Elena Atrián-Blasco

Lucie De Cremoux

Xudong Lin

Rufus Mitchell-Heggs

Laurent Sabater

Sébastien Blanchard

Christelle Hureau

Keggin-type polyoxometalates as Cu( ii ) chelators in the context of Alzheimer's disease

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Two Keggin polyoxometalates were used as new copper ligands to counteract the effects of Cu II (Amyloid-β) interaction. Their ability to remove Cu II from Cu II (Amyloid-β), to stop Cu II (Amyloid-β) induced formation of reactive oxygen species and to restore apolike self-assembly of Cu II (Amyloid-β) were shown.

Alzheimer's disease (AD) is a debilitating and devastating mental disorder and the most widespread kind of dementia. Due to the aging of the population and the lack of curative treatments, the number of AD patients will increase dramatically in the near future, putting the health care system under an incredibly huge pressure. 1 This underlines the need for alternatives to the current therapeutic options. AD is characterized by the presence of extra-cellular deposits of highly ordered supra-molecular assemblies of the Amyloid-β (Aβ) peptide, the so-called senile or amyloid plaques in specific regions of the brain. The Aβ peptide indeed belongs to the amyloid-forming peptides family that also include amylin and αsynuclein, involved in Type II diabetes and Parkinson's diseases respectively. 2 The main forms of Aβ peptides are made of 39 to 43 amino acids residues, with the 40 amino-acids sequence being the most studied. In addition to a N-terminal hydrophilic part (up to the 16 residues), the peptide is made of a central hydrophobic core encompassing residues LVVF and of a Cterminal hydrophobic part, both involved in the self-assembly ability of the peptide. This process known as aggregation finally leads to the formation of highly stable β-sheets rich supramolecular structures (amyloids) that gather into the senile plaques. 3 The 16 residues N-terminal sequence contains several metal binding residues (mainly histidine and carboxylatecontaining residues) involved in the coordination of d-block ions, mainly Copper and Zinc. 2 Based on that ability and the abnormally high level of Cu, Zn and Fe found in the senile plaques, and in line with many in vitro studies that have shown a role of metal ions in the modulation of Aβ self-assembly, metal ions are regarded as biologically relevant partner of amyloid formation and related toxicity. 2,4,[START_REF] Hureau | Alzheimer's Disease: Recent Findings in Pathophysiology, Diagnostic and Therapeutic Modalities[END_REF] In addition to the Aβ aggregation, oxidative stress is the other key event in the etiology of AD. Cu-bound to Aβ can catalyze the production of Reactive Oxygen Species (ROS) and thus may contribute to the overall oxidative stress observed in the disease. [START_REF] Cheignon | [END_REF] Polyoxometalates are oxoclusters of early transition metal ions that have found applications in various fields from catalysis to material science. They result from the polycondensation in acidic media of oxoanions such as WO4 2-, templated or not by poxoanions (PO4 3-, SiO4 4-, etc.). Furthermore, controlled basic degradation of complete POMs can lead to lacunary POMs, which are very efficient all-inorganic ligands. Among the vastly proposed biochemical and medical applications of polyoxometalates, [7][8][9] their interactions with peptides and proteins have been gaining interest over the last years. 10,11 For example, Prusiner and co-workers have studied the ability of different POMs, mainly Keggin-type (Scheme 1), to promote the aggregation of the toxic form of the prion protein. 12 The modulating activity of these polyoxoanions relies on their electrostatic interactions with the positively charged amino acids of the protein, while the size of the POM may also play a role. 13 While it has been proposed that various types of POMs could modulate Aβ aggregation via electrostatic interaction, 14- 16 Qu et al. reported that transition metal substituted POMs (MS-POMs) were even more efficient, presumably via additional coordination bond between metal ion and the histidine residues of Aβ. 17 While POMs, including MS-POM, were used to tune Aβ self-assembly, there is no report on the use of POMs as chelating agents (in the context of AD), although giant POMs were shown to alter Cu(II)Aβ and Zn(II)Aβ aggregation due to multiple weak electrostatic interactions with metal ions. 16 Up to now, no lacunary POM has been studied for its ability to host The thorough inspection of the UV-Vis but mainly the EPR spectrum of the complex formed by the addition of K 4-or Kℓ 8-to Cu(Aβ) slightly differs from that of the in situ generated KCu 6- (without Aβ). An additional small peak at 335 mT is indeed detected (Figure 1, panel C, blue vertical line). Linear combinations of the EPR spectra of Cu-Aβ and KCu 6-, which could mirror the incomplete removal of Cu(II) from Aβ by the POMs, failed to reproduce this EPR signal thus pointing strongly toward the formation of a ternary species. While deciphering its exact nature is beyond the scope of the present communication, we propose that this species corresponds to a complex between KCu 6-and Aβ, where Aβ may bind the Cu(II) ion on its sixth labile position. This is confirmed by the increase of the relative intensity of the peak induced by the presence of higher ratio of Aβ versus KCu 6-(Figure 1, panel C). This proposition is in line with the lability of the water molecule bound to the MS-POMs, 21 and with reported interaction of Cu(II) in MS-POM with the imidazole from histidine side-chains of Aβ peptides. 17 Since K 4-and Kℓ 8-can bind Cu(II) out from the Aβ peptide (although with different rates), their ability to stop the Cu(Aβ)induced ROS production was assayed. This was done with a well-established ascorbate (Asc) consumption experiment that mirrors the production of ROS. 22 Briefly, under aerobic conditions, Asc fuels the incomplete reduction of dioxygen and thus the formation of ROS. The Asc consumption is catalysed by the presence of Cu II or Cu II (Aβ) leading to the decrease of the absorbance at 265 nm (corresponding to ascorbate, ε = 14500 M -1 cm -1 ). Two experiments were performed, either starting from the +II redox state and including a short incubation with the POM (c.a. 60 s) or with the POM added in the course of Asc consumption (when absorbance equals 1), thus in presence of both Cu I and Cu II ions. Kℓ 8-can prevent the Asc consumption, both starting from Cu II and Cu II (Aβ) or when added in the course of the experiment. There is indeed no decrease in the Asc UV band in presence of Kℓ 8-(Figure 2, panel A). In contrast, K 4-is active only if incubated before Asc addition (Figure 2, panel B), in line with the longer time required to form the KCu 6-species. In addition to its participation to oxidative stress, Cu(II) also affects the aggregation path of the Aβ peptide. 23 This will be under focus in the coming section. The self-assembly of Aβ is a very complex and multi-step mechanism, which can be viewed as a nucleation-elongation supramolecular polymerisation process, in which secondary nucleation by formed amyloids takes a predominant part. 24,25 The low molecular weight intermediates present during the aggregation are currently regarded as the most toxic species (compared to the starting monomer and the final fibrils) and Cu II has been proposed to promote their formation. 26 To monitor Aβ aggregation, the thioflavin-T (ThT) fluorescence screening assay was used, 27 and the morphology of the aggregates formed was observed after 6 days using Transmission Electronic Microscopy (TEM). Note that as recently pointed out, due to the intrinsic auto-catalytic nature of the aggregation process, the reproducibility of aggregation experiments is fairly weak. 28 To secure robust results, we have thus repeated several times the aggregation assays and have changed several environmental parameters, mainly to avoid the so-called "batch-dependent" results. The results are described based on the data shown in Figure 3 that are representative of the trends observed in all the experiments, and the range of values of the key kinetic parameters of the 3 mathematically-analysed experiments are given in Table S1 (see also Figure S2 for an illustration of the fitting). The aggregation curves for the apo-Aβ peptide ‡ show the expected sigmoidal shape (Figure 3, black line) characteristic of amyloids formation, 25,28 with the time evolution of the fluorescence intensity being described by 𝐹(𝑡) = ∆𝐹 (1+𝑒 -𝑘(𝑡-𝑡 1/2 ) (Eq. 1) . t1/2 is the time necessary to reach half of ∆𝐹, the variation of ThT fluorescence intensity; k is the growth rate mirroring the formation of fibrils. Overall, in presence of Kℓ 8-or K 4-, the maximum of fluorescence intensity is increased by about 2 to 3fold and the t1/2 is increased by about 10 to 20% compared to Aβ (Figure 3, panel A). The important increase in the final fluorescence reflects the formation of more fibrils and/or the formation of more ordered fibrils inducing a higher enhancement of the ThT fluorescence. No statisticallymeaningful differences were observed between Kℓ 8-or K 4-in line with the quite rapid evolution of K 4-to Kℓ 8-compared to the time scale of the aggregation (Table S1). By TEM (Figure S3), the pictures show long and twisted Aβ fibrils in absence of POM; while in presence of POM, more abundant and longer fibrils are observed. In presence of Cu II , the aggregation curve shows a two-step aggregation path where the ThT fluorescence increases during the first five hours followed by a second illdefined and smooth sigmoid-like increase. The resulting fluorescence is weaker than in absence of Cu II by about 15%. This kinetic profile is reminiscent of published data on Cu II modulation of Aβ aggregation, 23 and mirrors the formation of less-ordered aggregates. In presence of Kℓ 8-or K 4-, the kinetic of the ThT fluorescence is dramatically changed with curves that resemble those obtained upon addition of Kℓ 8-or K 4-on the apo Aβ (Figure 3, panel B). In the TEM pictures, in presence of Cu II , the Aβ fibrils are shorter, thicker and less abundant than with the apo-peptide, but in presence of POM very long and thin fibrils are recovered. This indicates that the in-situ generated KCu 6-has a similar effect on Aβ aggregation than its apo counterparts, Kℓ 8-or K 4-, id est redirecting the Aβ aggregation towards more fibrillar species. Deeper inspection of the ThT fluorescence curves reveals that the t1/2 value of the Cu(Aβ) aggregation in presence of Kℓ 8-or K 4-is increased by about 20% (Table S1) compared to those of Aβ in presence of Kℓ 8-or K 4-. Hence, the in situ generated KCu 6-has a slightly increased ability to delay Aβ self-assembly effect compared Kℓ 8-(or K 4-). This may be linked to the difference in the net charge and in the possibility to form coordination bond between KCu 6-(but not Kℓ 8- nor K 4-) and some of the amino-acid side chains of the Aβ peptide. Under our working conditions, Kℓ 8-/ K 4-and KCu 6- interact with Aβ promoting the formation of highly ordered fibrils. This is different to previous data on other POM and MS-POM, which were shown to reduce ThT intensity by 40 to 80 % depending on the substituted cations. 15,17 The origin of such discrepancy has to be understood but will require an in-depth investigation of the modes of interaction of POM with Aβ peptide (currently under investigation) that is beyond the scope of the present communication, while the exact nature of the POM and different working conditions may be part of the explanation. The enhancement of amyloid formation is however highly reminiscent to what has been observed for linear polyphosphates, a biological polyanion. 29 The most remarkable effect of the studied POMs on Cu II modulated Aβ aggregation is their double mode of action. Indeed they (i) remove Cu II from Aβ preventing the formation of ill-defined Cu(Aβ) aggregates, regarded as the most deleterious with respect to AD, 30 and (ii) then the in situ generated KCu 6-reshapes the aggregation kinetic and species towards a better defined sigmoidal process and the formation of mature fibrils, respectively. Most of the ligands reported in the literature can restore apo-aggregation if they are able to retrieve Cu II from Aβ, 31 but have no additional effect. [EDTA] = 0.1 μM, [HEPES] = 50 mM, pH 6.9, T = 37 °C. The pH was controlled after the completion of the aggregation process and verified to be kept at 6.9 for all conditions. All the curves corresponding to this experiment (noted N°1, are given in the Supporting Information along with simulated curves).

