Sun protection has gained worldwide attention because repetitive overexposure to ultraviolet radiation can result in harmful effects on human skin, including sunburn, premature skin ageing, and in the worst case, skin cancer (Eckhardt and Rohwer, 2000; Sengupta and Blain, 2001). The diminishing stratospheric ozone layer, due to environmental degradation in the past few decades, combined with the modern outdoor-oriented lifestyles, are leading to unexpected levels of skin cancer (Davis, Capjack, Kerr, and Fedosejevs, 1997). Wearing Ultraviolet protective clothing is a simple way of practicing sun safety; however, regular cotton generally has very low ultraviolet protection and it is one of the most environmentally damaging crops despite of it is commonly used to make summer clothing. With the increased interest of public awareness related to sustainability and environmental issues, naturally colored cotton was recommended as it provides better ultraviolet protection than regular cotton. In addition, the production of naturally colored cotton is more environmentally friendly than regular cotton. Although several studies have been conducted on the UVR protection of naturally colored cotton, many questions regarding the factors that influence the UVR protection of fabrics remain unanswered. The primary purpose of the study was to examine the effects of perspiration application, weathering exposures, washing action of automatic home clothes washers, and repeated laundering on the UVR protection of a NC lightweight cotton fabric. In addition, five fabric property changes in the test specimen after the treatments of perspiration, weathering exposure, washing action, and repeated laundering (i.e., fabric count change, thickness change, weight change, color change and dimensional change) were included in this study to serve as secondary dependent variables to examine if the four treatment factors (i.e., perspiration application, weathering exposures, washing action of automatic home clothes washers, and repeated laundering) will cause changes in these five fabric properties, and if these changes will lead to changes of UVR protection of NC lightweight cotton fabric. Based on the purpose and objectives of the study, a split-plot repeated measures experimental design was used for the current study. In this study, the whole plot treatment was the weathering exposure, which contained three levels (i.e., semi-tropical climate without water spray, semi-arid climate, and standard conditioning), and the split plot treatments were the combinations of two treatment factors. In order to understand the effects of repeated laundering on the UVR protection and the five fabric properties, except for the control group, all test specimens were laundered after being treated with the three treatment factors (i.e., perspiration, weathering exposure, and washing action), and this process was repeated 15 times. The UVR protection (i.e., express in UPF value change in current study) and the five fabric properties of these treated test specimens were measured before laundering, and after each laundering cycle. The results of UPF value change showed that test specimens treated with perspiration had a lower change in UPF value than the specimens without treatment. The test specimens exposed to Florida condition had the most UPF value change, followed by Arizona and Standard textile testing conditions. A significant difference also found in test specimens that laundered in a traditional washer after ninth cycle and the UPF value decreased as the number of laundering cycle increased. However, test specimens that laundered in a front-loading HE washer showed no significant UPF value change. For the five fabric properties that listed in secondary objective, all four treatments significantly influenced fabric count, fabric thickness and fabric weight. However, perspiration treatment had no significant effect on the dimensional change in warp direction of test specimens, and washing action had no significant effect on the dimensional change in filling direction of the test specimen as well as both Delta E and Delta L of color change. For testing the relationship between the changes of the five fabric properties and UPF value change, Delta E and Delta L of color change had the highest correlation coefficient with UPF value change. Therefore, it is possible that the changes of these two properties caused by the four treatments and lead to the UPF value change. Future research is needed to confirm this relationship. In conclusion, of perspiration application, weathering exposures, washing action of automatic home clothes washers, and repeated laundering do have influence on the ultraviolet protection of the naturally colored cotton. The color change of the test specimens caused by these four treatments possible lead to the change of the ultraviolet protection of the test specimens. More studies are needed to confirm this relationship. / Ph. D.

