Ph.D Antonio Monari

Neither art nor science knows anything of moral approval or disapproval. Science is out of the reach of morals, for her eyes are fixed upon eternal truths.

Art is out of the reach of morals, for her eyes are fixed upon things beautiful and immortal and ever-changing."

Forewords

The main corpus of this manuscript will be devoted to the study, via computational modeling and simulations, of the photophysical and photochemical processes leading to the induction of DNA lesions. However, also in consideration of the fact that my scientific interests have evolved considerably during the course of my carrier, an exhaustive curriculum vitae will firstly be presented, highlighting not only the scientific achievements, but also the teaching activities, the supervision of students and post-doc, and the service duties for the community.

Afterwards, a more detailed presentation of the main scientific interests developed all along my carrier is provided. This will also allow to more easily retracing the evolution of my scientific interests during the course of the past years and the links still present with my previous main activities.

The main text is organized as follows: After a brief Introduction to state the general problem of DNA lesions inductions via photophysical processes, in particular focusing on the photosensitization mechanisms, three chapters will be presented: the first one will deal with the high level modeling of potential DNA sensitizers to unravel their inherent optical properties and photophysical active channels; the second one will deal with the interaction of photoactive drugs with DNA; while in the third the problem of understanding DNA repair mechanism will be tackled. Finally the Conclusions and the scientific project I intend to pursue in the next years will be presented in the last chapter.

• Elected Member of the Scientific Council of the Molecular Chemistry and Physics institute (CPM) of the Université de Lorraine (2012-2017) • Member of the Executive Board of the Lorraine Section of French Chemical Society (2015-) • Member of the Executive Board of the French Society for Photobiology (2017-)

Student Supervision:

• Co-supervisor of 5 Ph.D. students • Supervisor of 5 post-doc fellows • Supervisor or co-supervisor of 11 master and 6 bachelor internships.

Teaching Activity:

Principal Scientific Interests:

• Methodological Development in Theoretical Chemistry,

• Hybrid QM/MM methods,

• Molecular and non-adiabatic dynamics,

• Electronically excited states in photoactive systems,

• Photochemistry and photophysics,

• Time-resolved ultrafast spectroscopy,

• Environment effects in complex systems,

• DNA oxidative and photoinduced damages,

• DNA recognition and repair mechanism,

• DNA and molecular bases of epigenetics,

• Charge-transfer systems,

• Dye-sensitized solar-cells,

• Rational drug design,

• Photodynamic and light assisted therapy. After having obtained my position at the University of Lorraine I have been involved in the co-supervision and co-direction of six Ph.D. students:

Principal Collaborations in Nancy

Since I was hired in Nancy and after the completion of his first year Ph.D., I de facto assumed the cosupervision of Thibaut Very, who's Ph.D. was defended in November 2012, for his last two years (Director X. Assfeld). This implied a participation of 50%. After his Ph.D. defense Thibaut Very performed a first post-doc in the group of prof. Clémence Corminbeouf (EPFL Lausanne), followed by post-docs in the groups of Dr. Mario Barbatti at Max Planck Institute Mülheim, and of Prof. Valérie Brenner at CEA Saclay. At present Thibaut Very holds a tenured position as research engineering ("ingenieur de recherche") at the IDRIS computing center in Orsay.

The works performed under my co-supervision led to the publication of 9 cosigned articles in peer-reviewed journals, among the others Phys. Chem. Chem. Phys. and J. Chem. Theory Comp.

I was also the official co-supervisor of the Ph.D. of Thibaud Etienne (started in October 2012) with a participation of 50%, the supervisor being X. Assfeld. Thibaud Etienne defended his Ph.D in 2015 and was immediately hired with a post-doctoral contract in the group of Dr. Filippo de Angelis, University of Perugia, Italy. In 2017 Dr. Etienne secured a tenured position as Ass. Prof. ("Maître de Conférence") at the ICGM -University of Montpellier. Dr. Etienne was awarded the prize for the best Ph.D. thesis in 2017 by the French Network for Theoretical Chemistry (RFCT). The works performed under my co-supervision led to the publication of 14 cosigned articles in peer-reviewed journals, among the others Phys. Chem. Chem. Phys. and J. Chem. Theory Comp.

Since October 2015, I also assured the de facto co-supervision, with a quota comprised between 50% and 70% of the Ph.D. of Hugo Gattuso. Dr. Gattuso defended his Ph.D. in July 2017 and is since a post-doctoral fellow in the group of Prof. Françoise Demacle at the University of Liège, Belgium. The works performed under my co-supervision led to the publication of 23 cosigned articles in peerreviewed journals, among the others J. Phys. Chem. Lett., Nucleic Acids Res., Nature Sc. Rep.

Since 2018 I am co-supervisor of the Ph.D. thesis of Florian Cornu, presently in his second year, with a quota of 50%, the director being Dr. Stephanie Grandemange. The doctoral project of Mr. Cornu concerns the elucidation of the induction of epigenetic regulations via endogenous molecular agents in the framework of the development of novel epigenetic drugs.

I participated, with a quota of 20%, to the scientific supervision of Soraya Abtouche during the time she spent in our laboratory under her joint Ph.D. program between the Universities of Lorraine and Alger. Soraya Abtouche is currently holding a tenured position at the University of Alger. The works performed under my co-supervision led to the publication of 2 cosigned articles in peer-reviewed journals.

Finally I also participated again with a quota of 20% to the supervision of Nadjwa Drici Ph.D. student from the University of Oran, Algeria, during her 18 months stay at the Nancy department.

All along my activity I participated to the supervision or co-supervision of six bachelor students for their internship program:

• Roseline Esmilaire from Université de Lorraine in 2011.

• Kevin Baldini from Université de Lorraine June-July 2014

• Elodie Durand from Université de Lorraine March-June 2016

• The internship was valorized via a cosigned publication in J. Phys. Chem. Lett.

• Houda Ennaim from Université de Lorraine March-June 2017

• Yigitcan Eken from Bogazici Universitesi Istanbul June-July 2015

• Yigitcan Eken is at present Ph.D. student at the Michigan State University, the USA. The internship was valorized via a cosigned publication in J. Phys. Chem. C. • Busenur Aslanoglu from Bogazici Universitesi Istanbul January 2018, June-July 2018 I have supervised or co-supervised 11 Master students' internships:

• Thibaud Etienne, 2nd year Master, Université de Namur (2012)

• After the Master he successfully applied for the Ph.D. program under my co-supervision.

• Hugo Gattuso, 1st year Master, Université de Lorraine (2013)

Disseminations to General Public

I always considered that one of the missions of a researcher is to favor the dissemination of his results to a general audience and public. For this reasons I actively participated to disseminations programs oriented toward the general public and having as central subject the renewable energies and in particular the photovoltaic devices. These efforts have concretized in a public conference with visit of the laboratory that has been held in May 2013. Note that this initiative got a very good media coverage with an article and the cover on the regional newspaper and an emission on regional news. Moreover with the same spirit I participated to many stands held in Nancy and Metz during the year of chemistry (2011) illustrating our activities on the photoactive systems and I have constantly participated to the outreaching event the "fete de la science" programs.Last year I was also involved in the outreach conferences organized by the Faculty of Science students associations "Pretexte" that are based on the popular "A Pint of Science" format. Finally in 2017 I have presented scientific research and in particular DNA in a primary school of Nancy obtaining a large success.

Chapter 0 Summary of Research Activities

A large part of my research activities have been characterized by the interest for the development and applications of high-level quantum chemistry methodologies to elucidate the properties of electronic excited states as well as the electron and energy transfer phenomena.

In particular during the first period of my research activity (Ph.D. and post-doc experiences) I was mainly interested to the application and development of highly correlated wave function based methods. In this context one may cite for instance the development that I carried out for the calculation of polarizabilities and dispersion coefficients at the Full Configuration Interaction (FCI) level. [1][2][3][4][5][6] Besides FCI, I was mostly concerned with CAS-SCF methods as well as MR-CI and second order multi-reference perturbation theory (MRPT2) approaches.

From a methodological point of view I was particularly committed to the use of localized orbitals [7][8][9] with a double-fond interest. On the one hand localized orbitals can assure an easier and more chemically sounding definition of the active space in complex systems. Indeed the use of local orbitals allows to significantly reduce the size of the active space, compared to the one defined in the basis of canonical orbitals, without loosing physical informations and therefore getting a good representation of the static correlation.

On the other hand, the locality of orbitals can be exploited in the post-CASSCF procedure (namely in the MRCI) to assure a suitable truncation of the determinant expansions, whose huge size is often the limiting factor in the use of MRCI methods. Indeed, dynamic correlation being a local phenomenon if one disposes of an expansion basis whose functions (orbitals) are localized it is reasonable and correct to include in the CI expansion only those determinants arising from excitations involving spatially close orbitals. Indeed, the former strategy is so powerful that can, at least for some systems, achieve linear scaling.

It is also important to note that I have also been strongly interested, in the framework of different European programs, in the definition of standard formats to assure an easy exchange of data and information between quantum chemistry codes, so as to achieve a real interoperability between those different codes and methods. [10][11][12][13] The development of such methods allowed me to tackle some important scientific problems, mainly related to model systems to understand problems related with the material science domain. In particular I was able to retrieve the parameters of the model hamiltonian describing the magnetic coupling in poly-metallic systems, as well as the low energy spectrum of such clusters. 8,14,15 One particular class of magnetic systems I was interested to is the one composed of linear chains of closed shell atoms (for instance Beryllium). [16][17][18] Indeed, their ground state shows quasi-degeneracy between an open-shell singlet and a triplet state, and the high amount of static correlation necessitates the use of multireference wavefunction methods. Most notably I was also able to model the interaction of such aggregates with different surfaces and in particular graphene nano-ribbons. 19 It is worth to cite that my attention was also oriented toward the remarkable properties of graphene nanoclusters and in particular to their magnetic properties, as well as to the relation between the latter and the clusters' geometrical parameters. 20,21 I also implemented in different quantum chemistry codes the calculation of the so-called localization tensor that comes from modern theory of conductibility and properly defines the insulator or conducting properties of a given material. In this framework I was able to study the metal-insulator transition in some model systems, as well as in graphene nano-clusters. 20,[22][23][24] Finally the use of efficient multi-reference methods has allowed the study of electron transfer notably in the case of phthalocyanines dimer cations. 25 On that context let us cite that phthalocyanines form homeotropic liquid crystals that can act as molecular wires. Not only the process was well described in terms of the Marcus theory, but for the first time I evidenced the presence of a conical intersection that can strongly influence the electron transfer process in such systems.

After securing the position in Nancy, my scientific interests, although still closely focused on the study of the electronic excited states' properties, have partially switched to consider the influence of the molecular environment and in particular of the surroundings of the active chromophores, in modulating their response to light stimuli. 26 Indeed when a photo-active molecule is surrounded by a solvent or by a biological macromolecule (a protein or a nucleic acid for instance) its optical properties can change dramatically, as well as its capacity to undergo electron-or energy-transfer. In other words its photophysics and photochemistry will be strongly altered by the environment effects.

Such influence in triggering and tune the response to light can be due to a geometrical constrain of the surroundings, but also to the electrostatic interactions with the surrounding charges and permanents dipoles, and by the polarization of the surroundings due to the density charge rearrangement taking place in the photo-active site. [26][27][28][29][30] Obviously to reasonably tackle such problems the method of choice is the hybrid quantum mechanics / molecular mechanics (QM/MM), that, through a suitable partition of the complex systems, allows to take into account the electronic rearrangements on the active site as well as the influence of the surroundings. Notably in case of homogeneous media, i.e. isotropic solvents, the QM/MM method can be replaced by the use of a polarizable continuum (PCM) via self-consistent reaction field (SRCF) procedures. 31 Moreover, in the case of relatively big and complex chromophores the best compromise between accuracy and computational cost is certainly offered by DFT technique and by its Time Dependent extension (TD-DFT) to obtain the electronically excited states energies and properties.