In summary, our results provide new insights on the use of POMs in the context of AD. Their ability to coordinate Cu II out of the Aβ was first assessed: Kℓ 8-is readily effective, while K 4-needs first to lose one WO 4+ unit to form Kℓ 8-. Once the KCu 6-has formed a redox inert and stable complex is obtained. Both POM induce the formation of moreordered Aβ aggregates (higher β-sheet contents) including in the presence of Cu II . Hence the two POM studied here fulfil criteria to be appropriate Cu II chelators in the context of AD. Indeed preventing Cu II interaction with Aβ is of interest for metal-based therapy against AD, as this prevents the formation of toxic oligomers and the deleterious production of ROS. In fact, the POM studied "kill two birds with one stone" since they not only stop Cu(Aβ) ROS production but also redirect aggregation towards mature fibrils regardless the presence of Cu. Previous studies have shown that POMs do not produce toxicity and are capable of passing the blood-brain barrier. 17 Therefore, K 4-and Kℓ 8-studied here could be promising drug candidates. In addition, modulation of charge of the POM by playing with the central heteroatom and/or its shape can offer a huge diversity of optimization options. These variations and their effects both on prevention of Cu II -and Cu II (Aβ)-induced ROS and on the modulation of Aβ assembly are currently under study.

Dr. V. Borghesani is acknowledged for her participation in recording preliminary data. E.A.B., L. de C, L.S. and C.H. thank the ERC StG grant 638712, aLzINK for financial support.

Notes and references ‡ The shorter Aβ16 peptide has been used as a valuable model for Cu(II) binding to the full length Aβ40 or Aβ42 peptides for the experiments studying the coordination and the ROS production. The longer Aβ40 peptide has been used for the experiments involving aggregation. No differences between the short and full-length Aβ peptides have been observed for their binding Cu(II) coordination, binding affinity or ROS production.

a

  Scheme 1. K 4-, Kℓ 8-and KM 6-structures (M II is a d-metal dication, w = water molecule).

K 4 -

 4 and Kℓ 8-structures (Scheme 1) are composed of a central Si(IV) surrounded by four atoms of oxygen, which connect the central Si to twelve (K 4-) or eleven (Kℓ 8-) W(IV) cations each in an octahedral WO6 environment. Since K 4-is known to generate Kℓ 8-at pH > 4,[START_REF] Hervé | Polyoxometalate Molecular Science[END_REF] (and ref. therein) we probed this transformation under our working conditions. Evolution of the O to W charge transfer bands around 240 nm in a pH 6.9 HEPES buffer shows that this conversion takes place over 90 minutes (Fig.S1, A) while Kℓ 8-is stable (Fig.S1, B) in line with reported aqueous solution stability properties.[START_REF] Gumerova | [END_REF] In the presence of Cu(II), the formation of the [α-SiW11O39Cu(OH2)] 6-(KCu 6-) anion from K 4-or Kℓ 8-, respectively takes about one hour or is instantaneous as attested by the kinetic monitoring of the appearance of a new band at 253 nm corresponding to the generation of a MS-POM (Fig.S1, panels C and D). Coordination of Cu(II) to the POM anion is also confirmed by a broad and very weak d-d band with maximum at 865 nm (ε = 40 M -1 cm -1 ) and an EPR spectrum characteristic of a Cu(II) ion lying in a D4h geometry surrounded by five oxygen atoms from the POM skeleton and a water molecule (Figure1, A, and C), in line with literature.20 Once the ability of K 4-and Kℓ 8-to coordinate Cu(II) was checked, their ability to retrieve Cu(II) bound to Aβ was studied. The evolution of the characteristic d-d band of the Cu(Aβ) complex at 625 nm was followed by UV-vis spectroscopy after the addition of either Kℓ 8-or K 4-(Figure1, panel A and B). The observed full disappearance of the Cu(Aβ) signature indicates that both POMs can completely remove Cu(II) from the peptide to form the KCu 6-. While the removal has a time of half completion of 40 minutes with K 4-, it is instantaneous with Kℓ 8-. This was also observed by EPR, where K 4-completely removes Cu(II) from Aβ after a 3-hours incubation time while the EPR spectrum measured just after addition of Kℓ 8-to a Cu(Aβ) solution shows already signatures of the KCu 6-as a largely predominant species (Figure1, panel C).

Figure 1 .

 1 Figure 1. (A) UV-Vis spectra in the d-d region of Cu(II) + Kℓ 8-(a, dark green) and Cu(Aβ) + Kℓ 8--(c, green), (B) Kinetic monitoring of Cu(Aβ) + K 4-; (f, black) starting spectrum and (d, green) last spectrum, once the reaction is completed. Spectrum measured at 5 (brown), 20 (red), 40 (dark orange), 70 (light brown), 110 (dark brown) and 170 minutes (light green, d) after the addition of K 4-to Cu(Aβ). Conditions: [Aβ16] = [K 4-] = 500 μM, [Cu(II)] = 450 μM, [HEPES] = 100 mM pH 6.9, ℓ = 1 cm, the solution was continuously stirred and the temperature controlled at 25 °C. (C) EPR spectra of: Cu(Aβ) (f, black); Cu(Aβ) + K 4-at t0 (e, grey) and tf (d, light green); Cu(Aβ) + Kℓ 8-(c, green, at t0 or tf), Cu(II) + 4 equiv. (Aβ) + 1 equiv. Kℓ 8-(b, turquoise) and Cu(II) + Kℓ 8-(a, dark green). Spectra at t0 were taken just after the addition of Kℓ 8-or K 4-to the Cu(Aβ) solution, and after 1 day of incubation for tf. Conditions: [Aβ16] = [Kℓ 8-or K 4-] = 200 μM, [ 65 Cu(II)] = 180 μM, [HEPES] = 100 mM pH 6.9, 10% glycerol added as cryoprotectant.  = 9.47 GHz, T = 120 K, microwave power = 20 mW, Modulation Amplitude = 0.5 mT.

Figure 2 .

 2 Figure 2. Absorption (measured at 265 nm) monitoring of the effect Kℓ 8-(panel A) and K 4-(panel B) on Cu II and Cu II (Aβ)-induced Asc consumption. (A) dotted lines: data with Cu II (green) and Cu II (Aβ) (blue) incubated first with Kℓ 8-during 60 s; plain lines: data with Kℓ 8-added when Abs is at ca. 1 (indicated by arrows), during Asc consumption catalysed by Cu II (green) and Cu II (Aβ) (blue). Before POM addition the curves corresponds to Asc consumption by Cu II and Cu II (Aβ). (B) dotted lines: data with Cu II (purple) and Cu II (Aβ) (pink) incubated first with K 4-during 60 s; plain lines: data with K 4-added when Abs is at ca. 1, during Asc consumption catalysed by Cu II (purple) and Cu II (Aβ) (pink). [Aβ16] = [Kℓ 8-, K 4-] = 10 μM, [Cu(II)] = 9 μM, [HEPES] = 100 mM pH 6.9, ℓ = 1 cm, T = 25 °C.

Figure 3 .