Ce travail de thèse présente trois grands axes. Le premier axe traite de la synthèse de microsphères de polyméthacrylate de méthyle (PMMA) encapsulant des absorbeurs d’UV organiques (UVA) et des piégeurs de radicaux (HALS) nécessaires à la protection UV des finitions. Le second axe étudie un système d’encapsulation stimuli-sensible pour la libération des stabilisateurs de lumière. Le dernier axe a pour but de comparer l’efficacité des deux systèmes par rapport à leur protection UV dans une application de finition claire acrylique extérieure pour le bois. Dans le premier axe, des microsphères de PMMA contenant des UVA et des HALS ont été synthétisées par la méthode de séparation de phase interne. Les microsphères ont été ajoutées à une résine claire destinée à des applications extérieures, puis appliquées sur du bois d’épinette blanche. Plusieurs formulations ont été réalisées avec des composés libres ou des composés encapsulés. Certaines contiennent des absorbeurs UV et HALS libres, ce qui est comparable aux formulations commerciales disponibles sur le marché présentement, d’autres possèdent plusieurs concentrations différentes en composés encapsulés. La performance des finitions a été évaluée après un vieillissement artificiel. La comparaison permet d’évaluer l’utilité de microsphères avec des stabiliseurs UV dans des finitions claires extérieures pour le bois. Une bonne résistance des finitions contre les UV se traduit par une bonne tenue esthétique de la finition et du bois, un faible changement de couleur du bois ainsi qu’une faible dégradation chimique. Les analyses telles que la spectroscopie infrarouge à transformée de Fourier (FTIR), la microscopie électronique à transmission (MET) et la colorimétrie ont permis d’obtenir les résultats. Dans le second axe, le système est amélioré de façon à le rendre sensible à l’intensité lumineuse. L’utilisation d’un polymère changeant de conformation suivant le rayonnement peut permettre d’optimiser la libération des stabiliseurs UV. Le poly(1-(4-(3-carboxy-4-hydroxyphényl-azo)benzènesulfonamido)-1 ,2-éthanediyl) ou (PAZO) est additionné sur des supports en CaCO3 contenant les stabiliseurs UV. L’encapsulation des composés se fait par coprécipitation de carbonate de sodium (Na2CO3) et de chlorure de calcium (CaCl2). L’assemblage couche par couche permet de déposer des polymères chargés sur le support. Le PAZO est déposé après une couche de chlorure de poly(diallyldiméthylammonium) (PDADMAC). Les particules obtenues sont ajoutées à une formulation de finition claire complète. Les échantillons ont été placés en chambre de vieillissement accéléré. L’encapsulation a été démonté par spectrométrie photoélectronique des rayons X (XPS), MET et par analyse thermogravimétrique (TGA). La transition du polymère PAZO a été observée par colorimétrie. Dans le dernier axe, les deux systèmes ont été comparés en utilisation dans des finitions claires. Les films secs ont été placés en chambre de vieillissement artificiel pour tester leur efficacité. Après 1000 h de vieillissement artificiel, les films sont analysés et comparés au film initial. La technique d’imagerie en spectroscopie Raman permet de visualiser la distribution des protecteurs UV libres. L’analyse mécanique dynamique (DMA) a permis de calculer les modules mécaniques ainsi que la température de transition vitreuse (Tg) des films étudiés. Le dernier axe permet de relier la distribution des protecteurs UV et l’efficacité de protection du film. / The work presented in this thesis is divided in three research axis. The first axis regards the poly(methyl methacrylate) (PMMA) microspheres synthesis containing UV absorbers (UVA) and hindered amine light stabilizers (HALS). UVA and HALS work synergistically to prevent UV degradation in coatings. The second axis studies the synthesis of stimuli-sensitive encapsulation system for the tailored release of the UV stabilizers. The last axis compares the system efficiency in the case of UV protection for clear coat for exterior wood. In the first axis, PMMA microspheres with UVA and HALS were synthesized by the internal phase separation method. Microspheres are added to an exterior clear coat binder then applied onto white spruce panels. Some formulations are realized. Some contains free UVA and HALS similar to commercial paint and others have different concentrations in encapsulated compounds. Paint performance is evaluated after accelerated weathering. The comparison of formulations allow the evaluation of the efficiency of encapsulation of UV stabilizers inside the wood clear coat. The performance of coating consists on a good aesthetic resistance, a small color change and low chemical degradation. Obtained results are based on analysis with FTIR, TEM and colorimetry. In the second axis, the release of UVA is improved by the UV sensitive polymer. This polymer is able to change its own molecular conformation under UV rays. The poly(1-(4-(3-carboxy-4-hydroxyphenyl-azo)benzenesulfonamido)-1,2 -ethanediyl) or (PAZO) is deposited onto CaCO3 templates containing UVA encapsulated. The encapsulation is done by coprecipitation of Na2CO3 and CaCl2.The layer by layer allow the deposition of charged polymers. PAZO is adsorbed after a layer of poly(diallyldimethylammonium chloride) (PDADMAC). The obtained particles are added to a complete clear coat formulation. Samples are placed in an accelerated weathering chamber. The encapsulation is proved by analysis techniques of XPS, TEM and TGA. Polymer PAZO transition is observed by colorimetry. In the last axis of this work, the two encapsulation systems are compared using encapsulated UV stabilizers inside a formulation of wood clear coat. Dried films are placed in accelerated weathering chamber in order to test their efficiency. After 1000 h of artificial weathering, films are analyzed and compared to the initial state. The mapping Raman technique allows to visualize the free UV stabilizers distribution inside the dried film. DMA calculate mechanical modulus and Tg of studied films. This last axis allows to connect distribution of UV protectors and efficiency of coating protection.