On the other hand to tackle the problems of the QM/MM frontier when covalent bonds need to be cut, one can consider different strategies based on link atom, pseudo-potential or localized orbitals. I most often rely on the local self-consistent field (LSCF) technique 26,[32][33][34] that basically allows converging the SCF (or DFT) problem keeping one or more orbitals frozen. Practically one places a localized orbital, obtained from a model system, on the bond defining the QM/MM frontier and keeps it frozen and constantly doubly occupied. Notably enough the LSCF technique is much more general and can be used not only to treat the frontier but also to impose constraints on the wavefunction. 33 Finally, since complex (biological) molecules are quite flexible I also considered with a particular attention the necessity to use molecular dynamics (MD) to accurately sample the conformational space and thus take into account the effects of the vibrational degrees of freedom, as well as their coupling with the surroundings, on the optical properties as well as on photophysical responses. [35][36][37][38][39] Note that this influence, as I have evidenced in some important works on DNA photosensitization, can be extremely important and, therefore, cannot be neglected.

In addition to linear optical properties such as UV/Vis absorption and fluorescence spectra, I am also interested in the modeling of non-linear phenomena, such us two-photons absorption (TPA) that can be efficiently exploited both in the case of biological applications and in material sciences. The same protocol used for linear-absorption and based on a combined sampling of the conformational space and of the excitedstates manifold in presence of complex macromolecular environments has been used giving extremely satisfactorily results for different systems including polytiohphenes, 40 natural drugs, 41 or organic dyes. 42,43 The study of the structural modification in complex biological systems induced by lesions or by the interaction with drugs also stimulated the development of efficient protocol to model the circular dichroism spectroscopy. [44][45][46] Indeed, the latter is a very straightforward experimental technique characterized by a very high sensitivity to structural deformation. However, the very rich density of information of circular dichroism can be only hardly exploited directly and requires a one-to-one mapping with molecular modeling and simulation to provide atomistic resolution. Our protocol based once again on the combination of MD to sample the conformational space and QM/MM to obtain excited states properties, allows to reproduce the circular dichroism signal of macromolecular aggregates via the diagonalization of the Frenkel excitonic Hamiltonian.

This simple, and relatively computationally inexpensive technique has been successfully used to simulated the electronic circular dichroism of B-DNA, revealing some subtle and unexpected sequence effects, 44 as well as non-canonical DNA strands such as G-quadruplexes. 45 The modification of the circular dichroism signal induced by the interaction with external drugs and sensitizers has also been obtained and allowed to easily differentiate between competitive binding-modes. Recently, we have also presented a fully detailed analysis of the influence of the partitioning of the macromolecular chromophore into individual monomers, 30,47 in the case of nucleic acids and polypeptides, showing most notably the differential effects between hydrogen bonding and ππ-stacking.

The extensive methodological study of the properties of electronic excited states, and in particular of chargetransfer states, also pushed myself to reconsider the topological properties of the excited states electronic density and their rearrangement following an optical excitation. In particular, together with my coworkers I proposed a novel index (φ S ), based on the spatial overlap between hole and particle density in the excited state to numerically and semi-quantitatively clearly define the amount of charge transfer of a given excited state. It has to be noted that the derivation of the index being performed in the Hilbert space it avoids any possible inaccuracy due to density cancellation. [48][49][50] The method has been implemented in a freely available open-source code and starts to be relatively widely used in the community.

More recently in order to achieve a proper description of the photophysical and photochemical processes, also providing access to crucial information such as life-times and quantum yields I have turned my attention to the use of excited state non-adiabatic state-hopping techniques. 51,52 Indeed in this semi-classical approach while the nuclei are propagated over a classical (excited state) potential energy surface the conversions (hops) between the surfaces are stochastically allowed with a probability based on their respective couplings. More specifically, I have collaborated with the developers team of the "Sharc" code, from the University of Vienna, that has the particularity to be the only one allowing the inclusion of spin-orbit coupling and hence being able to describe intersystem crossing phenomena. 37 In particular we have participated in the inclusion of a proper TD-DFT QM/MM embedding in the "Sharc" code via its interfacing with the ADF quantum chemistry code. This recent achievement opens the way to the treatment of complex photophysical problems in non-homogenous environment providing an unprecedented comprehension of crucial phenomena related to material science development and to the treatment of crucial biological problems. As an example the photophysical properties of benzophenone, thiophenes dimers and Rhenium chromophores in solution has been revealed thanks to this strategy. 37 From a chronological point of view the applicative problems that I firstly dealt with was the computational modeling of dyes for dye-sensitized solar cells (DSSC). 42,[53][54][55][56][57] This project, strongly connected to the production of efficient renewable energy, has always been conducted through a very strong collaboration and synergy with experimentalists and in particular with the organic synthesis team in Nancy. It is oriented to the improvement of DSSC through rational molecular design. In particular the possibility to obtain a reliable computed spectrum coupled with the possibility to analyze the excited states in terms of electronic density reorganization allows us to qualitatively predict the capacity of the dye's excited state to inject electrons in the semiconductor and hence to produce an electric current. In particular the analysis of the excited state electronic density's reorganization, that takes advantage of the use of natural transition orbitals (NTO) 48,58 and the φ S formalism, is fundamental to obtain a first screening of good candidates to DSSC and to estimate the influence of different substituents as well as of different photoactive kernels.

Modeling and simulation also offer an unprecedented opportunity to analyze and rationalize the competitive deactivation pathways that occur in the dye's excited states and that strongly limit the efficiency of the device. More specifically I dealt with the study of novel inorganic systems, mainly Ruthenium organometallic complexes and porphyrine based bimetallic antenna dyads. 53,57 More recently I turned my attention toward all organic dyes, also tackling the problems related to their aggregation and dimerization and its influence on the spectral properties and on the injection capacities. 42,43,59 Notably, the formation of stable aggregates coupled with the presence of exciton formations and inter-monomer charge transfer has been clearly evidenced.

Finally and very recently, thanks to a multidisciplinar project merging modeling, organic synthesis and time-resolved spectroscopy, we proposed, characterized and synthetized iron-based organometallic complexes in which the thermal deactivation through metal-centered triplet states, is strongly hampered. [60][61][62][63][64][65] Therefore we obtained a long lived metal-to-ligand charge transfer and hence a non-zero light-to-energy conversion where the former complexes are used as DSSC dyes. 62 Our combined efforts has allowed not only to obtain one of the longest lived excited state of iron compounds but also, more recently, to finely characterize the complex photophysics related to the excited states deactivation, and in particular the role played by the different triplet state energy potential as well as the difference in their topology between different compounds and isomers. 65 This latter aspect far from being of interest only for DSSC field is instead of crucial fundamental importance in order to understand the photophysics of first row transition metals and hence potentially produce much cheaper light active or phosphorescentes materials.

Another fundamental aspect of my research interests concerns the study of excited state properties of biological macro systems. Such systems can be chromophores in proteins such as tryptophane in Human Serum Albumin, 26,66 or copper complexes in plastocyanin and azurin, 28,29 or coupled Rhenium and thryptophanes in artificially labeled proteins to be used for coherent electron transfer. 37 Most notably, in the case of the Rhenium sensitized protein and by applying a cutting-edge multiscale model combining classical and ab initio molecular dynamics, we unraveled the crucial role of the protein environment in providing metastable MLCT or charge-separate (CS) state via the participation of a nearby tryptophan. The clear identification of those states will allow rationalizing the complex and long-range photoinduced electrontransfer phenomenon taking place in such protein.

However, in the last years the system of choice in such domain for my research activities has been the study of the photophysical properties of small molecules interacting with nucleic acids and DNA in particular, i.e. the process of DNA photosensitization. For this reason, and to avoid useless repetitions, this aspect will not be treated in the present Chapter but will instead constitute the core of the present manuscript. [67][68][69][70][71][72][73][74][75][76] In addition to the production of DNA photolesions I have also started tackling oxidative lesions and their outcome. 77,78 In this context in particular I have studied the reaction of singlet oxygen with naturally occurring nucleobases such as guanine and adenine. We have been able to elucidate the mechanism of formation of the singlet oxygen adduct, identify keys intermediates such as endoperoxides moieties, and most importantly rationalize, in terms of transition state energies, the selectivity exhibited towards guanine over adenine. Furthermore and thanks once again to the combination of MD and quantum chemistry, we have been able to explain the observed difference in the product distribution between solvated and DNA embedded guanines. Indeed, the chemoselectivity has been shown to depend crucially on the different water accessibility in the two situations. 78 The enlargement of my domain of study related to DNA lesions production has also been instrumental in the opening of new research lines, in particular related to the development of anticancer therapeutic agents acting via a selective epigenetic regulation. This topic, that has already provided fascinating and promising results, and that have been tackled with an extremely multidisciplinary consortium allying modeling and simulation, organic synthesis, and cellular and molecular biology, cannot be treated fully in the present report due to confidentiality requirements, since a patent application is being deposited.

On the same spirit centered on the study of biological systems under external stress, I have also tackled the study of photosensitization and oxidative stress of lipid membranes also induced by electric fields. Oxidation and sensitization of biological membranes is indeed of fundamental importance being related to aging and to many diseases such as inflammations, neurodegenerative processes and cancer. The fine comprehension of its mechanism will therefore allow designing more efficient protective strategies such as improved antioxidant agents, or on the other hand (photo-)active drugs able to induce cellular apoptosis by inducing the membrane perturbation. To achieve such a comprehension we need a fine modeling of the kinetic of the different rate-limiting step reaction happening in this complex environment. Once again the use of a global strategy combining molecular dynamics with QM/MM 30 is necessary to provide a full picture of the different processes. While the explicit use of ab initio QM/MM molecular dynamics is necessary to have a good representation of the free-energy profiles involved in the different elementary reactions ultimately giving rise to oxidation.

Most notably I was able to follow thanks to classical MD simulations the penetration of different sensitizers such as porphyrines or natural occurring compounds 30,41 in the biological membrane, while the modulation of the singlet oxygen generation efficiency by the heterogenous lipid environment has also been assessed.

By using MD techniques I was already able to prove that one of the most common hypothesis invoked to interpret oxidation in biological membranes, i.e. the expulsion toward the aqueous interface of the propagating peroxyl radicals, does not hold and it cannot therefore be invoked as a membrane autoprotecting mechanism. 79 The fact that the radicals ensuring the chain-reaction propagation are located deep inside the membranes can be used to develop and design novel antioxidants that will show a better overlap with the reactive radical species and hence will terminate the chain reaction more efficiently.

Very recently we have studied the photosensitization of rhodopsin, a membrane protein involved in the vision mechanism, by the red/infrared absorbing chlorine-e6 chromophore. 80 We have evidenced the possibility of a cascade of energy-transfer through the different aminoacids finally reaching the retinal and inducing photoisomerization. The retinal photoisomerization induced by the reaction with singlet oxygen has also been shown as a possible reactive channel. These results can explain the observed phenomenon of the increased sensitivity to low light intensities observed in some phototherapy patients ("night vision").

Chapter 1 DNA Lesions and Their Biological Effects: The Role of Molecular Modeling and Simulation

The fundamental biological role of nucleic acids, and DNA in particular, as the repository of the genetic information, the regulator of its transduction and expression, and the vector of its replication is nowadays fairly well understood. However, to efficiently perform such a role the molecular constituents of DNA should possess a number of crucial properties. In particular, DNA constituents should experience a fairly high stability to avoid the insurgence and accumulation of lesions that ultimately could lead to mutagenesis and more generally to genomic instability. However, and form a more general point of view, it is important to moderate such a strong affirmation taking into account that, from a totally reductionist point of view, evolution itself may ultimately be tracked back to genomic mutation. Furthermore, one should also recognize that DNA is constantly exposed to a huge number of stress sources coming both from the cellular exogenous and endogenous agents. [1][2][3][4] As a non-exhaustive example one may cite the presence of reactive oxygen species (ROS), related to oxidative stress insurgence, and produced either by healthy or pathologic metabolic pathways, or by external agents such as drugs or ionizing radiations. 1,[4][5][6][7][8][9][10][11] Furthermore, DNA is also constantly exposed to the action of electromagnetic radiation that may also trigger its photophysics and photochemistry, especially after absorption of UVB radiations, and hence the accumulation of photoinduced lesions.