 3 Figure 3. Selection of representative ThT curves of the Aβ1-40 peptide (A) or the Cu(Aβ40) complex (B) in absence or presence of POMs. Panel A: apo-Aβ40 (black), Aβ40 + 1 equiv. K 4-(pink), Aβ40 + 1 equiv. Kℓ 8-(green). Panel B: Cu(Aβ40) (blue) formed at 0.9 equiv. of Cu(II), Cu(Aβ40) + 1 equiv. K 4-(light green), Cu(Aβ40) + 1 equiv. Kℓ 8-(light pink) and apo-Aβ40 (black) for direct comparison. Conditions: [Aβ40] = 20 μM, [Kℓ 8-or K 4-] = 0 or 20 μM, [Cu(II)] = 0 or 18 μM, [ThT] = 10 μM,[EDTA] = 0.1 μM, [HEPES] = 50 mM, pH 6.9, T = 37 °C. The pH was controlled after the completion of the aggregation process and verified to be kept at 6.9 for all conditions. All the curves corresponding to this experiment (noted N°1, are given in the Supporting Information along with simulated curves).

  /www.alz.org/alzheimers-dementia/facts-figures).

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Companies have been looking for new methods for treating toxic or refractory wastewaters; which can mainly be used prior to or after or in connexion with biological treatment processes.

This paper compares conventional ozone oxidation with activated carbon (AC) promoted ozone oxidation, which helps developing a mechanism involving HO • radical. For a compound which is quite easy to oxidise, like 2,4-dichlorophenol (2,4-DCP) conventional ozonation is efficient enough to remove the initial molecule. The mechanism involved mainly consists of an electrophilic attack on the aromatic ring, which is activated by the donor effect of the -OH group, then followed by a 1,3 dipolar cycloaddition (Criegee mechanism) that leads to aliphatic species, mainly carboxylic acids. Yet, the addition of AC, through the presence of HO • radical, enhances the removal of these species which are more refractory.

For a refractory compound like nitrobenzene (NB), with a de-activated aromatic ring because of the attractive effect of -NO 2 , conventional ozonation is inefficient. On the contrary, this molecule can be quite easily removed with AC promoted oxidation and it is found that the mechanism (electrophilic attack followed by a 1,3 dipolar cycloaddition) is quite similar to the one corresponding to conventional ozonation, but with less selectivity.

For both molecules, a mass balance has established that the by-products accounting for more than 75% of the remaining COD can be quantified. A significant part is composed of carboxylic acids (acetic, oxalic, etc.), which could afterwards be easily removed in an industrial wastewater treatment process followed by a final biological treatment step.

Introduction

Wastewater is often treated by biological routes with good removal efficiencies; however, toxic organic compounds cannot be reliably eliminated by these bioprocesses. Additional treatments, such as adsorption or chemical oxidation, must be used for nonbiodegradable molecules removal. Ozone is frequently used since it is a powerful oxidizing agent. The reactivity of the ozone molecule with unsaturated organics follows two main mechanisms [START_REF] Bailey | Ozonation in Organic Chemistry[END_REF][START_REF] Alvarez | The influence of various factors on aqueous ozone decomposition by granular activated carbons and the development of a mechanistic approach[END_REF]: electrophilic substitution and 1,3 dipolar cycloaddition, known as the "Criegee mechanism". In the particular case of aromatic compounds, the substituents of the aromatic ring play a major role in the actual mechanism. Yet all these reaction pathways are selective and unfortunately some organic compounds cannot be oxidised with a single ozonation step. In these cases, the use of advanced oxidation processes can be the best solution [START_REF] Andreozzi | Advanced oxidation processes (AOP) for water purification and recovery[END_REF][START_REF] Legube | Catalytic ozonation: a promising advanced oxidation technology for water treatment[END_REF][START_REF] Lin | Effective catalysts for decomposition of aqueous ozone[END_REF][START_REF] Beltran | Gallic acid water ozonation using activated carbon[END_REF].

Ozone decomposition in water is a well-known phenomenon. It can be described thanks to a cyclic mechanism involving several radical species and acido-basic equilibria [START_REF] Staehelin | Ozone decomposition in water studied by pulse radiolysis. II. HO • and HO4 • as chain intermediate[END_REF]. Hydroxide ions and hydrogen peroxide strongly participate in the initiation steps of this cycle (Eqs. ( 1) and ( 2)):

O 3 + HO -→ O 2 • -+ HO 2 • (1)
O 3 + HO 2 -→ HO • + O 2 • -+ O 2 (2)
The kinetics of the first reaction is quite slow (second order constant k 1 = 70 L mol -1 s -1 ) whereas the kinetic constant for the second reaction is much larger (k 2 = 2.8 × 10 6 L mol -1 s -1 ), thus hydrogen peroxide strongly enhances radicals production from ozone [START_REF] Staehelin | Decomposition of ozone in water: rate of inititation by hydroxide ions and hydrogen peroxide[END_REF]. The kinetics of ozone decomposition into HO • radical are generally well described with a simple first order law according to dissolved ozone concentration [START_REF] Stumm | Der zerfall von ozon in wässriger Lösung[END_REF]. The value of the corresponding kinetic constant (k C ) depends, among other parameters, on the pH of the solution, on hydrogen peroxide concentration or on the presence of catalytic materials.

In previous papers, we showed that ozone decomposition could be obtained through activated carbons (AC) [START_REF] Reungoat | Adsorption of nitrobenzene from water onto high silica zeolites and regeneration by ozone[END_REF][START_REF] Merle | Enhanced bio-recalcitrant organics removal by combined adsorption and ozonation[END_REF][START_REF] Merle | Comparison of activated carbon and hydrophobic zeolite efficiencies in 2,4-dichlorophenol advanced ozonation[END_REF] or zeolite Fig. 1. Experimental set-up. [START_REF] Monneyron | Regeneration of high silica zeolites via A.O.P.-a preliminary study about adsorbent reactivity toward ozone[END_REF][START_REF] Monneyron | A combined selective adsorption and ozonation process for VOCs removal from air[END_REF]. In particular, some authors assume that the formation of HO • radicals from activated carbons is due to their surface basicities [START_REF] Rivera-Utrilla | Ozonation of 1,3,6-naphatalenetrisulfonic acid catalysed by activated carbon in aqueous phase[END_REF]. Indeed, the basic-oxygen-containing functional groups and/or delocalised electrons of the graphene layers of AC can act as Lewis bases and form electron donor-acceptor complexes with H 2 O molecules (Eq. ( 3)). The hydroxide ion which is released will then initiate ozone decomposition

C C (graphene) + H 3 O + ↔ C + -CH (graphene) + H 2 O 2H 2 O ↔ H 3 O + + HO - (3)
The present work studies the influence of AC on the oxidation by ozone of two bio-recalcitrant compounds: nitrobenzene and 2,4dichlorophenol. The process efficiency is expressed as molecule removal rate, reaction mechanisms, by-products quantification and mass balance calculations.

Materials and methods

Oxidation runs were performed in a 2 L well stirred semi-batch reactor fed with ozonated oxygen (30 L h -1 , 30 g m -3 ) through a pipe placed below the stirrer. Ozone concentration was measured in the liquid phase (polarographic probe Orbisphere model 410/probe 31330.15xx) as well as in the gas inlet and outlet (UV absorbance analyser Trailigaz UVOZON TLG 200). In most experiments pH was not controlled.

Two microporous activated carbons (supplied by PICA Corp., France) called F-22 and S-23 were used with 1 g L -1 amounts. Their specific areas and mean pore diameter are, respectively 1000 m 2 g -1 -0.9 nm and 1300 m 2 g -1 -1.7 nm.

The experimental set up is shown in Fig. 1 and detailed physical properties of the activated carbons are listed in Table 1.

Nitrobenzene and 2,4-dichlorophenol were chosen to prepare synthetic polluted water. The initial concentrations of the two pollutants were 250 mg L -1 , i.e. 1879 mol L -1 for nitrobenzene and 1534 mol L -1 for 2,4-dichlorophenol. The concentrations of the pollutants and oxidation products (purchased from Acros Chemicals) were determined by High Performance Liquid Chro-

Results and discussions

Ozone decomposition with or without activated carbon

The decomposition rate of ozone was studied according to increasing concentrations of activated carbon F-22 and S-23. The kinetic constant of ozone decomposition k C was calculated through the mass balance for ozone dissolved in water (Eq. ( 4)). It was compared to results without materials and plotted in Fig. 2. The measurement method as well as the reference value obtained without AC, 1.1 × 10 -3 s -1 , are described by Lopez-Lopez et al. [START_REF] Lopez-Lopez | Influence of tbutanol and pH on hydrodynamic and mass transfer parameters in an ozonation process[END_REF]

k C = Q G ([O 3 ] G,in -[O] ∞ G,out ) [O 3 ] ∞ L V L (4)
with Q G : volumetric gas flow rate (m 3 s -1 ); [O 3 ] G,in : ozone concentration in inlet gas (g m -3 ); [O] ∞ G,out : ozone concentration in outlet gas at steady state (g m -3 ); [O 3 ] ∞ L : ozone concentration in water at steady state (g m -3 ); V L : liquid volume in the reactor (m 3 ). The k C constant evolution is proportional to the material concentration, in accordance with the results of Guiza et al. [START_REF] Guiza | Decomposition of dissolved ozone in the presence of activated carbon: an experimental study[END_REF]. With 10 g AC L -1 , the decomposition rate is multiplied by 5 for S-23 and by 7.5 for F-22.

