and for the region concerning the carbons linked to heteroatoms. Indeed, the highest content of aliphatic C is found for the Sample 3, that is the sample obtained with the lowest amount of H2O2 (0.0006 %wt), while the highest content of C linked to heteroatoms is obtained for the Sample 1 in which the quantity of H2O2 was the highest (0.002 %wt). These results indicate that: a) the Fenton treatment is able to made changes in the initial OFMSW matrix; b) HR-MAS 13 C NMR spectroscopy is effective in appreciating the variations that individual families of compounds undergo following the different treatments, in terms of reagents concentration used.

The experiments just described show that the NMR HR-MAS method provides relevant information on the composition variations of the organic fraction of the waste subjected to the Fenton treatment.

In order to better understand what happens to the various families of compounds during the oxidative process and to investigate the best reaction conditions, the HR-MAS NMR technique has been applied to the study of two oxidative processes, A and B, carried out in different reaction conditions as described in section 2.6.1. A total of 9 samples were collected for the two processes, 4 for process A and 5 for process B. Also for these samples, no alterations were observed in the profile of the 13 C HR-MAS NMR spectra but there are marked variations in the areas of the values associated with each region of the spectra.

Results for process A are reported in Table 2.9 and illustrated in Figure 2.45, while results for process B are reported in Table 2.10 and illustrated in Figure 2.46, for both process the values of the integrated areas are compared with the corresponding ones of initial OFMSW matrix (Sample 0).

Process A.

Table 2.9: Values of 13 C HR-MAS NMR integrated area, expressed as percentages, and their errors, calculated for the four families of carbon (aliphatic, heteroatoms-linked, unsaturated and carbonylic) for each samples of process A (Sample 4_3h, Sample 4_6h, Sample 4_9h and Sample 4_12h) and comparison with corresponding values of Sample 0. From the data reported in Table 2.9 and Figure 2.45 it can be deduced that the reaction proceeds according to two main steps. The first stage goes from zero time up to 9 hours: during this time, as the reaction proceeds, a decrease in the content of carbon atoms linked to heteroatoms is observed. Since the data present in the literature on organic waste, show that the amount of carbons linked to oxygen (C-O) is greater than the amount of any other C-X [START_REF] Chefetz | Chemical and Biological Characterization of Organic Matter during Composting of Municipal Solid Waste[END_REF], it is probable that this also occurs for our samples and, therefore, this decrease can be attributable, for example, to the oxidation of alcoholic groups, mostly present in sugars, to carbonyl groups. On the other hand, however, there is no substantial variation in the areas related to the carbon atoms of the carbonyl groups and this suggests that probably, in this period of time, further oxidation of carbonyl groups with loss of CO2 occurs. It should be emphasized that it cannot be deduced in any way from the recorded spectra if the CO2 produced comes from the newly formed or pre-existing carbonyl groups. Stage II occurs around 12 hours: at this time there is an evident increase in the area of carbon atoms linked to heteroatoms due, probably, to an oxidation of aliphatic carbons and, to a lesser extent, to the unsaturated ones. As an evidence of this, a decrease in the areas due to these carbon groups can be appreciated. From the data reported in Table 2.10 and from the relative distribution of Figure 2.46, it is possible to infer that the process B proceeds in the same way as for process A, but the two steps described above start and finish at different times. Indeed, the Stage I of the Sample 5 goes from zero time to 6 hours unlike the Sample 4 in which it continues up to 9 hours, while the Stage II two curves is that, although they have maxima at different angles, they are one mirror image of the other, just like the two enantiomers (Figure 3.9). By adopting our model to interpret the experimental data, we were able to obtaining not only the torsional curves displayed above, but also the -dependent absolute sets of the R and S order parameters as shown in Figure 3.10. By observing the Figure 3.10, it is immediate to realize that all the order parameters are very small (as expected, being the PBLG/CDCl3 liquid-crystalline phase a weakly-orienting solvent, as said above); moreover, the order parameters exhibited by the R enantiomer are, in particular, smaller than the ones of the S species.

Process B.