The danger for the DNA can be strengthen by the combined synergic effects between two or more stress factors, which will not only enhanced the occurrence of a given lesion but may even open totally new chemical pathways leading to the production of totally novel lesions. As a paradigmatic example one can cite the case of DNA photosensitization, 12,13 in which a drug absorbing in the visible or near UV, can interact with DNA and subsequently trigger complex photochemical channels leading to the insurgence of photolesions.

To contrast such a hostile environment evolution has adopted two strategies, on the one hand the chemical components of nucleic acids have been selected to maximize their stability, hence minimizing the insurgence of lesions. On the other hand, efficient and complex repair mechanisms have been put in place to reduce the accumulation of the lesions. 14 Finally, other signaling pathways are also used to trigger apoptosis, when the amount of DNA lesions becomes higher than a given threshold, and the viability of the cell become irreversibly compromised.

However, the accumulation of DNA lesions has in many cases been directly related to the insurgence of not only genomic instability but also to tumorigenesis. One particular important case is the one of DNA photolesions, which will be extensively treated in the following, and whose accumulation in the skin, also due to unprotected sun exposure, is known to be directly related to the apparition and development of malignant tumors such as melanoma. [15][16][17][18][19] On the other hand, the induction of DNA lesion represents also a widespread therapeutic strategy, in particular in the case of chemotherapy for malignant tumors. [20][21][22][23][24] In the framework of these approaches, one exploits the fact that tumor cells present a generally higher quantity of DNA lesions than healthy cells. Hence, administering drugs producing additional lesions to the DNA will push cancer cell to reach the threshold for the induction of apoptosis before the healthy ones. This in turn should lead to a selective annihilation of the tumor cells and hence the eradication of the lesion. We may remind in this context that one of the first, and still widely used, chemotherapeutic agents, cisplatinum, [25][26][27] is indeed relying on this principle and its mechanism of action is based on the formation of covalent bonds with the DNA backbone. 28 However, such a strategy is limited by the fact that healthy tissues are also affected in addition to the cancer cell, and hence chemotherapy agents are usually plagued with a number of heavy side effects, in many cases significantly affecting the quality of life of the patients. In order to enhance selectivity, and hence strongly reduce secondary effect, some strategies based on the combined action of drugs and light are nowadays exploited in the domain of photodynamic therapy [29][30][31][32][33] or light-assisted chemotherapy. [34][35][36][37] In these protocols a DNA interacting drug, that is non-toxic in the dark, is administered to the patient. Subsequently, light is applied to the area where the lesion is located to activate the drug via the absorption of an electromagnetic radiation and hence trigger photochemical processes that lead to the DNA photosensitization. [38][39][40][41][42] Obviously, in this case selectivity is dictated by the local application of light that is intended to be restricted only to the cancer lesion. Although attractive, this strategy is still of quite complicated practical application, indeed one has to tackle many different problems such as the necessity to have drugs absorbing in the red or infrared portion of the electromagnetic spectrum to assure the coverage of the so-called therapeutic window, and hence a good light penetration to treat non-superficial lesions. 43 The complexity of the scenario related to DNA lesions is already apparent from these preliminary considerations, however it should be underlined that, in addition, the chemical space spanned by the different lesions is extremely large, and comprises chemical modification happening at the backbone, together with nucleic base change. 2,6,44,45 In particular one can recognize single-and double-strand breaks, characterized by the cleavage of either one or both polymeric chains of the DNA double helix, respectively. [46][47][48][49] While single-strand breaks are more easily processed by the cells, double-strand breaks stand among the most dangerous lesions due to their strong impact in the replication and transcription processes and to their difficult repair. Both classes of lesions can be the results of ionizing radiation and the accumulation of ROS in particular OH radical.

Oxidative lesions may however results also in the chemical modification of the DNA bases, 44,50,51 this will in turn induce a perturbation of the hydrogen network patterns that lead to the selective pairing of the DNA strands and hence lead to aberrant replication processes. Due to the favorable oxidation potential, lesions involving purines, and guanine in particular, are the most common for this class, such as the widely celebrated 8-oxo-guanine resulting by the action of 1 O 2 . However, oxidative pathways may lead to other and more complex outcomes, and in particular to the production of the so called abasic sites in which the nucleobase is totally cleaved from the sugar moiety. [52][53][54] Most notably, one should consider that abasic site also represents an intermediate in the DNA repair processes, in particular in the case of oxidative lesions. 55 Intra-strand base dimerization is instead the main outcome of DNA photolesions, especially in case of direct UV absorption. Because of the more favorable topology and energetic of the involved excited states, pyrimidines, and thymine in particular will be the most vulnerable nucleobases with respect to photolesions. As far as thymine photodimers are concerned two main products should be taken into accounts: the so-called cyclobutane pyrimidine dimers (CPD) and the 64-pyrimidinpyrimidone photoproduct (64-PP). [56][57][58][59][60] While CPD are by far the most common; 64-PP is characterized by a very high mutagenicity making it extremely dangerous. In addition to dimerization, photo induced interstrand hydrogen transfer between Watson-Crick bases has also been identified as a pathway leading to DNA lesions via the formation of nucleobases tautomers. 61 In addition, one should also consider other less common, yet important lesions that can be produced especially by interaction with external drugs. 37,[62][63][64] Those include the formation of covalently bounded, and in some cases bulky, adducts via a coupling to either the backbone or to the nucleobases, which may induce marked structural modification. In addition, in some cases inter-strand cross-links may be produced, via the formation of a covalent bridge between the 5'-and 3'-strand. The effects of inter-strand cross-link in impeding replication are evident, and hence they represent particularly toxic lesions, usually rapidly inducing cell death. Inter-strand cross-link may also be produced photochemically, by the action of photodissociable drugs for example, 63 or may also be the final outcome of oxidative pathways.

Finally, a particularly intriguing class of lesions that are gaining more and more recognition is the one due to DNA protein cross-link, in which an oxidized nucleobase (guanine) forms a covalent bond with aminoacids, such as lysine or asparagine, whose lateral chain present an amine function. 44,65 The final outcome of such a process will be the occurrence of a very bulk adduct, composed by DNA and a covalently bounded protein, whose repair could be extremely problematic. The same chemical process may also lead to the interaction between DNA and primary amines, such as putrescine, spermine, or spermidine that are present in large amount in the cellular nuclei. 66,67 Furthermore, if one considers that DNA structure at rest is coiled around positively charged proteins, such as histones, characterized by a high density of lysines and asparagines, the relevance of such class of lesions cannot be underestimated.

Even though we have provided a classification of the different classes of DNA lesions, it has to be recognized that such lesions are rarely appearing in isolated spots on the DNA structure. Instead they may be concentrated in a relatively restricted area of the nucleic acids giving rise to the so-called DNA cluster lesions, [68][69][70] i.e. combination of two or more lesions appearing in between one or two helical turns. Cluster lesions are, in particular, extremely common in the case of the exposure to ionizing radiations, since the latter produce a burst of ROS in spatially concentrated regions. As compared to isolated ones cluster lesions may behave in a correlate way, and hence inducing structural deformation of the DNA structure that can significantly diminish the repair efficiency. 71,72 In addition the intermediate of the repair processes may results in double strand breaks making cluster lesions quite toxic DNA damages to be dealt with by the cells.

As already stated the actual toxicity varies considerably among the different classes of lesions, and, at first approximation, it depends on the complex interplay between frequency of occurrence, mutagenic power, apoptosis induction, and repair efficiency. Repair mechanisms in particular are necessarily quite complex to be able to deal with such different kinds of lesions and processes, and may vary considerably between different organisms.

In the case of lesions involving only one strand, the undamaged polymer may efficiently serve as a template to guide the repair process and minimize the errors. Two main repair pathways are known: base excision repair (BER) [73][74][75][76][77] is active in the case of damages localized on only one nucleobase, and involves glicosylase enzymes to eliminate the damaged base leaving an abasic site as an intermediate, that is subsequently excised and repair by DNA polymerase. Nucleotide excision repair (NER) [78][79][80][81] is instead used for bulky lesions or intra-strand dimers, such as photolesions. Portions of about 12-24 nucleotides are removed and substituted during the repair process that usually consists of three steps: recognition, excision both upstream and downstream of the lesion by endonucleases, and finally synthesis of the removed DNA. While in prokaryotic organisms NER is mediated by Uvr proteins, the mechanism is much more complex in eukaryotic systems and involves a larger number of protein, however the global mechanism is the same in the two classes of organisms.

Since both BER and NER pathways necessitate using the secondary strand as a template to correctly repair the damaged one, they are obviously not applicable in the case of double-strand breaks or interstrand cross-links. Instead, three different repair mechanism called non-homologous end-joining, microhomology-mediated end joining, and homologous recombination are available. The three mechanisms may act simultaneously, although they are characterized by different characteristic rates, and different accuracies, however the latter are generally lower than for single-strand lesions. 82,83 It is clear that one of the key factors to determine the repair rate will be the recognition of the damaged strand by the repair mechanism, hence the structural modification induced by different lesions on the canonical DNA conformations will also be crucial to understand such modifications. 71,84 However, in addition one has to take into account that the organization of DNA, especially in the case of eukaryotes is quite complex, with the nucleic acid coiled in the chromatin structure. 85,86 This organization leads to important consequences for the repair, indeed regions of high or low compaction may be characterized by different repair rates, while the chromatin organization may change in the course of the cellular cycles, and is also related to epigenetic signaling.

Even through this brief and non-exhaustive introduction, it appears that the world of DNA lesion induction and repair is a fascinating, yet extremely complex one. Moreover, to achieve a proper interpretation of all the different intertwined relevant phenomena a proper multi-scale approach is needed, that should be able to take into account all the different effects at molecular and systemic level. In particular, molecular modeling and simulation is fundamental to provide an atomistic and electronic resolution of the different phenomena taking place, and hence discriminate between different processes and different causes, as well as to predict or rationalize complex outcomes, and, in the long term, assist in the development of appropriate protective strategy or entirely novel drugs and therapeutic protocols. Indeed, since the discovery of its biological relevant role, and in particular after the determination of its structure in the fifties by the seminal works of Watson, Crick, and Franklin, the scientific community has witnessed a surge in the computational and experimental works devoted to DNA and to the production of its lesions.