This phenomenon can be explained either by the AC physical structure and also by the AC chemical composition which both influence the reactivity of the catalyst. The experimental measurements of surface groups according to Boehm method [START_REF] Boehm | Chemical identification of surface groups[END_REF] showed that both AC have basic sites on their surfaces: 157 equiv. g -1 for F-22 and 306 equiv. g -1 for S-23. These sites can enhance the decomposition of ozone by the reaction with chromene and pyrone groups, as shown in Fig. 3 [START_REF] Alvarez | The influence of various factors on aqueous ozone decomposition by granular activated carbons and the development of a mechanistic approach[END_REF]. Then, hydrogen peroxide provided by this reaction can react with remaining ozone to give hydroxyl radicals as explained in Section 1.

Moreover, material porosity is an important factor for the diffusion of ozone molecules to the active sites. Even if F-22 has a lower external specific area than that of S-23, it has a greater mesoporous volume, so the diffusion in F-22 is probably easier than in S-23, which has a microporous structure, and as a consequence the decomposition of ozone can be superior with F-22 than with S-23.

NB and 2,4-DCP ozonation

Fig. 4 shows NB and 2,4-DCP ozonation removal rate, with and without AC. First, it can be observed that none of the two media had any beneficial effect on 2,4-DCP oxidation rate. Indeed, 2,4-DCP is totally removed after only 1 h single or combined oxidation process. On the other hand, the presence of activated carbon allows a lower ozone consumption: it is lowered by 31% with F-22 AC and by 11% with S-23 AC. Concerning NB, the presence of activated carbon slightly enhances the oxidation rate: a total removal occurs after 3.5 h single ozonation whereas only 2.5 h treatment are necessary with AC. The performances of the two media are quite similar, ozone consumption is reduced by 4% with F-22 and by 12% with S-23.

The difference of reactivity with ozone itself can be easily explained by the molecules mesomery. Indeed ozone direct pathways, Criegee addition or electrophilic attack, are improved when the aromatic ring is strongly activated. The -NO 2 substituent of NB is a deactivating group whereas the -OH substituent of 2,4-DCP is an activating group. Also the -Cl group is a deactivating group since ozone reacts faster with phenol than with 4-chlorophenol (4-CP). However, Benitez et al. [START_REF] Benitez | Rate constants for the reactions of ozone with chlorophenols in aqueous solutions[END_REF] showed that the more phenol is substituted with -Cl groups, the faster ozone oxidation occurs. Thus 2,3,4,6-tetrachlorophenol is more quickly oxidised than phenol. A strong substitution on the aromatic ring probably changes the reaction mechanism, for example nucleophilic substitution onto -Cl groups could take place [START_REF] Rivera-Utrilla | Ozonation of 1,3,6-naphatalenetrisulfonic acid catalysed by activated carbon in aqueous phase[END_REF]. These statements explain the faster ozonation rate for 2,4-DCP than that of NB.

In the presence of activated carbon, NB oxidation kinetics is enhanced whereas when 2,4-DCP is used the removal is not any faster. Single adsorption cannot explain this behaviour since the adsorption kinetics is relatively slow for both molecules [START_REF] Merle | Comparison of activated carbon and hydrophobic zeolite efficiencies in 2,4-dichlorophenol advanced ozonation[END_REF]. Thus the difference in NB removal rate is clearly attributed to a catalytic effect of activated carbon. Indeed, NB is rather refractory to direct ozonation, so radical oxidation with CA being less selective, clearly enhances the oxidation kinetics. For 2,4-DCP, direct ozonation kinetics is faster and thus is not improved through the radical mechanism. Finally, for both molecules, the radical route probably leads to a better yield for the oxidizing agent resulting in a reduced ozone global consumption.

Oxidation mechanisms of NB

Ortho-nitrophenol (o-NP), meta-nitrophenol (m-NP), paranitrophenol (p-NP) and acetic acid, which are four NB ozonation by-products, were quantified using HPLC. o-NP and m-NP were equally produced with maximum concentrations reaching 14-22 mol L -1 after 30 min experiment. The corresponding curves are not showed here for lack of space. p-NP concentration was twice higher with 40 mol L -1 after 1 h experiment. At last about 1330 mol L -1 acetic acid were formed after 1.5 h experiment (Fig. 5). The presence of meta-nitrophenol can be explained by NB resonance. Indeed, the -NO 2 substituent linked to the aro-matic ring is meta directing and deactivating for an electrophilic attack. The simultaneous presence of o-NP and p-NP in the solution can be attributed either to another mechanism or to a low selectivity in the position of the electrophilic attack. This can occur when the stability of the o, m and p-transition states are very close [START_REF] Rivera-Utrilla | Ozonation of 1,3,6-naphatalenetrisulfonic acid catalysed by activated carbon in aqueous phase[END_REF]. However, after 1 h experiment, the amount of nitrophenols only accounts for 10% of the oxidised NB. Thus the electrophilic attack is not the only pathway in NB ozonation and the Criegee reaction occurs too. Indeed carboxylic acids such as acetic (Fig. 5) or oxalic acids are produced. Unfortunately the quantification of oxalic acid was unsuccessful because of interferences with nitrates, also produced. Finally, a pathway for NB direct ozonation is suggested in Fig. 6.

The addition of activated carbons slightly improved the removal rates for the four by-products. Hence, the radical route increases reaction rates without much changing the nature of the byproducts. The reason why radical efficiency is higher is probably due to less selectivity.

A global mass balance was performed through two COD estimations. The actual COD was measured, and the part of COD due to NB was calculated from NB concentration. The difference between the two values stands for the actual by-products COD, plotted in Fig. 7a [START_REF] Andreozzi | Advanced oxidation processes (AOP) for water purification and recovery[END_REF]. Alongside this experiment, a theoretical COD value for the by-products was calculated from the measured by-products concentrations, as showed in Fig. 7b. The actual COD was higher than the theoretical COD, which indicates that about 25% of the byproducts were not quantified. Again it should be pointed out that the monitoring of oxalic acid was unsuccessful.

For a single ozonation, 130 mg COD L -1 of by-products were detected after 4 h experiment whereas only 30 mg L -1 were observed in the AC/ozone combined process. Thus, the addition of AC greatly enhances the oxidation rates of the by-products com-pared to those in single ozonation. This also explains the gain in global ozone consumption and clearly justifies the interest of a radical route for NB oxidation.

Oxidation mechanisms of 2,4-DCP

Eight 2,4-DCP ozonation by-products, listed in Table 2, were monitored. Fig. 8 shows the results for two of them: an intermediate aromatic species (3,5-DCC) and a final product (oxalic acid). The aromatic intermediates reached their maximum concentrations between 15 and 30 min experiment. The resultant values were: 3,5-DCC (56 mol L -1 ), 4,6-DCR (2 mol L -1 ), 4-CC (14 mol L -1 ), 2-CHQ (76 mol L -1 ) and catechol (118 mol L -1 ). The first two molecules are generated through the electrophilic attack of ozone on 2,4-DCP. The amount of 3,5-DCC is higher because the -OH group activates the aromatic ring in position 6 while the -Cl group has a smaller influence on position 5. Then, the two mono-chlorinated compounds are probably formed through the electrophilic substitution (called the "ipso effect") of ozone on the -Cl group position. The difference in the concentration of these two species was not explained. At last the presence of catechol cannot be easily explained, but however, it is certainly obtained after several elementary steps. Finally, the aromatic molecules are opened following the Criegee reaction, and carboxylic acids are formed. Up to 1000 mol L -1 acetic acid was generated in the first 30 min and was subsequently totally removed after about 2 h. On the other hand, oxalic acid was continuously formed during the 2 h and reached 889 mol L -1 before slowly being removed from the solution until the level of 222 mol L -1 recorded after 8 h experiment. Finally a pathway for 2,4-DCP ozonation is proposed in Fig. 9.

The addition of activated carbons had no noticeable effect on the removal of 2,4-DCP and its by-products. Yet, there seems to be a slight increase in oxalic acid production in the presence of S-23 media. This may be due to the production of dissolved organic species (either oxalic acid or other ones such as formic, acetic or glyoxylic acids) resulting from the oxidation of the AC itself but this hypothesis still has not been verified. Nevertheless, this difference is not really significant, as oxalic acid amounts to very little in the global mass balance (Fig. 10). Whatever the oxidation route, about 150 mg L -1 COD was generated after 1 h experiment, followed by a slow decrease during the 7 remaining hours. Thus, the radical route does not improve 2,4-DCP oxidation, the direct ozonation kinetics is too fast to be enhanced by activated carbons, at least for the AC concentrations (1 g L -1 ) used in this study.

Conclusions

The feasibility and interest of adding activated carbon to ozonation process was studied in this paper, in order to develop a hybrid process (combining at the same time and in the same device adsorption and oxidation) for refractory molecules treatment. The results showed the ability of the activated carbons to decompose molecular ozone and to create radical species, which were much more efficient for oxidation as they are non-selective. For organic pollutants which are easy to oxidise by ozone, like 2,4-dichlorophenol, the removal rate was not significantly enhanced, but a smaller ozone consumption was noticed. For organic pollutants much more refractory to ozone oxidation, like nitrobenzene, the development of a radical route dramatically increased the removal rate and the ozonation kinetics became faster.