Samples preparation and experimental procedure

The conformational study performed on the two enantiomers of TA, was carried out using an experimental approach different from the one adopted in the case of the 4FMBOH for which two different anisotropic samples were prepared, one for each enantiomer. Indeed, in the case of TA, a single anisotropic sample was prepared by dissolving a racemic mixture of the two enantiomers, R-TA and S-TA, in the lyotropic liquid crystalline phase composed by PBLG/CDCl3. In this case the spectra obtained are given by the superposition of the spectra generated by each enantiomer and, since the technique used allows to identify and label the signals of each enantiomer only if the profile of one of the two is known, it is not possible to associate the signals obtained from the anisotropic racemic sample to the enantiomer that generated them. For this reason, the observables extracted were assigned to two generic enantiomers A and B and only later, through a trial and error process during the conformational analysis, it was possible to assign the experimental dipolar couplings to the correspondent enantiomer.

As states equations 1.32 and 1.33, to have access to the RDCs it is necessary to know the values of 𝐽 𝑖𝑗 . Thus, the first experimental step involved the preparation of an isotropic solution of the TA. TA acid was purchased from Aldrich in the form of a racemic mixture (RS-TA) and it was used for sample preparation without further purification. Then the isotropic sample (RS-TAiso) was obtained by simple addition of 0.0116 g of RS-TA to 0.6588 g CDCl3 into which the molecule was immediately dissolved. The preparation of the anisotropic sample (RS-TAaniso), on the other hand, required a more elaborate procedure (Canet et al, 1995): it was obtained by dissolving 0.0900 g of RS-TA in 0.5556 g of CDCl3 and 0.1094 g of PBLG was added to this solution, directly in an NMR tube. This led to obtain a sample with a concentration of 14.5% wt in PBLG. This sample was heated several times up, centrifuged back and forth to give a very homogeneous birefringent phase. In order to verify that the solution was actually anisotropic, the 1D 2 H spectrum of the deuterated solvent was recorded. In the spectrum obtained, a singlet is visible if the phase is isotropic and a doublet if the phase is anisotropic. It is important that no isotropic domains are in the sample and this is detectable by the absence of the peak that is located in the centre of the doublet due to the quadrupolar splitting. PBLG (150000 Da < MW < 350000 Da), and CDCl3 (99.96 %D), were purchased from Aldrich and used for sample preparation without further purification. The composition of the prepared samples is summarized in Table 3.3. are the width at half height of each Gaussian curve. In addition, according to the AP approach, it is necessary to describe the molecular fragment in terms of interaction tensors (see paragraph 1.2.2) and in our case 4 uniaxial components are needed 𝜀 2,0 𝑗 , two to describe the aromatic ring: 𝜀 2,0 𝑆-𝐶5 and 𝜀 2,0 𝐶5-𝐶6 ; two to describe the propionic subunit 𝜀 2,0 𝐶1-𝐶2 and 𝜀 2,0 𝐶2-𝐶3 . Once defined the 𝑃 𝑖𝑠𝑜 ({𝜙 }) function, fixed the geometry and described the molecular fragment in terms of 𝜀 2,0 𝑗 , we tried to reproduce the experimental 𝐷 𝑖𝑗 𝐴,𝐵 (𝑜𝑏𝑠) by suitably iterating over the 4 orientational parameters 𝜀 2,0 𝑗 , over the conformational term 𝜙 𝑚𝑎𝑥 and parametrically varying ℎ 𝜙 up to achieve an acceptable RMS value. From the spectral analysis some 𝐷 𝑖𝑗 (𝑜𝑏𝑠) remained unassigned to a specific enantiomer, at this point of the study, these constant were tested to both of enantiomers with a trial and error process and were assigned to the species which gave the lowest RMS value. It should be noted that, since it is not known the species A which enantiomer corresponds to, whether R or S, and the same is valid for B, in the iterative process for a given geometric structure, both data sets have been tested in order to reach the lowest RMS possible. It was relatively easy to assign the RDCs to the correct enantiomer using this methodology because in case the RDCs and geometry do not match, very large RMS values were obtained (higher than 7).