Indeed, the calculation of the properties of isolated or hydrogen coupled nucleobases, used as model systems to elucidate the electronic effects responsible for the stability and selectivity of the DNA strands have been and still are actively pursued. As an example we may cite the study, performed using energy decomposition analysis tools, of the role of cooperativity in hydrogen bonds in the case of canonical and non-canonical structures such as guanine-quadruplexes. [87][88][89][90] In addition, the study of isolated systems performed at a high quantum chemical level has also allowed for a constant development and improvement of force fields for molecular dynamics simulations, whose quality is nowadays well established and allows for a proper modeling and simulation of the most relevant structural parameters of DNA in different environment. [91][92][93][94] Most importantly, such a development has also allowed a good representation of the complex interactions taking place between nucleic acids and proteins, either for compaction, replication, or repair. [95][96][97] The theoretical study of the induction of DNA lesions has most of the time been conducted on model systems composed of the isolated nucleobase. 50 Most notably, it has to be noted that such a protocol was indeed directly mimicking the experimental studies that where also conducted on model system to reduce the complexity of the problem. 44 If such a strategy has allowed significant breakthroughs in the comprehension of DNA reactivity to oxidative stress, more recently the crude approximation underlying the approach has shown its limitations, once again both from an experimental and theoretical point of view. 50 Indeed, environment dependent distributions of reaction products between solvated nucleotides and DNA have been observed, leading to the recognition of the fundamental role of the molecular environment in tuning the overall process. The development of efficient hybrid quantum mechanics/molecular mechanics (QM/MM) algorithms and protocols has allowed including the environment effects in the study of oxidative DNA lesions. [98][99][100][101][102][103] Also the bases for the differences in reactivity between the different environment have also been pinpointed and ascribed from the one hand to a much less flexible environment in the highly organized DNA polymeric structure, and to the other hand to different solvent accessibility between the isolated and DNA embedded nucleobases that may preclude the opening of some reactive channels. 51,100 The same scenario is also globally applicable to the computational study of DNA photolesions, which has usually proceeded via the analysis of the behavior of the isolated chromophores. In particular, the molecular bases of the DNA nucleobases' photostability have been assessed and related to the easy accessibility of the conical intersection mediating the deactivation to the ground state, compared to the reactive channel involving base photo-dimerization to produce either CPD or 64-PP. [104][105][106][107][108][109][110][111] Most notably the easy access to the conical intersection and the decay has also been confirmed by different non-adiabatic molecular dynamics studies. Even if globally coherent with the results provided by the time-resolved spectroscopy the use of an oversimplified model does not allow to fully rationalizing the processes, in particular debates are still open on the relevance of hydrogen transfer in the DNA nucleobase and its coupling with conical intersection regions. Furthermore, once again both theoretically and experimentally, the reactive channels involving the nucleobase triplet state have been considered, showing an alternative channel also leading to the production of CPD lesions. 112,113 Note that, although non-adiabatic dynamic trajectories including spin-orbit coupling have shown that the decay to the low lying triplet states represent a non-negligible relaxation pathways for photoexcited nucleobases, 114 triplet photoreactivity is particularly relevant in the case of photosensitization since a number of sensitizers undergo intersystem-crossing and then induce a triplet-energy transfer to purines, and thymine in particular.

The computational study of photosensitization mechanisms is however made more complex by the necessity to identify the interaction mode between DNA and the sensitizers, as a consequence the role of the environment becomes even more crucial. 50,115,116 On this respect it is also worth citing that usually many different competing interaction modes between a drug and DNA can exist and that the different conformers may give raise to very different coupling between the multichromophoric systems, and hence being characterized by different photophysical processes. 117,118 In addition, as compared to the situation for protein/drug interactions, it is evident that the studies aimed to provide precise free energy profiles are much less common, and in most cases rely on the use of continuum solvation models. 119,120 Also the computational studies of the excited states manifold for such a systems are somehow scarcer than the ones concerned with the direct photoreactivity. However, the careful design of precise model systems, mimicking the extended nucleic acid case, also coupled with the studies in DNA and even cellular environment have allowed to underline many different photosensitization mechanisms, such as triplet energy transfer. 13,121 In addition, and more recently, the effects of DNA lesions acting as internal photosensitizers have also been clearly demonstrated. 122,123 Finally the use of QM/MM algorithms has also allowed achieving some clear insights into DNA repair mechanisms, in particular obtaining highly precise and statistically converged free energy profile for the reactions of repair enzymes with damaged oligonucleotide sequences, also including photoactivated mechanisms in the case of bacterial systems. [124][125][126][127][128] However, less systematic studies are devoted to the one hand to the global structural deformations induced by the different class of lesions on the DNA structure and their consequence on the repair mechanism and from the other hand on the coupling between cluster lesions and their effect on recognition and repair. To answer such a question an efficient and effective sampling of the conformational space of DNA strands, both solvated and in interaction with repair enzymes, should be performed. 71,84 Hence it is necessary to switch the focus from the electronic effects (reaction free energy) to the structural and dynamic perturbation.

In the next chapters of this manuscript we will focus on the DNA photolesions induction via the photosensitization mechanism, and on the elucidation of the effects of different types of lesions on the repair efficiency. We will pursue a systematic and multiscale approach that will gradually increase the complexity of the problem: starting from the photophysics and photochemistry of isolated compounds, we will then consider their interaction with DNA, to finally treat the influence of complex lesions on the repair machinery. The methodological developments that have been necessary to allow such a process will also be briefly presented. As it will be underlined in the following, we aim to provide a full multiscale study of the DNA lesions and repair processes that will allow to overcome many of the present day limitations, explicitly taking into account the effects of the environment and the influence of dynamic and structural effects, while still providing an high-level description of the photophysical processes into play.

Chapter 2 The First Step: Understanding the Photophysics and Photochemistry of Relevant Chromophores

In order to understand the effects produced by photoexcited dyes and drugs on biological systems, obviously, one has to first provide a full characterization of their intrinsic photophysical and photochemical behavior, as well as of their linear, and eventually non-linear, optical properties. 1,2 Form a molecular modeling and computational point of view, such a characterization need to be as precise as possible, and more importantly should take into account a fully detailed description of all the possible effects playing a role in tuning the response to the light stimuli, including the electronic density reorganization, its evolution upon relaxation, and the effects of the interplay with the chromophore's surroundings. This in turn calls for a fully multiscale treatment taking into account on a proper footing the sampling of the conformational space and the adequate treatment of the electronic structure of the excited states.

In this chapter those aspects will be taken into accounts and will be summarized presenting some relevant examples and original theoretical methods including also the use of non-adiabatic statehopping dynamics to take into account the excited state relaxation pathways.

Section 2.1 The Topology of the Excited States

The population of an electronic excited state can be defined as the sudden reorganization of the electronic density happening without any rearrangement of the nuclear position (Franck-Condon approximation). Hence, to properly define the excited states nature and to offer a physically sounding insight on their properties it is important to provide their description in terms of the density rearrangements as compared to the situation of the ground state. More specifically, this description should take into account both the reorganization in terms of orbital occupations (Hilbert space), and the changes in the spatial regions associated to electron rich or electron poor densities (Cartesian space). Indeed, in many cases, the so-called charge transfer (CT) states, i.e. states in which the electron density is displaced from one region to another of the molecular system, are associated with crucial photo-induced functions, such as electron-or energy-transfer, 3 electron injections in solar cells, 4 or luminescence quenching. 5 Hence, such states require a proper characterization as well as a possibly quantitative, or semi-quantitative, estimation of their charge-transfer nature. This is also crucial due to the usually problematic treatment of CT states, for instance using TD-DFT methods, that requires particular attention and the use of proper exchange-correlation functionals. 6,7 However, when taking into account such a task one faces two important problems that should be tackled. Because of the multideterminant nature of the excited state wavefunction, that is evident both in the case of ab-initio and TD-DFT solutions, the excitation cannot, in many cases, be described by a simple transition from an occupied to a virtual orbital; in contrast several orbital couples are needed to properly describe the transition. This fact makes the description of the density reorganization quite cumbersome and may even prevent a clear and unambiguous definition of the excited state nature. 8 On the other hand, the definition of a charge-transfer state is usually mostly based on qualitative observation of the orbitals, hence lacking a quantitative rigor and in some instance making the analysis extremely tedious, especially when one wants to take into account a sampling of the conformational space.

For this reasons, in the last years I worked, in the framework of TD-DFT, on the development of orbital transformations based on the NTOs formalisms that allows to concentrate all the physical relevant information on the transition between one occupied/virtual orbital couple. 9,10 Note that, although strongly related to the approaches based on the density differences between ground and excited states, our strategy has the advantage of keeping the information related to the orbitals' phase.

I also developed a quantitative measure of the charge-transfer nature of a given excited state, based on the spatial overlap of the attachment and detachment density matrices (φ S ). 10 Note that the overlap being calculated on the Hilbert space, any spurious effect due to density cancellation in the Cartesian space is avoided. Moreover, and in contrast with other indexes based on the distance measurement, 11 the spatial overlap does not fail in the case of centrosymmetric chromophores. The original, and the most used, formulation of the φ S index is based on the unrelaxed densities calculation at TD-DFT level, however an analysis of the effects of the density relaxation, and in particular the induced decreasing of the charge-transfer by back-polarization and the breaking of the π-σ symmetry have also been considered and are presented in the following.

Section 2.2 Exploring the Ground State Conformational Space

The good representation of the absorption spectra and in general of the optical properties of different chromophores necessitates taking into account the coupling between the electronic excitations and the vibrational movements, i.e. the vibronic coupling. Indeed, reproducing a spectrum from the equilibrium geometry of the chromophore, even if taking into account the different possible conformers, is not sufficient to reproduce the correct observed band-shapes, and most importantly can induce significant shifts in the calculated absorption or emission wavelengths. 1 Those aspects can be easily understood considering that the nuclear vibrations may induce symmetry breaking, driving intensity towards formally forbidden transitions, such as the n-π* ones. Moreover, large scale movements, such as out-of-plane vibrations of conjugate aromatic systems, may act differentially on the ground or the excited state stability and hence result in a non-negligible shift of the maximum absorption or emission wavelengths. 12,13 The formal quantum treatment of the vibronic effects would require the calculations of the Franck-Condon coupling, i.e. the overlap between the vibrational wavefunctions of the ground and the excited states. Even if some algorithms allowing such a treatment exist, their practical use is sometimes complicated and unstable. [14][15][16][17] In addition large-scale non-harmonic vibrational movements can usually be treated in the limit of the classical approach, hence allowing other strategies.

In particular, we examined the possibility to explore the conformational space of different chromophores, in solution or in complex environment, by means of classical molecular dynamics. The final spectrum will then be constructed as the convolution of the vertical transitions obtained from a set of randomly chosen snapshots to assure a meaningful statistical sampling. This strategy allows to easily taking into account, at QM/MM level, the complex molecular environment, but it necessitates the parameterization of force fields. An alternative solution, that we also examined, is based on the sampling of the phase space by the calculation of a Wigner distribution 18 from the harmonic vibrational frequencies. Obviously, in this case one needs to preliminary obtain the equilibrium geometry while the solvent can be best treated with continuum models. In the latter case the extension to the calculation of emission properties is straightforward 13 only necessitating the geometry optimization and the calculation of vibrational frequencies on the excited state potential energy surface (PES).

In the following we present some examples showing the crucial role of the inclusion of vibrational and dynamic effects in the determination of the optical properties of important chromophores. We will also clearly show that our strategy allows a straightforward extension to the calculation of non-linear optical properties such as two-photons absorption (TPA). Furthermore, the exploration of the potential energy surfaces can also be used as a starting point to preview the most important paths on the excited states' PES leading to the appearance of important photophysical properties.

Section 2.3 Let the States Evolve: (Non-Adiabatic) Excited State Dynamics

As we have seen in the previous section, from the one side the exploration of the conformational space is crucial to rationalize the optical properties of complex chromophores, and on the other side the elucidation of the photophysical phenomena is a non-trivial procedure that in many cases necessitates considering different and competitive pathways.

These considerations are even more pertinent in the case of the study of the photosensitizations of biological compounds that usually requires the population of the triplet manifold via intersystem crossing. 19 Furthermore, the evolution of the excited states happens usually on vibrational hot surfaces and is hence accompanied by a strong solvent relaxation to accommodate the energy dissipation. From an experimental point of view, the excited state evolution is studied by means of time-resolved and pump-probe spectroscopies that monitor the temporal evolution of specific spectroscopic signature, such as excited state aborption (ESA), stimulated emission (SE), or photoproduct absorption (PA). Their analysis and the required fitting to kinetic models allows to access excited state life-times as well as quantum yields and to propose meaningful relaxation mechanisms and pathways via the identification of key, short-lived, intermediates. The possibility to achieve a one-to-one mapping between molecular simulation quantities and time-resolved computational data would allow bringing an unprecedented molecular and electronic resolution to the spectral determination of the life-time and the kinetic models.