In addition, the analysis of by-products obtained during oxidation enabled an understanding of the major steps in the oxidation mechanisms of the various molecules. Classical mechanisms of electrophilic substitution and dipolar cycloaddition explained most of the sub-products formations. The same by-products have been found with or without AC, but the reaction rates are enhanced by the presence of activated carbon.

Finally, considering the ability of HO • radicals to remove an extremely wide range of refractory organics, including trace pollutants and emerging compounds, it appears that this AOP process, coupling activated carbon and ozonation, could be a good candidate for an additional treatment after biological treatment, above all for strongly refractory to ozone molecules oxidation.

  Influence of activated carbons on the kinetics and mechanisms of aromatic molecules ozonation T. Merle a , J.S. Pic a, * , M.H. Manero b , S. Mathé a , H. Debellefontaine a a Université de Toulouse, LISBP, UMR INSA/CNRS 5504 & UMR INSA/INRA 792, 135 Avenue de Rangueil, 31077 Toulouse Cedex 4, France b Université de Toulouse, LGC, UMR CNRS 5503, IUT GCGP, 137 Avenue de Rangueil, 31077 Toulouse Cedex 4, France a b s t r a c t

Fig. 2 .

 2 Fig. 2. Ozone decomposition constant versus activated carbons concentration (pH 7, [O3] G,in = 30 g N m 3 , 25 • C) S-23 ( ) and F-22 (᭹).

Fig. 3 .

 3 Fig. 3. Basic group (chromene up and pyrone down) reaction with ozone, leading to hydrogen peroxide.

Fig. 4 .

 4 Fig. 4. NB and 2,4-DCP removal by single ozonation ( ) and combined ozonation with S-23 ( ) and with F-22 (᭹).

Fig. 5 .

 5 Fig. 5. NB ozonation by-products versus time: single ozonation ( ), ozonation with S-23 ( ) and with F-22 (᭹).

Fig. 6 .

 6 Fig. 6. Suggested reaction pathway for nitrobenzene direct ozonation.

Fig. 7 .

 7 Fig. 7. Evolution of nitrobenzene by-products COD, estimated from (a) actual COD and NB concentration and (b) by-products concentration: single ozonation ( ), ozonation with S-23 ( ), ozonation with F-22 (᭹).

Fig. 8 .

 8 Fig. 8. 2,4-DCP oxidation by-products versus time: single ozonation ( ), ozonation with S-23 ( ), with F-22 (᭹).

Fig. 9 .

 9 Fig. 9. Suggested reaction pathway for 2,4-dichlorophenol direct ozonation.

Fig. 10 .

 10 Fig. 10. 2,4-DCP by-products COD removal, estimated from (a) actual COD and 2,4-DCP concentration and (b) by-products concentration: single ozonation ( ), ozonation with S-23 ( ),with F-22 (᭹).

Table 1

 1 Characteristics of the activated carbons.

S-23 F-22

Specific area (m 2 g -1 ) 1374 1005 Microporous volume (cm 3 g -1 ) 0.56 0.38 Mesoporous volume(cm 3 g -1 ) 0.05 0.10 Mean pore diameter (Å) 17.00 9.00 Granular diameter (mm) 1.46 1.00 matography using a HP 1050 series chromatograph equipped with a Diode Array Detector (Agilent Technologies HP 1200) and a C-18 column (EQUISORB ODS2 5 m). Samples were first passed through a 0.45 m filter before analyses in order to remove undissolved compounds. Table 2 lists the different chemicals which were monitored and gives their maximum absorbance wavelenghts () and kinetic diameters (). The chemical oxygen demand (COD) of the solutions was measured by Hanna Instrument test tubes (COD MR Reagent).

Table 2

 2 Molecules characteristics of nitrobenzene, 2,4-dichlorophenol and ozonation by-products.

	Molecule name	(nm)	(Å)	Stoichiometric ratio
				COD/molecule (mg O2 mg -1 )
	Nitrobenzene (NB)	267	5.9	1.951
	o-Nitrophenol (o-NP)	274	8.1	1.612
	m-Nitrophenol (m-NP)	274	8.1	1.612
	p-Nitrophenol (p-NP)	317	6.7	1.612
	2,4-Dichlorophenol (2,4-DCP)	286	-	1.178
	3,5-Dichlorocatechol (3,5-DCC)	202	-	0.447
	4,6-Dichlororesorcinol (4,6-DCR)	202	-	0.447
	4-Chlorocatechol (4-CC)	202	-	0.537
	2-Chlorohydroquinone (2-CHQ)	202	-	0.537
	2-Chlorobenzoquinone (2-CBQ)	254	-	0.673
	Catechol (C)	202	-	0.644
	Oxalic acid (OA)	202	-	0.177
	Acetic acid (AA)	202	-	1.066

Acknowledgement

The authors thank French National Agency (ANR) for financial support -Program Precodd, project "PHARE".


Jouneau Stéphane
email: stephane.jouneau@chu-rennes.fr

Mallorie Kerjouan

Ronan Thibault

Prof Stéphane Jouneau

To be malnourished or not to be malnourished: that is the question!

Take home messages:

Physicians in charge of patients with chronic respiratory diseases, including chronic pulmonary aspergillosis, should be aware of the high prevalence of malnutrition. It is underdiagnosed and yet it is associated with worse prognosis. Malnutrition could be easily diagnosed.

Correspondence

In the European Respiratory Journal, Thomas Maitre and colleagues analysed 17'290 cases of chronic pulmonary aspergillosis (CPA) retrieved between 2009 and 2018 in the French nationwide administrative hospital database (PMSI) (1). They demonstrated high rates of mortality, almost one third at one year. In multivariate analysis, they showed that mortality was increased in patients with malnutrition with the highest hazard ratio (HR): 2.49, 95% CI 2.05-3.04. Malnutrition, present in 33.4% of patients, was extracted from the database using the ICD-10 code. This implies that the malnutrition diagnosis should have been overtly diagnosed and collected in patients' charts or discharge reports. Because the study used the National health database, it was not possible for the authors to report on which criteria was diagnosed malnutrition. We could speculate that body mass index (BMI) below 21 kg/m² was probably the main criteria used, since it is the commonest diagnostic tool and the cut-off value for chronic respiratory diseases such as COPD patients (2). The mean BMI has been recently shown as even below this threshold, (20.8 ± 4.9 kg/m²) in a recent cohort of CPA patients (3).

As it is well known that malnutrition is highly underdiagnosed at hospital as well as in the community setting (4), we could speculate that the prevalence of malnutrition could have even been higher than 33.4% if a systematic and multimodal assessment of malnutrition was performed. In 2019, new international guidelines for malnutrition diagnosis have been published by "The Global Leadership Initiative on Malnutrition" (GLIM) (5) to increase the screening and the treatment of malnutrition all over the world. The GLIM guidelines recommend assessing nutritional status by integrating five criteria: BMI, weight loss, loss of muscle mass (by bioimpedance analysis for example) or function, and reduced food intake, using semi-quantitative tools, or the presence of an inflammatory disease such as CPA.

Undoubtedly, the use of a full assessment of malnutrition could have made identifying more malnourished patients, and may have strengthened the relation between malnutrition and mortality. Therefore, based on the latter and Thomas Maitre's paper, and as we proposed in IPF patients (6), the respiratory care community should propose a more systematic and integrative screening of malnutrition in all patients with chronic respiratory diseases [START_REF] Jouneau | Malnutrition in idiopathic pulmonary fibrosis: the great forgotten comorbidity![END_REF]. It is needed to better understand the relation between malnutrition and clinical outcomes, and optimize patient's care. It could be as simple as assessing weight and height at the first visit, in order to calculate BMI, assessing food intake (e.g. by using the Simple Evaluation of Food Intake (SEFI®) which is a 10-point visual analogue scale) [START_REF] Bouette | A visual analogue scale for food intake as a screening test for malnutrition in the primary care setting: Prospective non-interventional study[END_REF], measuring weight and calculating weight loss at each visit. By improving the awareness of health caregivers and stakeholders towards malnutrition, we would improve patients' quality of life and clinical outcomes as shown in COPD patients [START_REF] Collins | Nutritional support and functional capacity in chronic obstructive pulmonary disease: a systematic review and meta-analysis[END_REF][START_REF] Pison | Multimodal nutritional rehabilitation improves clinical outcomes of malnourished patients with chronic respiratory failure: a randomised controlled trial[END_REF].

1. Maitre T, Cottenet J, Godet C, Roussot A, Abdoul Carime N, Ok V,

  et al. Chronic pulmonary aspergillosis: prevalence, favouring pulmonary diseases and prognosis. Eur Respir J. 2021;58(2). What are the best indicators to assess malnutrition in idiopathic pulmonary fibrosis patients? A crosssectional study in a referral center. Nutrition. 2019;62:115-21.

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	50 50	47.
	51 51	4.	Kondrup J, Allison SP, Elia M, Vellas B, Plauth M, Educational, et al. ESPEN guidelines for
	52 52	nutrition screening 2002. Clin Nutr. 2003;22(4):415-21.
	53 53	5.	Cederholm T, Jensen GL, Correia M, Gonzalez MC, Fukushima R, Higashiguchi T, et al. GLIM
	54 54 55 55 56 56 57 57	criteria for the diagnosis of malnutrition -A consensus report from the global clinical nutrition community. Clin Nutr. 2019;38(1):1-9. 6.
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Jouneau S, Kerjouan M, Rousseau C, Lederlin M, Llamas-Guttierez F, De Latour B, et al.