Once all the parameter and variables were optimized, an acceptable reproducibility of the experimental values was obtained (RMS = 0.92 Hz for R isomer and RMS = 0.85 Hz for S isomer). The curves obtained for the two enantiomers 𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑅 and 𝑃 𝑖𝑠𝑜 ({𝜙 }) 𝑆 were sampled between 0° and 360° in steps of 2° and are shown in = 1 -0.3 = 0.7).

General conclusions

The aim of this Ph.D project, carried out in cotutorship between Università della Calabria and Université Paris-Saclay, was to explore different applications of NMR Spectroscopy in metabolomic analysis of natural matrices and in pharmacological field. Today, NMR spectroscopy is used in a variety of ways and among them metabolomics applications emerge.

Metabolites results of internal genetically determined processes as well as external environmental factors, for this reason the study of the set of small molecules, substrates, intermediates and products of cell metabolism, can provide important knowledge about the studied organisms (cellular mechanisms, mutations, aging, diet, stress, pathologies, geographical origin, cultivar peculiarities.). In addition, each metabolite is a bioactive molecule that is able to interact with endogenous ligands and receptors. These interactions are predetermined by the configuration and the conformations of the flexible species involved, hence the importance to perform a conformational studies and configurational discriminations of these bioactive flexible species in parallel with the characterization of the metabolome itself.

In the first chapter of this Ph.D thesis theoretical bases of the NMR spectroscopy were provided together with its application into the two fields investigated during the project, i.e. the NMRbased metabolomics characterization of natural matrices or extracts and the orientational, structure and conformational distribution study of small flexible chiral solutes with pharmacological activity. In particular, after the description of the project and its main objectives, in the first chapter a brief overview of the NMR spectroscopy basic principles has been provided. The section continued by giving a general description of metabolomics science and the scientific approaches available, then the main analytical platforms and the most common processing and pre-processing tools needed to perform the data analysis were presented. This chapter ends with the presentation of the NMR methodology used to obtain conformational and configurational information on a flexible solute dissolved in partially ordered chiral media by using the experimental residual dipolar couplings (RDCs). Specifically, an overview of the basic tools of NMR in partially ordered media was given, the pulse sequences able to provide the useful RDCs and the theoretical model adopted in order to convert the experimental data into the structural, orientational and conformational informations.

In the second chapter the NMR-based metabolomics applications explored during the Ph.D.

were presented. In the first study described, NMR spectroscopy was applied in order to study the metabolic profile of Calabrian fresh orange and clementine juices and to follow any metabolic variations that the fresh juice undergoes during the industrial thermal process in order complex waste. In addition, this HR-MAS approach has also proved suitable for monitoring the oxidative processes based on the Fenton reaction to which the initial waste matrix was subjected. Indeed, the results obtained highlight the qualitative/quantitative modifications of the different types of carbon atoms as a function of both concentrations of Fenton reactants and duration of the oxidative process. To our knowledge, this case study represents the first example in which the HR-MAS approach has been successfully applied to the organic fractions of MSW.

Finally, the advantages in terms of rapidity, simplicity, and diversity of available information makes this technique very attractive for further studies on different matrix of OFMSW (Organic Fraction of Municipal Solid Waste) and also to monitor other degradation/transformation processes to which organic waste can be subjected.

In the third chapter the results obtained from the application of NMR spectroscopy in the second research field investigated were presented. In particular, during the years, NMR application in weakly ordered chiral phases have extensively proved to be able to furnish RDCs which, if treated with an appropriate theoretical model, can lead to obtain structural, conformational and orientational information of flexible molecules. This methodology has been applied to a pair of enantiomers in order to investigate their individual conformations and to understand the different solute-solvent interactions that the two species, R and S, form in a chiral solvent. In particular the first study presented in this chapter is referred to a simplest case consisting of the R and S enantiomers of the 4-fluoro-α-methylbenzyl alcohol (4FMBOH) molecule, a single rotor species. The example reported in this section proved the unique power of NMR in liquid crystalline phases in exploring molecular conformational equilibria in order to let emerge, for the first time in our knowledge, differences in the torsional distribution curves and orientational order parameters of R and S enantiomers dissolved in a chiral non-racemic aligning solvent. In particular, the results obtained showed that the two enantiomers are characterized by a conformational curve composed by two maxima equally populated and the two curves obtained, one for each enantiomer, are one the mirror image of the other. In addition, the AP-DPD theoretical model applied, known to be a solid approach to treat the experimental data for both highly and weakly oriented molecules, it seems to be appropriate for treating as well the chiral features of enantiomers by studying their conformational behavior.