In the following, we will firstly illustrate, via the use of the paradigmatic case of benzophenone, the effects of solvent relaxation on the ultrafast spectral responses. 20 The evolution of the benzophenone excited state is followed via QM/MM Born-Oppenheimer molecular dynamic on the S 1 state potential energy surface. In particular, we will underline an important reinforcement of the hydrogen-bond networks taking place in the excited state, that is coherent with the excited benzophenone increase in basicity, and that give raise to important spectroscopic signatures at long wavelengths both in the linear and 2D spectra. 21,22 Subsequently, we will show how the use of state-hopping dynamics, especially including arbitrary coupling, 23,24 and most notably the spin orbit coupling (SOC) allows to define a proper kinetic model for the efficient, quasi-unitary, intersystem crossing of benzophenone. 25 Most notably we will show that the efficiency of the process is due to the quasi-degeneracy of the S 1 , T 1 , and T 2 states. 26 More importantly, we also show that the triplet population proceeds via an equilibrium between T 1 and T 2 with the latter being a fully accessible state and not a simple intermediate, accounting for about 10% of the total population. This finding will also be of extreme importance to unravel the photosensitization processes of benzophenone as it will also be illustrated in the following chapter. Furthermore, the experimental estimated life-time, that have been erroneously attributed to T 1, will be corrected taking into account both the quasi-degeneracy of the two triplet states and their different ESA probabilities, that ultimately lead to the total masking of the processes involving T 1 by the brightest T 2 .

Chapter 3 DNA Photosensitization Processes

DNA photosensitization 1,2 is a fundamental problem since it can lead to photoinduced energy-or electron-transfer ultimately resulting in irreversible lesions of the nucleic acid, and hence in deleterious outcome for the cells and the living organisms such as mutation or death. Furthermore, as photosensitizers usually absorb at longer wavelengths than the DNA nucleobases, they enlarge the harmful spectral window of the solar light.

Hence, understanding DNA photosensitization is not only a challenge for molecular modeling but is also crucial to identify novel protection strategies against DNA threats, and also to rationally develop novel potential phototherapeutic drugs.

Photosensitization is indeed a complex process that necessitates high-level and cutting-edge computational and experimental methods to be correctly treated. Indeed, after light absorption the chromophores may evolve through a number of competitive pathways, most notably including intersystem crossing leading to the triplet manifold population. Subsequently, the triplet state manifold can directly transfer energy or electrons to the DNA nucleobases (type I photosensitization) or induce the formation of the high reactive singlet oxygen that will then oxidize the DNA (type II photosensitization). 1,3 However the former is only an extremely simplified scenario and other complex photochemical pathways can come into play including hydrogen transfer processes 4 or coupled proton electron transfer that can lead to strand breaks or tautomerization. 5 Moreover, one should take into account that all those complex processes happen in a non-homogeneous environment that can significantly alter the drug photophysics and photochemistry and therefore cannot be neglected. Furthermore, in most of the cases the aggregates between photosensitizers and DNA are not precisely characterized and their structure should be modeled by using appropriate molecular dynamics techniques.

In the following, I will present examples in which different photsensitization pathways have been fully unraveled using molecular modeling and simulation with the appropriate multi-scale protocols. In particular, we will show how the interplay between structural features, chromophores coupling, and allowed photochemical pathways is fundamental to fully unravel the complex processes of the photosensitization.

Section 3.1 Ruthenium Complexes and the Light Switch Effect

Ruthenium complexes are known to interact with DNA via intercalation (i.e. slipping between two base pairs). The interaction with the environment produces a very impressive change in their spectroscopic properties giving raise to the so called "light-switch effect". 6,7 Complexes that are not luminescent in water become extremely luminescent when DNA is added, furthermore the behavior can be completely reversed by a small change in the ancillary ligands, with luminescence being now quenched by DNA. Even more interestingly in the latter case the quenching is accompanied by a photo-induced charge transfer from a DNA base (most probably guanine) to the Ruthenium complex leading to a irreversible oxidative lesions that can be exploited in phototherapy. 8 In the following we will present the QM/MM determination of the spectroscopic properties of Ruthenium complexes embedded in DNA, showing that our strategy is able to correctly reproduce the experimental results. 9,10 The analysis of the vertical transitions has also shown a quite complex mixture of metal-to-ligand (MLCT) charge transfers coupled with inter-and intra-ligand charge transfer. Interestingly, by enlarging the QM region to include two DNA base pairs, although the convoluted spectrum is overall quite similar to the one in which only the Ruthenium complex belongs to the QM region, several transitions show an important amount of charge transfer from guanine or adenine to the Ruthenium complex (up to 0.5 electrons, already in Franck-Condon region). 11 Noticeably, our result represents the first evidenced photo-induced charge transfer taking place from excited singlet states between DNA and Ruthenium complexes.

Subsequently, we present the rationalization of the light-switch phenomena based on the change in the nature of the triplet MLCT states produced by the DNA environment. 12 Indeed, while water solution favor the population of MLCT states centered on the dark ligands, DNA induces population of triplet states whose spin density is reorganized on the bright ligands, therefore justifying the luminescence.

Section 3.2 Organic Photosensitizers: Find the Right Interaction Mode

Even if the excited state manifold, and hence the ensuing photophysics, of organic compounds can appear much simpler as compared to the ones of organometallic dyes, one should consider that while crystallographic structure of the DNA/sensitizer adducts exist for the latter, 7 they are almost absent for the former. 2 Hence, the first problem one has to solve is the structural resolution, also considering that non-covalent interactions may lead to several competitive binding modes.

In this case, the use of molecular modeling and simulation becomes crucial to propose reasonable interacting modes that will further allow the study of the photophysic phenomena. Indeed, it is obvious that different binding modes will produce different coupling between the involved chromophores, i.e. the sensitizers and the DNA nucleobases that can lead to totally different photochemical outcomes.

In the following, we will present the molecular dynamic study of benzophenone interacting with DNA duplexes. Most particularly, we will identify a competition between a classical, i.e. minor groovebinding, and a totally novel interaction mode. The careful analysis of the latter called double insertion, and characterized by the ejection of a full base pair from the DNA double-helix, also allowed to identify the energetically favorable interactions that explain its stability despite the huge helical deformation. 13 However, a simply qualitative estimation of the binding-mode is not entirely satisfactorily to interpret the experimental results, and to extrapolate a population ratio of the two modes. Hence, again in the case of benzophenone, I resort to high-level biased sampling molecular dynamics techniques, such as alchemical transformation free energy perturbation, 14,15 to obtain an estimation of the different binding free energy. Although, some difficulties due to the flexibility of the DNA were encountered we were able to clearly preview a significant thermodynamic preference for the minor groove-binding. 16 In order to proove the generalizability and robustness of our free-energy protocol we will also present its application to the very well known DNA-binder netropsin, for which experimental binding free energy are known. 17 In this case, instead of an alchemical transformation, and partially to avoid the problems related to the DNA flexibility, we turned towards the use of the extended adapted biased force (eABF) 15 methods with geometrical transformations, that allowed to overcome many of the problems experienced so far.

Section 3.3 Organic Sensitizers: Transfer Processes and Singlet Oxygen Activation

Once the structure of the DNA/drug adduct has been determined, one should take into account the possible sensitization mechanisms happening between the excited photosensitizers and the DNA nucleobases. In this Section we will mainly consider triplet-triplet sensitization, based on energytransfer involving thymine, and type II photosensitization involving the production of singlet oxygen from the sensitizer triplet manifold.

Again taking into account benzophenone interacting with DNA, we will present a QM/MM study clearly unraveling the energy-transfer mechanisms leading to triplet-triplet energy transfer, i.e. the most favorable photophysical channel for this drug. 18 Indeed, our study is recognized as one of the first direct computational observations of DNA photosensitization mechanisms. It is important to note that while we preview an easy triplet transfer from both T 1 and T 2 in the case of double insertion, only the transfer channel involving T 2 is open in the case of minor groove binding. As already pointed out in the previous section, 16 the latter represents the most favorable interaction mode, and hence once should answer the question on how photosensitization can indeed proceed from the T 2 state. This fact can however be explained by our non-adiabatic dynamics of benzophenone that has unequivocally shown that T 2 is persistently populated and should be regarded as a real active spectroscopic state rather than as an intermediate. 18 Furthermore, we will also present the mechanisms leading to the environmentally controlled production of singlet oxygen in the presence of DNA by the naturally occurring palmatine drug. 19,20 The latter environmental tuning is driven by the modulation of the energy level of a singlet chargetransfer state, whose low energy in the case of water solution leads to its population ultimately resulting in the deactivations and impeding intersystem crossing and the population of the triplet manifold.

Section 3.4 Non-Canonical Photosensitization Mechanisms

In addition to the most classic and celebrated sensitization mechanisms involving either energy-and electron-transfer, or singlet oxygen activation, one should also consider the possible opening of entirely different photochemical channels, including hydrogen abstraction 4 or sensitizers photodissociation that can also lead to the production of irreversible DNA damages.

In this section we will concentrate on the hydrogen abstraction by photoexcited benzophenone either from the sugar backbone or the DNA nucleobase. In particular, we will show how, once again, the complex interplay between structural and electronic factors will govern the outcome of the reaction. Indeed, while the hydrogen abstraction is energetically favored, and should proceed with only a small barrier in solution, the DNA structure acts keeping the benzophenone sufficiently far away from the reactive hydrogen, hence strongly limiting the occurrence of the former reaction. 21 As a second important example we will also consider the case of non-steroidal anti-inflammatory drugs (NSAIDs), such as ketoprofen and ibuprofen, that can indeed produce DNA sensitization either via triplet-triplet transfer, for ketoprofen, or via the easy photodissociation of their carboxylic group ultimately leaving a very reactive carbon radical in close proximity to the biological structures. [22][23][24] Those results, that have also been confirmed via cellular assays, point to the necessity to define specific protective strategies in particular concerning the use of topic application of NSAIDs followed by sun exposure.

Finally, we will also consider the possibility that DNA lesions, such as 6-4 Photo-Product (64-PP) may also act as internal sensitizers or "Trojan horse". 25 Most specifically we will show that the 64-PP displaces the DNA absorption towards the red and is subsequently able to induce triplet-triplet transfer with nearby thymines. 26 Our works confirms experimental observations previously reported 25 and suggests the possible occurrence of clustered photolesions whose repair could then be extremely complex.

Chapter 4 Understanding Complex DNA Lesions Recognition by the Repair Machineries

Although a necessary first step, understanding the production of DNA photo-or oxidative-lesions is by far not sufficient to rationalize the molecular bases of the biological outcomes induced by such damages, and hence understanding the danger they can represent. Indeed, and once again in a very simplified scheme, one should at least take into account the interplay between DNA damages production, mutagenic potential, and repair. [1][2][3] As a rule of the thumb, a lesion that is very easily repaired by the cellular machineries, even if produced very frequently, will not be accumulated, and hence will not be related to genome instability, mutation, or cellular death. 4 On the other hand, lesions that are hardly repaired but that are characterized by the blockage of the replication fork will most probably induce cell death but will not lead to mutations, or even to cangerogenesis.

As said the previous scenario is indeed a very simplified one, not taking into account all the complex pathways active in cellular environment and related to signals, epigenetic genes expression, and crosstalks that can be modified by the presence of DNA lesions. [5][6][7][8][9] In addition, in many cases DNA lesions are not isolated, on the contrary they can accumulate in socalled cluster lesions, i.e. in close spatial proximity in the DNA sequence. 10,11 This accumulation can be due either to the "Trojan Horse" effect of some photo-lesions, 12,13 as described in the previous Chapter, or to the production of reactive oxygen species, by either photosensitization or ionizing radiations, in spatially limited hot-spots. 14,15 The presence of DNA cluster lesions has been shown to significantly alter the repair efficiency, with different effects depending on the type of lesions, the repair machinery (BER or NER) involved, and the organism. 2,11,[16][17][18] In many cases the lesions recognition, that is usually the necessary triggering event of the repair process, is exerted considering the specific structural deformation induced in the DNA structure by the lesion itself. 19 This is particularly true for bulky lesions processed by the NER system. The presence of clusters can induced unexpected coupling between the lesions resulting in totally different structural outcomes that may, hence, prevent a good recognition.

In this case the use of molecular dynamics simulations may provide the necessary atomic resolution to rationalize the different structural outcomes of complex DNA lesions, while also providing hints on the modification of the DNA/repair protein interface and hence the influence on the recognition patterns.