Conflict of interest: none. Funding: none.


Rui Li
email: rui.li@orange.com

Bertrand Decocq
email: bertrand.decocq@orange.com

Anne Barros
email: anne.barros@centralesupelec.fr

Yiping Fang
email: yiping.fang@centralesupelec.fr

Zhiguo Zeng
email: zhiguo.zeng@centralesupelec.fr

Bruno Castanier

Marko Cepin

David Bigaud

Christophe Berenguer

Complexity in 5G Network Applications and use cases

Keywords: 5G Network, resilience quantification, resilience metrics, network applications, complex system, vertical requirements

come   L'archive ouverte pluridisciplinaire

Introduction

The telecommunication domain keeps evolving rapidly since its birth. From the first generation cellular network to the newest generation, every one of them brings convenience to daily life and work. As the key to the future technology ecosystem, the fifth generation (5G) of mobile telecommunication network is designed to be a faster, stronger, better and smarter telecommunication network than ever before.

By upgrading existing technologies and incorporating new technologies, 5G networks are without a doubt a promising solution for future telecommunication needs.

In the Radio Access Network (RAN), new radio technologies are applied to 5G networks. Terminals are eligible to use both 5G and 4G frequency bands and connect to both 4G and 5G antenna. The utilization of orthogonal frequency-division multiplexing (OFDM) allows multiple communication channels to coexist, and thus it is possible to treat high frequency and low frequency bands at the same time to obtain both higher bandwidth and wider coverage. Intelligent antennas using "massive MIMO" are implemented which can further improve network capacity (IEEE 802.11ad, 2012;[START_REF] Patriciello | NR-U and IEEE 802.11 Technologies Coexistence in Unlicensed mmWave Spectrum: Models and Evaluation[END_REF]. Besides, the beamforming technology ensures to transmit signal in a specific direction where it is useful to users rather than sending in all directions, such that less interference is created and less energy is consumed.

The 5G core network (5GC) becomes a service based architecture. In this software designed architecture, each Network Function (NF) is delivering "services" to other NFs to access control plane functionalities, subscriber or network data repositories through an interface of a common framework (3GPP TS 23.501, 2021;[START_REF] Mademann | The 5G system architecture[END_REF]. To deliver services more dynamically, 5G networks adopt the techniques such as Network Function Virtualisation (NFV) and Software Defined Networking (SDN).

By introducing the concept of slicing into the network, it is possible to create different virtual networks for different services. In 5G, such network will be a dedicated slice providing tailored network capabilities and network characteristics according to the requirement from the customers by respecting specific rules without disturbing the rest of the network outside the slice. Multiple users, if permitted, can connect to one same slice. One user equipment, if needed, may have access to multiple slices at one time. A network slice subnet represents a group of network functions that form part or complete constituents of a network slice. A network slice subnet may contain for example instances of Core Network functions only, or instances of Access Network functions only, or any combination thereof (3GPP TS 28.530, 2021).

Edge computing is a generic term encompassing a variety of different approaches to put computing and storage resources at the edge of the network close to the customer rather than in remote datacenters. Initially, this notion was introduced and used for mobile networks, hence the term Mobile Edge Computing (5G Smart, 2020). Later, the European Telecommunications Standards Institute (ETSI) defined the term Multi-access Edge Computing (MEC) as a generalization of Mobile Edge Computing to any network (ETSI, 2019). Some latency-sensitive network application functions can be deployed on MEC servers near the RAN or even at the macro base station. Therefore, some of the data will be stored and proceeded in distributed edge cloud services.

From an End-to-End perspective, these technologies break the boundary of different parts of network resources. From a multi-layer perspective, with NFV, a virtualization layer is added into the network architecture. Thus, 5G becomes a complex system and even a system of systems.

Combining the afore-cited technologies, 5G networks are going to greatly reshape the domain not only by its performance but also by offering a transition from a "horizontal" service delivery model toward a "vertical" service delivery model [START_REF] Banchs | A 5G mobile network architecture to support vertical industries[END_REF]. The former model provides identical services to all customers, while the latter provides tailored and personalized services for certain industry sectors. Such "vertical" delivery model introduces new scenarios and application use cases to 5G networks.

International Telecommunication Union (ITU-R, 2015) classifies 5G services into three categories: enhanced mobile broadband (eMBB), massive machine-type communications (mMTC), and ultra-reliable and lowlatency communications (URLLC). Virtual or Augmented Reality, one of eMBB use cases, which constantly transfers a huge amount of data could benefit from 5G high speed network connection. In the example of smart fabrication, a massive machine-type communications service, the network slicing may help the factory to check its production by connecting IoT equipment to a dedicated slice. Autonomous-driving, as a typical URLLC service, its massive data can be calculated in real time at the edge with the help of 5G networks.

With the digital and technological transformation, 5G can be applied to more and more scenarios, nevertheless, it also creates a resilience challenge not only for the service providers but also for all the supported industries or verticals. The more technologies are integrated into the system, the more potential risks. Furthermore, 5G becomes a complex system, one single failure, if not fully fixed, could propagate from one single point to a series elements of the system. Thus, it is crucial to study the complexity of such network system by comprehending each part and each layer of it. Only when we have a full understanding on how 5G networks are composed and how a Network Service is established, can we analyze the risk and resilience of the system.

The paper is structured as follows: in Section 2 we explain 5G complexity from an End-to-End perspective by decomposing each part of the End-to-End service; Section 3 is devoted to presenting a multilayer perspective of 5G network, where the Network Function Virtualisation is mainly discussed; in Section 4 we briefly describe the process of setting up a service using the technologies that we introduce; Section 5 takes on the resilience challenge by introducing adverse events in telecommunication network and recent works on performance evaluation in 5G system; Finally we conclude the paper by providing suggestions for further research in Section 6.

The complexity from the End-to-End perspective

Terminals

A Network Service normally starts from an end-user device. These devices are called terminals or user equipment (UE). Since 5G will be widely applied in telecommunication, variant terminals will be connected to the network. These terminals could be smart phones, vehicles, IoT terminals, etc. Among them, some terminals require simultaneous connections to multiple services. More specifically, a typical application would be the case where a smartphone plays an online football match for the user while in the background the device tries to get a push notification from mail service. In other cases, some devices may require the same service at the same time. Such scenario commonly happens in a factory. When an environment change is detected, all connected sensors will report this abnormal data or the extracted information to the central server at the same time. In the multiple services situation, the isolation between Network Services must be guaranteed to meet the requirement of each one of the services. In the massive devices access situation, the system must be resilient enough to cope with a potential congestion in the data processing and transport.

Radio access network

For a wireless terminal, to transfer data from the UE to the Radio Access Network (RAN), the data will be firstly received by the antenna embedded in Radio Unit (RU). Baseband Unit (BBU) connected to the RU will then transport a baseband frequency before sending the data to the edge or Core Network. Open RAN as a future generation of RAN chosen by 5G network, provides a standardized interface between RU and BBU as well as a standardized interface between BBUs to cooperate with multiple vendors. With virtualized RAN (vRAN), the BBU can be virtualized on multiple NFV platforms and be shared with operators (see Section 3.1 for more details on Network Function Virtualisation). BBU can be divided into multi parts. The first part, distributed unit (DU), takes charge of real-time BBU scheduling functions, while the second part, centralized unit (CU), completes the non-real-time BBU functions. Some software parts of BBU will be placed together with RU. CU and DU can be deployed flexibly, namely co-located with RU, in edge cloud or regional datacenter. Virtualisation and standardized interface make the most of open interfaces by enabling sharing CU and DU with multiple vendors (Wind River, 2017).

Transport network

To ensure a highly reliable and good performance network, Transport Network plays a crucial role. Transport Network includes the fronthaul of remote units, the backhaul of base stations, optionally a midhaul between the distributed and centralized units, and the backbone between core datacenters. Different transmission technologies are used for each part of Transport Network, for example: dark fiber for fronthaul and midhaul with direct connections between the nodes (RU to DU and DU to CU respectively), WDM rings for backhaul and backbone networks. Network Slicing can be based in the first step on VLAN/VPN for each transport segment (called basic soft-slicing or logical isolation between slices), and later on new technologies like Segment Routing-Traffic Engineering (SR-TE) for enhanced soft slicing with specific performance or designed per type of slice, and in the third step on Flexible Ethernet (FlexE) or Time Sensitive Networking (TSN) for hard slicing where the slices are fully isolated with guaranteed services performance. For resilience purposes, the IP network, from the Edge to the Core Network, is doubled and relies on WDM rings, and is able to react in 50 milliseconds in case of failure.

Core network

In 5GC, one of the most important characteristics is the separation of the User Plane (UP) functions from the Control Plane (CP) functions (3GPP TS 23.501, 2021). UP functions mainly take care of traffics forwarding while the CP functions manage the authentication, network slice selections, etc. The principal advantage of such separation is being able to flexibly scale the CP functions independently on UP functions in case of traffic peak vice versa. Another benefit lies in the flexibility to separately deploy CP functions so that some functions can be deployed, according to the requirement of the use case, in a centralized datacenter or a distributed one close to the RAN. The flexibility in scaling and deployment completely makes 5G networks more complex than the last generation. • Authentication Server Function (AUSF).

Core functions

• Access and Mobility Management Function (AMF).