Since chirality become a crucial problem when dealing with bioactive species, such as drugs for example, the second study carried out regarded a more complex species of which only one enantiomer is active as anti-inflammatory drug: the tiaprofenic acid (TA). The molecule consists of two cooperative torsions and one independent torsion, in the study presented the attention was focused on the conformational behaviour of the independent torsion involving the

Conclusioni generali

L'obiettivo di questo progetto di dottorato, condotto in cotutela tra l'Università della Calabria e l'université Paris Saclay, è stato quello di esplorare diverse applicazioni della spettroscopia NMR. Al giorno d'oggi la spettroscopia NMR viene comunemente utilizzata in diversi settori e tra questi le applicazioni nel campo della metabolomica occupano sicuramente una posizione di rilievo. I metaboliti presenti in un determinato sistema biologico sono il risultato di processi interni geneticamente determinati ma sono anche fortemente influenzati da fattori ambientali esterni. Per tali ragioni, lo studio dell'insieme di queste piccole molecole, substrati, intermedi e prodotti del metabolismo cellulare, può fornire importanti informazioni sull'organismo studiato (meccanismi cellulari, mutazioni, invecchiamento, dieta, stress, patologie, provenienza geografica, cultivar).

In aggiunta, ogni metabolita è una molecola bioattiva in grado di interagire con ligandi e recettori endogeni. Queste interazioni sono predeterminate dalla configurazione e dalle conformazioni della specie flessibile coinvolta e da qui ne risulta l'importanza di eseguire studi conformazionali e discriminazioni configurazionali di specie flessibili bioattive in parallelo con la caratterizzazione del metaboloma stesso.

Nel primo capitolo di questa tesi di dottorato sono state fornite le basi teoriche della spettroscopia NMR e contemporaneamente è stata illustrata la sua applicazione nei due campi di ricerca esplorati durante il progetto, ovvero la caratterizzazione metabolomica di matrici o estratti naturali e lo studio orientazionale, strutturale e conformazionale di piccoli enantiomeri flessibili biologicamente attivi. In particolare, dopo la descrizione del progetto e dei suoi principali obiettivi, nel primo capitolo è stata fornita una breve panoramica dei principi di base connessi alla spettroscopia NMR. La sezione è proseguita fornendo una descrizione generale della metabolomica e degli approcci scientifici comunemente utilizzati, quindi sono state presentate le principali piattaforme analitiche e gli strumenti di elaborazione necessari per eseguire l'analisi dei dati. Il capitolo si conclude con la descrizione della metodologia NMR utilizzata per ottenere informazioni conformazionali e configurazionali relative ad un soluto flessibile disciolto in mezzi chirali parzialmente orientanti mediante l'utilizzo degli accoppiamenti dipolari sperimentali (RDC). Nello specifico, è stata fornita una panoramica degli strumenti di base della spettroscopia NMR in mezzi parzialmente ordinati, sono state presentate le sequenze di impulsi in grado di fornire le RDC necessarie per tale tipo di studio ed è stato introdotto il modello teorico adottato per ottenere le informazioni strutturali, orientative e conformazionali a partire dalle osservabili sperimentali.

qualità industriale e, in aggiunta a ciò, l'NMR è una tecnica potente che può essere applicata nello studio di specie complesse chirali e flessibili permettendo di aggiungere nuovi tasselli per comprendere il meccanismo d'azione di farmaci e la diversa attività biologica degli enantiomeri, aprendo così la strada a nuove intuizioni.

première fois à notre connaissance, des différences dans les courbes de distribution de torsion et des paramètres de l'ordre orientationnel de énantiomères dissous dans un solvant faiblement orienté chiral. En particulier, les résultats obtenus ont montré que les deux énantiomères sont caractérisés par une courbe conformationnelle composée de deux maxima également peuplés et les deux courbes obtenues, une pour chaque énantiomère, sont l'image miroir l'une de l'autre.