In the following we will present some high-level molecular dynamics study on the behavior of cluster lesions, and photolesions in order to provide the elucidation of their repair, or more precisely recognition, mechanisms and understand the specific pathways and the influence of the lesions.

Section 4.1 Repair of Clustered Abasic Sites

As a first case we will present the influence of clustered abasic site (AP) on their repair processing. Let us remind that the latter lesion, characterized by the excision of an entire nucleobase, is a frequent outcome of oxidative stress by reactive oxygen species, and may also result as an intermediate of the initial base excision repair process. 20 It has been reported that the processing of cluster abasic site may be extremely different as compared to the repair rate of the single damaged strand. 21,22 In particular it has been shown that in the case of bacteria such as escherichia coli closed-by abasic site may reduce the repair efficiency of more than 40%. This situation is different in the case of human repair protein, which is characterized by a globally higher efficiency, but for one specific cluster whose repair is even lower than the one for the corresponding bacterial system. Molecular dynamics of the solvated DNA strands containing a single or clustered abasic sites have allowed to correlate the important deformations to the repair rate in the case of bacterial enzymes. 23 However, in the case of the human systems one need to explicitly take into account the interaction between the damaged poly-nucleotide and the repair enzyme, since the latter is exerting much stronger structural constrains as compared to the bacterial system. In particular we have evidenced that the problematic case for the human repair machinery, can be correlated to the interference of the secondary lesion with the recognition region of the enzyme producing a global destabilization of the protein/DNA aggregate inducing also important allosteric modifications. 24

Section 4.2 Repair of Photolesions

The most important and common DNA photolesions, produced either via direct absorption of UV light, or after triplet-triplet photosensitization, are the so-called cyclobutane pyrimidine dimers (CPD) or the 64-PhotoProduct (64-PP). 1,25 Both lesions involve mainly pyrimidine, due their higher photoreactivity, and produce intrastrand dimerization of π-stacked nucleobases.

However, CPD appears as extremely repair resistant and their accumulation in the cell is quite known. CPD are mostly related to the blocking of the replication fork, and as a consequence their mutagenic potential is relatively low. On the contrary, 64-PP is much well repaired but is also extremely mutagenic. 26,27 We performed µs scale molecular dynamics, including parallel tempering, in order to unravel the molecular bases of such a behavior. 28 Indeed, CPD strands are extremely rigid and their behavior is almost undistinguishable from the ones of undamaged DNA, hence their recognition by the repair machinery is extremely complicated. On the contrary, 64-PP is so flexible to give raise to real polymorphisms with a constant shifting between very different conformers. If their structural modifications explain the more facile recognition by the repair enzymes, its flexibility also allows escaping the replication fork blockage and hence explains its mutagenicity potential. It is important to note that 64-PP polymorphism also include both high-and low-bent structure, and hence allows to reconcile conflicting experimental observation between NMR and Förster Resonance Energy Transfer (FRET) techniques that were pin-pointing one or the other conformer.

Even if CPD are globally worse repaired than 64-PP, their process rate is not uniform. 26 More specifically, thymine dimers are much worse processed than the corresponding cytosine. To understand such a behavior we performed molecular dynamic simulation of a CPD containing strand in interaction with the NER DDB2 unit, responsible for the recognition. It definitively turns out that the specific protein/DNA interactions taking place with the thymine dimers are less strong than the ones exerted with the cytosine one leading to a better recognition of the latter. (Note that this work is presently submitted and we present the final version of the manuscript).

Chapter 5 Conclusions and Scientific Project

Also by considering all the results, and the different strategies, presented in this manuscript I think one can clearly assume that molecular modeling and simulation have nowadays achieved a sufficient enough degree of maturity allowing to clearly answer to relevant chemical and biological questions, also providing an unprecedented molecular, and even electronic, resolution. 1,2 Indeed, the complex pathways leading to DNA lesions productions can be rationalized, taking into account the different environmental effects tuning and conditioning the processes. As an example we may cite the elucidation of the light-switch effect of Ruthenium complexes interacting with DNA 3,4 or the clarification of the environment dependent production of singlet oxygen by some phototherapeutic drugs. 5,6 More importantly, the accuracy and the complexity achieved by the simulation methods allows to take into accounts, at least partially, also the different complex pathways that characterize complex biological environments, with most notably the interplay between DNA and repair enzyme in defining the toxicity of a given lesion. [7][8][9] Up to a certain extent, we can consider that the prediction concerning the use of the computational microscope 10 to elucidate chemical problems has been entirely fulfilled and even overcome. Furthermore, the computational microscope 10 has achieved even an electronic resolution, 11,12 thanks to the phenomenal development of the QM/MM methods, and on the other side the temporal scale has also increased considerably. This in turn, may, among the other aspects, help in a totally novel approach to the rational design of novel therapeutic strategy and drugs, for which modeling does not confine only to the screening of the relationship between structure and reactivity as usually provided by molecular docking. Instead, it becomes a fundamental actor in the definition of the scientific problem and in bringing the necessary answers, also providing a necessary and complementary point of view compared to the experimental determinations. In parallel, the treatment of more complex phenomena will also call for the necessity to take into account the complex networks of communication and cross talks coming into play.

It is clear that such an advancement calls for many challenges both from the methodological and the applicative side. Indeed, a totally novel relationship between researchers of different domains will be necessary, and a fully interdisciplinary approach should be pursued involving theoretical and computational chemists, experimental chemists, and biologists. Once again, I believe that such a strong interdisciplinary environment will certainly produce, at medium-or long-term, a major gamechange in the chemical and biological sciences frameworks. Furthermore, and in addition to the novel and interdisciplinary relationship, a strong methodological development will also be necessary to pursue the advancement and answer to the most challenging questions. 1,13 This will include an algorithmic development to increase the computational efficiency, but will also necessitate entirely novel methodological development especially for what concerns the problem of the statistical sampling, the treatment of complex and crowded environment, 14 and the treatment of considerable volume of data. In particular, considering the last point the possibility to resort to modern machine learning, or deep-learning, algorithms [15][16][17] will be crucial for the development of highly precise force field or for the elucidation of all the possible connection that characterize biological phenomena and that should be taken into accounts.

Keeping those general considerations in mind, and on the basis of my competences and my personal interests the core of my scientific project can be summarized as follows:

"To provide a fully multiscale modeling of photophysics and photochemistry in complex (bio)environment, including a relevant sampling of the initial conditions and the elucidation of the outcomes of the light-induced perturbations."

Obviously photo-induced phenomena include linear and non-linear absorption and emission, but also all the energy-and electron-transfer processes as well as photochemistry.

If QM/MM protocols, especially when coupled with MD, allow to efficiently model the absorption spectra and analyze the nature of excited states, 2,5,18,19 the emission spectra, 20,21 especially because of the necessity to consider the relaxation of the environment, are still much more challenging. A possible strategy could be to perform a systematic parameterization of force fields for the different excited state to achieve a good sampling of the excited state conformational space via MD and subsequently perform QM/MM on selected snapshots to obtain emission wavelengths.

In the framework of photochemistry, electron-transfer [22][23][24][25] represents a very challenging problem since the presence of complex environments necessitates reconsidering the usual Marcus theory. At the same time electron-transfer also represents a case for which DFT can suffer sever drawbacks 26 even with the most recent functionals. On this aspect, I am presently working on the combination of free energy techniques with biased QM/MM dynamics to provide energy profiles for the ground and the involved excited states along the most relevant coordinates. Obtaining relevant energy profiles, while combining a good electronic structure modeling and a conformational sampling, will allow reconsidering Marcus problem and the most relevant parameters such as the electronic coupling and the reorganization energy. This topic is presently carried on in the framework of a French-Austrian ANR consortium and is applied to the case of a Rhenium sensitized protein experiencing extremely efficient and long-range electron-transfer. 19,[27][28][29][30] The possibility to use localized self-consistent field (LSCF) 12,[31][32][33] methods to obtain constrained states will be also exploited to facilitate the treatment of electron-and energy-transfer phenomena. Indeed freezing orbitals on different molecular fragments will allow us to obtain a sort of diabatic states mediating charge-or energy-transfer processes. Note that this can be considered an alternative way to achieve a constrained DFT (cDFT) [34][35][36] solution. Most notably these constrained states will not suffer from the problem of the too large delocalization typical of DFT. Subsequently it will be also possible to use the diabatic states as the basis for a configuration interaction (CI) formalism to obtain the corresponding adiabatic states. Most notably, the LSCF constrained DFT formalisms will be much more general than the usual constrained DFT techniques. Indeed, it will also allow to correctly representing, in combination with the pseudo-CI combination of the diabatic states, all the kind of conical intersections and non-adiabatic points eventually characterizing the potential energy landscape, even those not related to charge-or spin-transfer phenomena. As an example one can imagine the possibility to treat at DFT level conical intersection characteristic of double-bond isomerization. As a consequence, it is evident that the aforementioned methodological development will pave the way to a novel use of DFT for the study of computational photophysics and photochemistry.

Another direct applications of these new approaches will be the complete modeling of 2-dimensional electron spectroscopy (2DES), [37][38][39][40][41][42][43] which could be directly applied to the study of photosensitized DNA. 44,45 Indeed, 2DES, recently extended to the visible and UV wavelengths domain, allows to directly observe all the transfer phenomena, thanks to the information embedded into the off-diagonal peaks, and therefore will shed light on many fundamental chemical and biochemical relevant processes. Moreover, since 2DES is very sensitive to the coupling between different chromophores, and hence to their relative orientation, it could be efficiently used as a probe to monitor the different conformations undertaken by the DNA/photosensitizer complexes. However, the rich density of information contained into 2DES spectra makes their direct exploitation quite complicated. For this reason a one-to-one mapping with the results stemming from molecular modeling is mostly welcomed, and calls for an important role of the theoretical chemical community. 41,42,[46][47][48] Indeed, to achieve the proper simulation of 2DES in complex systems an important methodological development will be required in particular concerning its extension to the (Time Dependent-)DFT framework, that can again take advantage by the use of LSCF to impose constrains on the electronic density, and hence access relevant high-lying, or doubly excited states. I also plan to strongly invest myself in the development of more efficient state-hopping dynamics algorithm 49,50 allowing for the efficient treatment of problem in which an energy barrier in the excited state prevents an easy accessibility to the non-adiabatic points driving the photochemical phenomena. More in detail I plan to combine the use of biased techniques such as adaptive biased force (ABF) 51,52 with non-adiabatic dynamics, in the spirit of what is done to recover the free energy landscapes for ground state problems concerning either conformational flexibility or chemical reactivity. However, a proper statistical post-process will be necessary in order to keep the time resolution giving access to excited state life-times that represents one of the key results of the non-adiabatic dynamics. Such advancement will certainly allow treating completely unexplored area of photochemistry and photophysics. The methodological development will be integrated in largely popular MD codes, such as for example NAMD, 11,53 taking profit of the existence of biased MD algorithm, 54 and also facilitating the diffusion and wide spreading of the novel techniques, that I believe could contribute to define a novel paradigm in computational photochemistry.

In the following years I will continue the study of DNA photosensitization and photolesions production and outcome. As stated all along the present manuscript, DNA photosensitization requires to consider the structural and dynamic properties of the aggregates as well as their photophysics and photochemistry, moreover spectroscopical signatures able to discriminate between different competitive non-covalent DNA/sensitizers [55][56][57] complexes will be looked for and properly modeled to help and guide the experimental resolution of the different structures.