• Data Network (DN), e.g. operator services, Internet access or 3rd party services. • Network Exposure Function (NEF).

• Network Repository Function (NRF).

• Network Slice Specific Authentication and Authorization Function (NSSAAF). • Network Slice Selection Function (NSSF). • Policy Control Function (PCF). • Session Management Function (SMF).

• Unified Data Management (UDM).

• Unified Data Repository Function (UDF).

• Application Function (AF). To avoid ambiguity in the paper, NF service refers to part of functionality of a Network Function that can be consumed by other NF, while Network Service defines a set of NFs connected together that facilitate a network operation.

Edge computing

Edge computing is an optional solution for 5G networks. The presence of Multi-access Edge Computing (MEC) reduces some Network Service latency as well as the network contention, resulting in a better service experience for end-users.

Apart from signaling services, a user equipment may also interact with other Network Functions such as third party application functions. By introducing edge computing, these functions are able to be hosted in a decentralized cloud. The main advantage of edge computing lies in the possibility to deploy such decentralized MEC cloud close to the UE's access point of attachment similar to the distributed deployment of some control plane NFs. It is indeed possible for a MEC to be deployed at the RAN edge, in a distributed datacenter or even in a centralized datacenter depending on the service requirement. Edge computing or MEC is not a new technology. To enable the interaction between MEC system and 5GC control plane, the design approach taken by 3GPP allows the mapping of MEC onto Application Functions (AF) (ETSI, 2018). MEC can thus interact with the 5G system using Network Exposure Function (NEF) that provides information from external application to 3GPP network, or directly with the target 5G NFs if permitted (ETSI, 2020). Edge computing could be a suitable solution for URLLC type scenarios, e.g. collection and analysis of a large amount of information from massive IoT devices such as connected sensors. The application running on a MEC host deployed on the RAN edge could process the data locally and extract the useful information to the central server. Integrated with MEC system, 5G networks need to be resilient to guarantee the Core Network Function availability and to ensure the connection between MEC and 5G Core Network.

Service function chaining

To deliver an End-to-End service, various Network Functions are required. A service function chain (SFC) defines an ordered set of Network Functions and ordering constraints that must be applied to packets and/or frames and/or flows selected as a result of classification and/or policy to deliver such an End-to-End service. The mechanism of building such function chains and forwarding packets, frames or flows through them is called service function chaining (ITU-T, 2016). From an End-to-End perspective, a SFC defines how a Network Service is implemented. Since the Network Function Virtualisation is applied in 5G networks (see Section 3.1 for more details), the SFC becomes Virtualized Network Function (VNF) chain (see Figure 3 an example of two service function chains). To allocate SFC request on NFV Infrastructure is challenging. The VNF instances should be hosted at the server with enough resources. Some specific rules may define the isolation or co-location of VNF instances. VNFs may have specific behaviors: Some VNFs can be load balancers, thus parallel processing is allowed; Some VNFs may have multiple outputs (next VNF) depending on the attribute of input traversing traffic; Sometimes, the traffic arrived at a VNF cannot be processed immediately, it has to be queued. Taking into consideration all these constraints, the placement of SFC is a complex problem. A lot of works address this problem by proposing different approaches including resolving a shortest path problem [START_REF] Martini | Latency-aware composition of virtual functions in 5G[END_REF], integer linear programming (ILP) [START_REF] Baumgartner | Mobile core network virtualization: A model for combined virtual core network function placement and topology optimization[END_REF] or mixed-integer linear programming (MILP) [START_REF] Dietrich | Network function placement on virtualized cellular cores[END_REF].

The complexity from the Multilayer perspective

The End-to-End perspective illustrates horizontally the complexity when delivering a Network Service. The multilayer perspective reveals vertically the complexity while orchestrating and managing a Network Service.

Network function virtualisation

With more and more new services joining the network, the resource allocation and service maintenance become a bottleneck for improving the service performance. In 5G networks, the Network Function Virtualisation (NFV) technology is proposed to solve the problem. This technology reforms the network architecture by separating software from the hardware with the help of virtualisation [START_REF] Chiosi | Network Functions Virtualisation: An Introduction, Benefits, Enablers, Challenges & Call for Action[END_REF]. Virtualizing Network Functions enables flexible distribution of hardware resources to improve the service performance and rapid launch of innovative services to generate new revenue sources. It is also an enabler for the formerly mentioned flexible deployment of several 5GC Network Functions, e.g. AMF, and co-locating them with the access network and thus eliminating long-distance data transport [START_REF] Han | Network function virtualization: Challenges and opportunities for innovations[END_REF]. All core NFs and some access NFs are targeted to be virtualized in 5G networks in some scenarios (ENISA, 2020).

With virtualisation, the physical Network Functions become Virtualized Network Functions (VNFs). These VNFs can be deployed on virtual machines (VMs) or containers. The former is a traditional virtualisation environment while the latter is lighter-weight and more agile. The infrastructure resources including storage, compute and network are virtualized and useable for virtualized layer. Instead of allocating a fixed amount of physical resources, VNFs can allocate dynamically virtual resources according to the service request and traffic in realtime.

More specifically, NFV adopts a three-tier architecture (ETSI, 2014) as shown in Figure 5. At the top is the operation layer, with Business Support Systems (BSS) and Operations Support Systems (OSS) to support various End-to-End telecommunication services. Some processes covered by OSS/BSS include: network management, service delivery, fulfilment, assurance, and billing. Lower down is the Network Service and Network Function layer. Inside this layer, the Virtualized Network Functions are managed by Element Managements (EMs). EM's role includes security management, fault management for the exposed Network Function services provided by VNFs. At the bottom lies the NFV infrastructure (NFVI). Storage and compute are two main physical hardware resources which are normally pooled. Another physical resource is networking devices including routers and links. The virtualisation layer abstracts the hardware resources and decouples the VNF software from the underlying hardware, ensuring a hardware independent lifecycle for the VNFs. For the majority of current deployments, the virtualisation layer in an NFVI comprises a hypervisor to partition physical servers into VMs and a network controller, typically a Software-Defined Network (SDN) controller, to help partition the physical network that connects the physical servers into multiple virtual networks interconnecting groups of VMs. While the vast majority of NFV deployments is still based on hypervisor technologies, container-based virtualisation (a.k.a. Operating System (OS) virtualisation) is gaining momentum and might become the norm for 5G. Containers provide an isolation capability that allows multiple VNF instances to share the same host OS while virtual machines require a separate guest OS for each VNF instance. VNFM) is in charge of the life cycle operations as well as performance, fault and configuration management of a VNF. Specifically, the managements include instantiation, heal, operation (changing the state), information modification, changing connectivity, scaling and termination. Each VNF manager serves one or multiple VNFs according to the network design. The third block, Virtualized Infrastructure Manager (VIM), involves all life cycle operations of a virtualized resource. Concretely, a VIM controls and manages the interaction of a VNF with physical and virtualized resources including compute, storage and network. Similar to VNFM, multiple VIMs can be deployed in the network.

VNF deployment

VNF can be VM based or container based depending on the choice of technologies. Traditional VMs virtualizes an underlying computer while the container is lighter, containing the code and dependencies needed, taking less time to image and to run an application.

A VNF is composed of one or multiple VNF components (VNFCs) (ETSI, 2014). A VNFC is a software entity in charge of different functionalities or data bases (in 5G network). Thus, a VNF is mapped to one or several VMs or containers in NFVI servers. It is worth noting that even though there could be multiple VNF components belonging to a same VNF instance, they are not necessarily deployed all on the same host.

Example of operations in Network Service

Scaling is a typical action to manage a Network Service. Scaling can be categorized into two classes: horizontal scaling and vertical scaling. The former includes scaling in and out, which refers to a process where one or more instances are removed or added. The latter action includes scaling up and down, which refers to a process of adding or releasing resources to or from an existing instance.

Network Service level scaling out and in are important operations during the management of Network Services (3GPP TR32.842, 2015). They may be triggered from OSS, by an operator manually or by some Network Managerlevel functions (e.g. Load Balancing) automatically. Then NFVO will receive the request to scale out or in a Network Service instance. For a Network Service, scaling is necessary when one or several of the actual VNF instances in the network is/are overloaded or too redundant for the Network Service. The scaling out in the Network Service level can be done by scaling out or scaling up concerning VNF instances. The service level scaling in can be done by scaling in or scaling down the corresponding VNF instances. Precisely, Network Service can be scaled out (or in) by expanding (contracting) some of existing VNF instances or by instantiating new (terminating existing) VNF instances. When there is a resource change involved, NFVO will also send a request to VIM to allocate the changed resources.

The VNF level scaling can be triggered from NFVO, VNFM, EM, OSS, or manually by the operator. This scaling concerns the management of VNF components. The scaling request will be received by VNFM and it performs the VNFC instantiation or termination procedure for horizontal scaling. In vertical scaling, VNFM requests an update of the resources for VNFC instances.

In case of threat or failure, an operation can be triggered from different levels of the network. When an overload situation happens on a server, this will cause malfunction on VNFCs relying on it. After failure or overload detection, multiple entities of the network may react competitively as depicted in Figure 6. In case of Kubernetes container, Kubernetes hypervisor may decide an instantiation of one or multiple related Pods (a group of containers) on available servers. VNFM may scale out or up the concerning VNFC. NFVO may also change the deployment flavor and apply a rule such as instantiating VNF or increasing the capacity allocated to the concerning VNF. The 5G network mechanism at the VNF level may decide to reduce the number of messages sent to the VNF hosted on the overloaded server or redirect the messages. All these decisions are helpful to some extent but not complementary. It is necessary to determine upstream the best entity which must make the decision according to the situation to avoid a bad decision making situation worse. If we find ourselves in a case of a signaling storm, deploying new instances of VNF or VNFC can impact the datacenter at large by saturating again the servers, whereas actions at the 5G network level would have made it possible to resolve the problem.