More specifically I will particularly be interested with sensitizers active in two-photon absorption (TPA), [58][59][60][61] whose cross-section will be modeled by QM/MM procedures. 62 This will be motivated by the fact that TPA will allow to significantly increase the absorption wavelength, hence reaching the therapeutic window, while also increase the spatial selectivity thanks to the non-linear nature of the process. Furthermore, we plan to computationally study and suggest photodynamic therapy agents whose photochemistry will not involve the activation of singlet oxygen but instead a direct reaction with DNA or cellular components. This will allow treating solid tumors, known to develop hypoxia, 63 more efficiently and will most probably allow to propose novel lead compounds for anticancer treatments. In this contest non-canonical photosensitization pathways, in particular leading to photodissociation will be privileged through the use of psoralene 64 or binitroimidazole 65 core units, while the TPA activity will be brought by specific antennae assuring a high cross section. The latter could be achieved for instance by the sensitization with novel compounds based on polythyophene core that has shown remarkably high TPA cross sections in the therapeutic window. 59,60 The interaction of the sensitizer, both free and labeled with TPA antennae, with DNA will be considered, also taking into account the calculation of binding free energy. Furthermore, linear and non-linear optical properties, in presence of the complex DNA environment, will be properly assessed following our established QM/MM protocol. 66,67 The photochemical pathway leading to the labile group photodissociation and hence to the production of reactive radical will also be assessed and properly quantified. Finally, from the one side the reaction of the photoproduct radical with DNA components will be studied, looking mainly for interstrand cross-links formation, and finally the structure of the covalently damaged DNA strand will be monitored via classical MD, also in order to preview the possible consequences on the repair process. This complete strategy will allow us to propose the rational design of novel non-oxygen dependent phototherapeutic drugs, active in TPA.

Furthermore, the use of the newly developed non-adiabatic dynamics protocol will allow to produce for the first time a fully time-resolved picture of the photosensitization process providing life-time and quantum yield of all the relevant processes involved in the photosensitization. The same protocols could also be used for the study of directly induced photo-lesions, in particular contributing to clarify some still open questions related to the influence of hydrogen transfer in the energy relaxation of photoexcited DNA.

However, the sensitization and photochemistry itself only represent a first step in the full process starting with light absorption and ending with cells death. The influence of different DNA environments on the lesion production from one side, and on the lesions structure and repair rates on the other side, need to be taken into account. In this context I especially plan to unravel the influence of complex DNA photo-induced lesions on the nucleotide excision repair (NER) machinery 68 that is still far from being fully understood. Furthermore, most of the time DNA is not free in eukaryotic cells, but is coiled in the crowded nucleosomal environment, 69,70 where is enrolled around histones. Nucleosome from the one side allows the compaction of the DNA to let it accommodate in the limited space of the nucleus. However, from the other side, nucleosomal environment is also fundamental in providing crucial epigenetic signals, through its compaction or the reversible modification of their proteins. 71,72 In the context of DNA damages the nucleosomal environment will open important questions relative from the one side on the structural modifications induced by the DNA lesions in the more constrained histone's environment, and on the other hand to the accessibility by the repair protein. 73,74 Some preliminary studies on a 64-PP containing strand in nucleosomal environment have clearly shown that the polymorphism of the nucleic acid is strongly reduced by the protein mechanical and electrostatic constraint. I plan to perform high-level full-atom molecular dynamics to unravel those structural modifications taking place in case of different lesions. However, considering the size of the system it will be necessary to turn towards a coarse grain description 75 that will allow to access the relevant time-scales needed to provide a reasonable description of the conformational space. The preliminary full-atom molecular dynamics, in addition to provide a first atomic resolution of the process, will also serve as a base for the benchmarking of the coarse-grained force field. Coarse grain model will also allow simulating the approach of repair enzymes to nucleosomal DNA, in particular evidencing the different accessibility between coiled and linking regions of the nucleic acids.

Finally, the interaction between DNA and nucleosomal proteins will also allow us to clarify from the one side the possible emergence of DNA/protein cross-links 76,77 that are an outcome of oxidative lesions, and from the other side the change in the photophysical landscape induced by the highly positively charged nucleosome in particular with respect to the relaxation of charge-transfer state that may lead to hydrogen transfer and tautomerizations. In particular, those latter aspects will also be studied on a simplified model, i.e. the coiled bacterial DNA, in which the compaction is exerted by the much smaller histone-like HU proteins, [78][79][80][81] sharing the same high density of positive charge with eukaryotic histones and also providing a net positive curvature of the nucleic acid. Finally, the nucleosome modification, such us compaction, induced by the lesions will also be considered in the framework of possible epigenetic modifications 82 that could be induced by the accumulation of unrepaired DNA lesions.

The systematic improvement of the parameterization, as well as the treatment of more complex phenomena and the nucleosomal organization, will naturally lead to a mesoscopic description of coiled and damaged DNA, via most notably the use of elastic networks approaches. 83,84 The mesoscopic, and elastic network parameters will also be directly obtained from the molecular dynamics simulation. This later aspect will also take benefit from collaborations to be developed in the framework of the new LPCT department, and will allow to bridge the gap between the atomistic and macroscopic description of biological macrostructure, adding to the generality of the present project.

The epigenetic effect of DNA lesions is, in my opinion, a key factor that is in many aspects not sufficiently understood today. Hence, I plan to study the interactions of transcription factors, [85][86][87] i.e. the proteins responsible for triggering the cascade leading to the gene transcription, with DNA promoting regions, i.e. non-coding highly conserved DNA sequences, This will allow understanding the molecular factors inducing the activation or suppression of a specific gene, in presence and absence of DNA lesions. Promoting regions and transcription factors directly related to cangerogenesis will be particularly considered, and the influence of lesions on the recognition of the promoting region will be rationalized, also taking into account the influence on the allosteric modification of the transcription factor upon DNA binding that represent the first triggering of the transcription signal. [88][89][90] All the project development will be realized with a constant collaboration between theory and experiments. In particular the relationship with time-resolved spectroscopy groups are obvious and fundamental in order to fully characterize all the photoinduced processes. Furthermore, results from structural biology will be beneficial to understand the structural modification of damaged DNA and their influence with the DNA/protein interaction pathways. On the same spirit cellular and molecular biology assays will be necessary to allow the rationalization of the phenomena taking place at cellular level and all their possible interplays.

The combination of methodological development, cutting-edge applications and constant relationships with experimentalists and other theoreticians, also nurtured by the participation to and the coordination of national and international projects, will assure a high level, original and uniform treatment of photo-induced and oxidative phenomena in biological environment. This will unravel the mechanisms underlying many fascinating phenomena and will undoubtedly foster significant development in the design and realization of (bio-inspired) novel smart materials.

One of the positive points of writing an extended manuscript such the HDR is that one is forced to trace back its evolution, both professional and personal, and in doing that one may also reconsider all the persons that have crossed his own path for a short or longer period. And obviously, one may so acknowledge the important traces they have left and they way they have contributed in shaping the road and influence the directions taken.

The number of persons that should be cited here is enormous; I know from the beginning that the effort to name them all is somehow below any reasonable hope. I nonetheless give a try and asking for their comprehension from the forgotten one.

In the first place I would like to thank all the members of the committee who have accepted to judge my work and participate to the defense. All the members are wonderful and inspiring colleagues with whom I sometimes had many stimulating scientific discussions, I am pretty sure we will go on with them in the future. Some of the committee members are also good friend of mine, and this make myself even more proud to have them here.

All this work has been done, and the project will be realized, in the framework of the new LPCT department. I am extremely happy to be part of this new adventure, and shaping the birth of our theoretical oriented unity, it is fascinating and inspiring and allows to significantly enlarge the horizons and to build great novel human relationships. I have had, and I will have the pleasure, to work, more or less intensely, with the most part of you and I consider this an honor and a privilege.

Obviously, some special thanks and thoughts should be sent to the people with whom I work more extensively. I will always thank Xavier for his supportive attitude, his good mood, his scientific discussions, all the beer we have shared, and especially for all the autonomy he has always wanted me to take. I warmly thank François and Christophe for the great projects we are building together, and also for actively participating in the transition allowing me, a former Full-CI guy, to approach the extensive sampling of the millions atoms world.

Chiara, how many time since we first met during our Ph.D., and how far the scientific path has brought each one of us. And especially what a great pleasure to go on working with you.

There is one person without whom this whole laboratory will not exist or will simply fall apart. Séverine will never be thanked enough for all her efforts, for her supporting mood, her friendship, and for always being there when she is needed.

Fabien and Christian are always ready to answer to my strange requests to open accounts or make VPN access for students whose names cannot sometimes be spelled. They are good friends and an important part of this department.

All the work that has been reported here would simply not have been possible without the participation of the wonderful Ph.D. students and post-docs who I add the great honor and the privilege to supervise or supervise. Thibaut, Thibaud, Agi, Julian, Hugo, Marco, Elena, Toni, Cécilia, Florian, you make an incredible dream team, your successes are one of the things that make me more proud, but I am specially honored to call you good friends of mine.

In addition I also had the opportunity to supervise the work of different Master and Bachelor students or foreigner Ph.D. visitors. Some of them are still working on their formation, some have chosen to go on with science, and some have taken other paths. I nonetheless thank them all for their work, their attitude and their efforts: my Turkish connection (Burcu, Taylan, Yigitcan, Esma, Özlem, Öykum, Büsenur, Oghuzan, Basak, Rengin), Elodie D., Elodie G., Laurent, Elise L., Tom, Maryline, Angelo, Nicola, and many others.

In Nancy I also have had the opportunity to work with great colleague from the experimental chemistry (Philippe, Marc, Cristina) and the biology (Stéphanie) department. Their works, our discussions, and all their experiences have been fundamental to achieve some of my success, that is why I think them greatly and I hope our collaborations will go on. Even if I am sometimes scared when I realize that I now understand the biologist language.

Science is never a one-man-show conducted in its golden isolation; instead it is a matter of exchange and sharing of experiences and collaborations. And through collaborations one sometimes develops also friendship. I have had the great opportunity to develop impressively inspiring collaborations in France or elsewhere of whom I am particularly proud; to cite just a few; Daniel, Inaki, Giampaolo, Saron, Viktorya, Andrea, Leticia, Eric, Cathrine, Jean-Luc, Thierry. And of course a special thank is devoted to Chantal, who bears the huge responsibility of having introduced me to the DNA world. I hope she is not regretting it too much.

Obviously, there is a person who owns a particular place. I will never thank enough Elise D. for having built a so strong scientific collaborations that some people were indeed thinking we had a Nancy-Lyon Labex; her role is fundamental in almost everything that is related in this manuscript. But especially I thank her for her friendship and her support that indeed mean a lot to me.

Since the last couple of years I also had the opportunity to explore the secret background of the French Research system by serving in the "Comité National". It is an incredible enriching experience that I will also look at with pleasure and proud. A large part of this is also due to the fact that I can share it with my partners in crime for the 13 th and 51 st sections. It is always great to meet in you in Paris, either in the windowless bunker of the CNRS or in front of a glass.

Nancy is special for many reasons. In this campus one can end the working day sharing a good Belgian beer between teachers and students in a friendly atmosphere. For this the glorious 40-yearsold Phi-Science student association should be held responsible and should be thanked. They should also be thanked for naming a topological index to describe the excited-state behavior (φ S ).

The other reasons is that in no other place I would have meet such good friends and inspiring persons to hang out together, share one experiences and difficulties, and holding on together. Antoine, Ilke, Daniel, Lucie, Hatice, Gamze, Maura, Sara, Lea, Magda, Benjamin, Sébastien, Andreea, Fernanda, Audrey, Alekos, Dorota, and all my students and post-docs; you are great friends and you made me feel home everywhere I was. I could have never foretold the evolution of my scientific career during my Ph.D., but when I now look back I understand that for what I have achieved I should thank my supervisor Gianni, for the scientific formation he has given me and for letting me taking my choices in total autonomy. Stefano, on the other hand was the one who convinced me to move to France I could never thank him enough.

Finally, many thanks to my family to have always supported me, even when I made bad choices, and allowed me to achieve what I wanted, even when it was not so easy for them.

  Large scale molecular dynamics, free energy methods, DNA lesions Principal External Collaborations • Elise Dumont (ENS Lyon, France) o Theoretical modeling of DNA lesions and repair • Jean-Luc Ravanat (CEA Grenoble, France) o Characterization of DNA lesions • Chantal Daniel (Universié de Strasbourg, France) o Theoretical photophysics of metal complexes • Stefan Haacke (Université de Strasbourg, France) o Time resolved and 2D electronic spectroscopy • Leticia Gonzalez (University of Vienna, Austria) o Non-adiabatic molecular dynamics • Marco Garavelli (Università di Bologna, Italy) o Computational 2D spectroscopy • Daniel Roca Sanjuan (Universidat de Valencia, Spain) o Theoretical photophysics and photochemistry • Saron Catak (Bogazici Universitesi, Istanbul) o Computational chemistry and photophysics • Alex Georgakilas (University of Athens, Greece) o Complex DNA lesions and their biological effects • Giampaolo Barone (University of Palermo, Italy)

• Stephanie Grandemange (CRAN) o Development of novel anticancer drug and biological effects • Philippe Gros, Cristina Cebrian Avila (L2CM) o Photophysics of iron compounds, solar energy conversion • Xavier Assfeld, Mariachiara Pastore (LPCT) o Theoretical phochemistry and photophysics • François Dehez, Christophe Chipot (LPCT) o o Non-canonical DNA structures 5 Main Publications: • Dumont E., Wibowo M., Roca-Sanjuan D., Garavelli M., Assfeld X., Monari A. "Resolving the Benzophenone DNA-Photosensitization Mechanism at QM/MM Level" (2015) J. Phys. Chem. Lett. 6 576. I.F. 9.353 • Gattuso H., Durand E., Bignon E., Morell C., Georgakilas A. G., Dumont E., Chipot C., Dehez F., Monari A.* "Repair rate of clustered abasic DNA lesions by human endonuclease: molecular bases of sequence specificity" (2016) J. Phys. Chem. Lett. 7, 3760 I.F. 9.353 • Monari, A., Rivail, J. L., Assfeld, X. (2013) "Theoretical Modelling of Large Molecular Systems. Advances in the Local Self Consistent Field Method for Mixed Quantum Mechanics/ Molecular Mechanics Calculations" Acc. Chem. Res. 46 596 I.F. 20.268 • Marazzi M., Mai S., Roca-Sanjuan D., Delcey M. G., Lindh R., Gonzalez L., Monari A. "Benzophenone Ultrafast Triplet Population: Revisiting the Kinetic Model by Surface-Hopping Dynamics" (2016) J. Phys. Chem. Lett. 7 622 I.F. 9.353 • Dehez F., Gattuso H., Bignon E., Morell C., Dumont E., Monari A.* "Conformational Students' Supervision (Ph.D. and internships)

  • After the Master he successfully applied for the Ph.D. program under my co-supervision. The works of his Master have been the object of a cosigned publication in Theor. Comp. Chem. • Laurent Chbibi, 2 nd year Master, Université de Lorraine (2013) • The works of his Master have been the object of a cosigned publication in Dyes and Pigments • H. Taylan Turan, Master degree of Bogazici Universitesi Istanbul (2015-2016) • After his Master degree he secured a Ph.D. scholarship at the University of Basel, Switzerland. The works of his Master have been the object of a cosigned publication in J. Phys. Chem. C • Esma B. Boydas, Master degree of Bogazici Universitesi Istanbul (2016-2018) • The works of his Master have been the object of two cosigned publications in Theor. Chem. Acc. and J. Phys. Chem. C • Ozlem Sengul, Master degree of Bogazici Universitesi Istanbul (2016-2018) • The works of his Master have been the object of two cosigned publications in Theor. Chem. Acc. and J. Phys. Chem. C • Elodie Gonis, 1 st year Master, Université de Lorraine (2017) • Cecilia Hognon, 1 st year Master and 2 nd year Master, Université de Lorraine (2017-2018) her works make the object of 3 submitted publications. In addition Cecilia Hognon also secured a Ph.D. scholarship at the Université de Lorraine. • Athena Pactor, 1 st year Master, Université de Lorraine (2018) • Elise Lognon, 1 st year Master, Université de Montpellier (2018) • N. Oykum Avci Master degree of Bogazici Universitesi Istanbul (2018-)Also thank to the active role in securing external funding I had the opportunity to (co)-supervise 5 post-docs fellows

	• University of Alger, Algeria
	o Soraya Abtouche (2013)
	• Université Paul Sabatier Toulouse
	o Adrien Göttle (2014)
	• Université de Strasbourg
	o Stephan Despax (2014)
	• University of Palermo, Italy
	o Angelo Spinello (2016)
	• University of Alcala de Henares, Spain
	o Cristina Garcia Iriepa (2016)
	• University of Valencia, Spain 2
	o Antonio Francés Monneris (2017), Kiryl Zynojev (2017)
	• University of Bern, Switzerland
	o Mariam Nazari Haghighi (2018)
	Teaching Activity:
	• Antonio Frances Monneris, April 2017-
	o The works of Dr. Francés-Monneris are, at the moment, the object of 2 published articles,
	while 3 others are submitted.

• Agisilaos Chantzis, November 2011-October 2012 o The works have made the object of 3 co-signed publications including J. Chem. Theory Comp. After a post-doc in the group of Prof. Jacquemin at the University of Nantes Dr. Chantzis is presently post-doc fellow at the Max Planck Institute In Mülheim, Germany. • Julian Garrec, February 2013-January 2015 o The works have made the object of a publication in J. Phys. Chem. Lett. At the end of his post-doc fellowship Dr. Garrec was hired with a tenured track position at ENSTA ParisTech. • Marco Marazzi, November 2015-November 2017 o The works of Dr. Marazzi have resulted in the publication of 15 co-signed articles in peerreviewed journals including J. Phys. Chem. Lett., J. Chem. Theory Comp., and Nature Sc. Rep. Dr. Marazzi is now holding a tenure track position in Alcala de Hénares University in Spain. • Elena Zvereva, November 2016-February 2017 o The works of Dr. Zvereva have been published in 2 publications, a third being currently in preparation. Dr. Zvereva is presently finishing her maternity leave. Participation to Ph.D. committee I have participated as an examiner or an external expert to a total of thirteen Ph.D. committees both in France and in Europe: • Université de Lorraine Nancy, 5 o Thibaut Very (2012), Sajda Noureen (2012), Thibaut Duchanois (2015), Thibaud Etienne (2015), Hugo Gattuso (2017) I started to be involved in teaching activity during my Ph.D. course in particular concerning subjects related to general chemistry and computer science.

I also continued to be involved with such a commitment in particular through a teaching assistant (ATER) position that I secured at the University of Toulouse in 2009-2010. After my recruitment in Nancy, I obviously continued to exercise my statutory teaching duties. It is however important to precise that thanks to the CNRS "chaire d'excellence" program during the first 5 years my teaching duties were limited to 64 hours per year.

A list of the main teaching activities is given in the following:

• University of Bologna (Italy). Bachelor in Chemistry and Chemical Engineering

• General Chemistry 1 st Year • Université de Toulouse (France) Bachelor and Master in Chemistry • General Chemistry 1 st year Bachelor • Thermodynamics 3 rd year Bachelor • Electrochemistry 2 nd year Bachelor • Spectroscopies 3 rd year Bachelor • Molecular Modeling 1 st year Master • Université de Lorraine (France) Bachelor and Master in Chemistry and Biological Sciences • General Chemistry 1 st year Bachelor • Electronic Structure 1 st year Bachelor • Practical analytical chemistry 1 st year Bachelor • Advanced Theoretical Spectroscopy 2 nd year Master • Photophysics and Photochemistry 2 nd year Master • QM/MM Methods and Molecular Modeling 2 nd year Master

Comme tout système biologique l'ADN est constamment exposé à un grand nombre des facteurs de stress qui peuvent induire de réaction chimique ou photochimiques et donc produire des modifications de sa structure, il s'agit la des lésions de l'ADN. Le rôle biologique de l'ADN étant le maintien, le stockage, et la transduction de l'information génétique il est apparent comme sa stabilité chimique soit essentielle pour assure la survie des organismes vivants. Par conséquences les cellules ont développé des stratégies pour contraster l 'accumulation des lésions de l'ADN, typiquement via l'établissement des mécanismes de réparations complexes mais très efficaces. Il est en effet évident que la toxicité d'une lésion dépendra en fine du rapport entre sa fréquence, la facilité de réparation et les effets induits vis-à-vis des processus de réplication ou de transduction. La compréhension, à l'échelle électronique et moléculaire, des phénomènes liés à la production de lésion de l'ADN va aussi permettre de rationaliser les bases moléculaires subjacentes à des nombreuses pathologies tels que les cancers, et par conséquences de proposer des stratégies de protection ou thérapeutiques efficaces. Il faut aussi rappeler que les mécanismes complexes entrant en jeu dans la production et la réparation des lésions de l'ADN, constituent aussi un vrai défis pour les méthodes de modélisation moléculaire et simulation, et vont donc aussi entrainer un véritable développement méthodologiques, notamment pour permettre un traitement véritablement multiéchelle du problème. Dans le cadre du ce manuscrit nous nous focaliserons sur les lésions photoinduites, en particulier produite par photosensibilisation, c'est à dire induite par l'action d'un composé photoactivable interagissant avec l'ADN. Nous présenterons des études, centrés sur des cas paradigmatiques, prenant en comptes tous les différents effets mis en jeu. En particulier nous traiteront les propriétés optiques et photophysiques des sensibilisateurs isolés, leur interaction avec l'ADN, et les effets de l'environnement macromoléculaire sur la modulation du paysage photophysique. Pour conclure nous nous intéresserons aux phénomènes de réparation de l'ADN, en particulier dans le cas des lésions complexes produisant des réorganisations structurelles importantes. Nous ferons appelle à un large panel des méthodes, pour permettre la description fine des différents phénomènes qui inclurons le dynamique classique et non-adiabatique, les méthodes d'énergie libre, ainsi que les protocoles hybrides de type mécanique classique/mécanique quantique. De plus nous présenterons le développement des nouveaux descripteurs pour permettre une caractérisation de la topologie des états excités

Acknowledgements

submitted to the Lorraine and Aquitaine Regions • Reviewer for the Hungary National Research Agency • Reviewer for the Polish National Research Agency • Reviewer for the Czech Republic Research Agency • Reviewer for the French ANR • Reviewer for the European Research Council (ERC) QMC techniques for the study of complex molecular systems (2011-2015) • ANR and DFG (Germany) Mit-LOW: Study of the metal-insulator transition in low-dimension systems (2012-2015Photo-excited electron transfer in Biological systems (2016-2019) • ANR IronSol (2016-2020) • ANSES (France) Marfem: Effect of external electric fields on the oxidation of biological lipid membranes (2013-2016) • Industrial contract with iS2 sas. Development of novel dyes for dye-sensitized solar-cells (2012-2016) • PEPS Mirabelle Anticancer Iron Made: AIM (2015) • Grand-East Regional Council Grant IDEA: Iron DNA Demethylating Agents (2015) • Bilateral France-Turkey Project "Rationalizing dye sensitized solar cells and water splitting mechanisms" (2015-2017) • Grand-East Regional Council Grant Post-doc position (2018) • Member of the COST in Chemistry Action "Biomimetic Radical Chemistry", "Molecules in Motion" and "StableNextSol" Administrative duties: • Elected member of the French CoNRS National Committee ("Section 13") for Physical Chemistry (2016-) • Elected member of the the French CoNRS National Committee ("CID51") for numerical methods in life sciences (2017-) • Responsible of the "Light Matter Interactions" group of the Theoretical Physics and Theoretical Chemistry Department (LPCT) of the Université de Lorraine (2017-) • Member of the Scientific Council of the Theoretical Physics and Theoretical Chemistry Department (LPCT) of the Université de Lorraine (2017-) • Member of the Department Council of the Theoretical Physics and Theoretical Chemistry Department (LPCT) of the Université de Lorraine (2017-) • Elected member of the Department Council of the SRSMC laboratory of the Université de Lorraine (2013-2017)

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