Procedures of setting up a slice

5G networks enable a variety of Network Services. With the use of network slicing, the application requirements become more challenging and heterogeneous in order to better serve the vertical industry (ITU-T, 2018; Foukas et al., 2015). A use case from vertical industries may be translated into several Network Services in 5G networks. To set up these Network Services, several procedures such as service selection, resource allocation, are indispensable for Mobile Network Operator (MNO). A detailed example of procedures may be as follows:

• Expression of need and service selection: description of the service and associated requirements referring to an existing catalog for solution

Resilience challenges in 5G network

Resilience is a relatively new field in system engineering that drew great attention over the last decade. Resilience could be defined as the ability of a system to prepare and plan for, absorb, recover from, and more successfully adapt to adverse events. (National Academies Committee on Increasing National Resilience to Hazards and Disasters, 2012).

From ENISA Telecom Services Security Incidents 2019 Annual Analysis Report, system failures, human errors and natural phenomena are the three main causes of telecommunication incidents (ENISA, 2020). More than half of the telecom security incidents were caused by system failures. The most common system failures are hardware failures and software bugs. This type of adverse events often happens on one single element, however other elements of the communication network may also be victims as a result of failure propagation due to interdependencies in telecommunication networks [START_REF] Martins | Interdependence between power grids and communication networks: A resilience perspective[END_REF]. Human errors normally result from an imperfect system design or a wrong configuration. For natural phenomena, the main characteristic is the broad effect in scope. For example, a blizzard would possibly result in multiple network nodes and links failures in the affected region. Other kinds of failures including malicious attacks are rare compared with the three main failures, thus are not the focus in our approach of resilience challenges in 5G network.

A resilient 5G network should be able to offer services with high Quality of Service (QoS) at all time regardless of the adverse events. QoS is the ability of a service to comply with quality requirements and service level as agreed (or targeted) with the end user. The QoS is often interpreted into important performance parameters of the telecommunication system, typically referred to Key Performance Indicators (KPIs) [START_REF] Kukliński | Key Performance Indicators for 5G network slicing[END_REF]. As 5G is supposed to be a vertical service delivery model, the challenge of resilience in 5G systems resides in the fact that KPIs and service requirements vary from one case to another. These KPIs may include latency, availability, throughput, etc. In the use case of tele-action in power systems, the End-to-End service latency is the main KPI, which should be less than 50 milliseconds in order to guarantee the functioning of the grid network (ENEDIS, 2020). In another use case of autonomous vehicles in manufacturing environments, network availability should be higher than 99.90% and network End-to-End latency should be lower than 10 milliseconds (5G EVE, 2018).

To evaluate the performance and resilience of 5G Network Services, recent works focus on two types of situations, namely in the design process and system recovery process. With the NFV environment, Service Function Chain (SFC) becomes the carrier of Network Services. Then the problem is to evaluate and optimize the SFC deployment. In the SFC design process, it aims to prevent failure before it happens by analyzing for example necessary redundancy on each element of the chain and verify the KPIs on the chain as well as the redundant ones. In the recovery process, it is important to analyze the cost and time to provision the backup elements in the chain. Given the complexity of 5G, the problem needs to be solved by taking into consideration of both End-to-End and multilayer perspectives. On the other hand, the multi-layer perspective emphasizes the complexity of SFC management. The operations such as network element scaling and failure recovery should be modeled. The competitive actions from different layers as presented in Section 3.3 will also be a challenge for resilience analysis.

The mainstream researches optimize the SFC deployment while subjecting to the resources, placement and performance constraints. These optimizations are mostly based on ILP or MILP models. The optimization goals vary from one to another, such as minimizing the bandwidth usage across the network [START_REF] Qu | A reliability-aware network service chain provisioning with delay guarantees in NFV-enabled enterprise datacenter networks[END_REF], minimizing the total cost of deploying all network slice requests [START_REF] Silva Coelho | On the impact of novel function mappings, sharing policies, and split settings in network slice design[END_REF], minimizing the cost of protecting the SC against single failure (Carlinet et al., 2020), jointly minimizing the overall deployment cost and service delay [START_REF] Leivadeas | Vnf placement optimization at the edge and cloud[END_REF]. These works simplify the Transport Network and Core Network and neglect the complex RAN. Only a few of them consider the system recovery process and these models often neglect the interdependency between each layer, the existence of MANO, the interaction between NFV-MANO and VNFs. For example, in the recovery process, the failure on MANO actually will block the VNF level or service level scaling.

Some recent works based on Petri Net have studies the recovery process [START_REF] Rui | Petri Net-Based Reliability Assessment and Migration Optimization Strategy of SFC[END_REF], the decomposition of VNF [START_REF] Mauro | Availability modeling and evaluation of a network service deployed via NFV[END_REF], Network Function behaviors [START_REF] Schneider | Specifying and analyzing virtual network services using queuing petri nets[END_REF], NFV-MANO structure (Tola, et al., 2019) in the telecommunication network. The models based on Petri Net and its extensions seems to be a promising tool in Network Service evaluation since it can better describe the complex 5G network. Petri Nets' main attraction as a modeling formalism is how the basic aspects of concurrent systems are identified both conceptually and mathematically [START_REF] Bonet | PIPE v2. 5: A Petri net tool for performance modelling[END_REF]. The marking of the state of a Petri Net model shows the state of the telecommunication system. The transition of a Petri Net model represents an action, e.g. scaling, failure and recovery. Its extensions such as Colored Petri Nets, Timed Petri Nets and Stochastic Petri Net enrich the capacity of the model and make it possible to measure the performance of the Network Service, e.g. availability and latency.

Conclusion

In this paper, we have introduced the complexity in 5G networks from both End-to-End and multi-layer perspectives. Some use cases are given to further explain the complexity in setting up a Network Service. By implementing new technologies, in particular NFV, 5G networks are becoming more flexible but also more complex. Therefore, more and more resilience challenges are awaiting to be taken before introducing 5G networks into new scenarios.

As we noted, modeling the network and its complexity is an important step for evaluating Network Service performance. We propose Petri-Net as a promising tool for the 5G network performance analysis. The work on modelling 5G networks to evaluate the resilience of an Endto-End service related to verticals, is in progress.

ACKNOWLEDGMENT

This work is funded by Orange in the framework of the Chair on Risk and Resilience of Complex Systems (CentraleSupélec, EDF, Orange, SNCF)

Figure 1

 1 Figure 1 shows the End-to-End architecture in 5G network. It includes in general terminals, Next generation Radio Access Network (Ng-RAN), Transport Network, 5G Core Network, Data network. In a 5G use cases such as autonomous vehicles, more than one Network Service may be needed. A Network Service may traverse all or only part of the aforementioned elements.

Fig. 1 .

 1 Fig. 1. 5G End-to-End Architecture.

Figure 2

 2 depicts a 5G network architecture. The upper part of the architecture shows the 5GC Control Plane which uses Service-based interfaces. The 5GC Control Plane consists of the following Core Network Functions (NF).

Fig. 2 .

 2 Fig. 2. 5G Service-Based Architecture. Mobile Signaling is the engine of Mobile Networks Control Plane. As the Core Network adopts a service-based architecture, the signaling network elements are designed in form of Control Plane Network Functions. Each NF can thus expose a set of services called NF services to the service-based interface. Thus, these NFs can be both a consumer where they seek to consume the NF services provided by other NFs and an NF service producer where they provide their exposed services to NF service consumers. Each NF can provide multiple NF services for different NF consumers and can consume NF services from multiple service providers.To avoid ambiguity in the paper, NF service refers to part of functionality of a Network Function that can be consumed by other NF, while Network Service defines a set of NFs connected together that facilitate a network operation.

Fig. 3 .

 3 Fig. 3. Two VNF Chains in one network.

Figure 4

 4 Figure 4 shows an example where two service function chains are deployed in the network. Both SFC 1 and 2 start from consuming VNF 1 and VNF 2. Since these two chains serve different services, they consume different VNFs separately thereafter. SFC 1 utilizes VNF 3, 5 and SFC 2

Fig. 5 .

 5 Fig. 5. Network Function Virtualisation architecture. NFV Management and Network Orchestration (MANO) takes charge of the management of NFVI and orchestrates the allocation of resources needed by the Network Services and VNFs (ETSI, 2016). NFV MANO includes three functional blocks. NFV Orchestrator (NFVO) is responsible in general for the life cycle operations of a Network Service. NFVO functions can be

Fig. 6 .

 6 Fig. 6. Four competitive reactions of different network entities to face one overload situation.

  From the End-to-End perspective, a SFC should include the Radio Access Network, Core Network, Transport Network, and the Edge Computing and Data Network if applicable. While modeling the network, we should be capable to answer how is RAN deployed and where are RU and BBU deployed; what are the Control Plane Core Network Functions engaged in this SFC; what technologies are used on Fronthaul, Midhaul, Backhaul and Backbone; what are the elements co-located in the network.