Hazard Analysis Critical Control Point (HACCP) regulations for juice manufacture require the application of a process that will result in a 5-log reduction (99.999%) of the pertinent pathogen in the juice being processed. The use of ultraviolet (UV) light, as an alternative to traditional thermal processing, has been adopted by some juice processors as a means of meeting the HACCP 5-log performance standard. However, little research had been performed to determine the effect of UV when used in combination with antimicrobial agents that are commonly added to juice products. Therefore, the objectives of this work were (1) to determine if chemical preservatives and ultraviolet light have a combined effect on the reduction of Escherichia coli in apple cider, and (2) to determine the influence of adding chemical preservatives at different points in the processing of juice (i.e., either prior to or after ultraviolet light processing) on the reduction of Escherichia coli in apple cider. In this study, refrigerated (4°C) pasteurized apple cider that contained no added preservatives was inoculated with E. coli ATCC 25922, a surrogate strain for E. coli O157:H7, and exposed to UV (peak output: 254 nm). The following chemical preservatives were added to apple cider either prior to or after UV exposure: dimethyl dicarbonate (75 and 150 ppm), hydrogen peroxide (75 and 150 ppm), potassium sorbate (1000 and 2000 ppm), and sodium benzoate (1000 and 2000 ppm). Following UV exposure and chemical preservative application, inoculated juices were stored at 4°C for 72 hours. Samples were collected prior to and immediately after UV exposure and at 24, 48, and 72 hours of storage. At each sampling point, juice portions (0.1 ml) were serially diluted in peptone diluent (0.1%) and surface plated onto Tryptic Soy Agar (TSA). Counts of the bacterial colonies were made 48 hours after incubating plates at 35°C. Overall, reductions of E. coli were greater in cider treated with preservatives after UV processing than when preservatives were added prior to UV processing (P < 0.05). Furthermore, dimethyl dicarbonate and hydrogen peroxide were more effective than potassium sorbate and sodium benzoate in reducing E. coli populations in conjunction with UV (P < 0.05). When added prior to UV exposure, potassium sorbate was the least effective, allowing for the greatest survival (P < 0.05). This study describes the use of UV in conjunction with hydrogen peroxide and dimethyl dicarbonate as an effective method for producing a 5-log or greater reduction of E. coli O157:H7 in apple cider. / Master of Science

No / Erythema occurs in human skin following excessive exposure to ultraviolet radiation (UVR), and this is in part mediated by the vasodilator prostaglandin E2 (PGE2). While keratinocytes are a major source of this pro-inflammatory eicosanoid, epidermal melanocytes (EM) also express some of the cellular machinery required for PGE2 production. The primary aim of this study is to determine whether EM can produce PGE2 and so potentially also contribute to UVR-induced skin inflammation. Furthermore, we investigate the likely pathway by which this PGE2 production is achieved and investigate whether PGE2 production by EM is correlated with melanogenic capacity. Primary cultures of EM were established from nine normal healthy individuals with skin phototype-1 (n=4) and 4 (n=5), and PGE2 production and melanogenic status were assessed. EM produced PGE2 under baseline conditions and this was increased further upon stimulation with arachidonic acid. Moreover, EM expressed cytoplasmic phospholipase A2, cyclooxygenase-1 and cytoplasmic prostaglandin E synthase. However, no EM culture expressed cyclooxygenase-2 under baseline conditions or following arachidonic acid, UVB- or H2O2 treatments. PGE2 production in response to UVB was highly variable in EM cultures derived from different donors but when pooled for skin phototype exhibited a positive correlation only with SPT-1 derived EM. Interestingly, PGE2 production by EM in response to UVB showed no correlation with baseline levels of melanin, tyrosinase expression/activity or tyrosinase-related protein-1 expression. However, there was an apparent negative correlation with baseline expression of dopachrome tautomerase (DCT), a melanogenic enzyme with reported anti-oxidant potential. These findings suggest that EM have the potential to contribute to UVR-induced erythema via PGE2 production, but that this response may be more related to oxidative stress than to their melanogenesis status. / The Wellcome Trust

No / High personal UVR doses can be gained during leisure activities, causing intense self-resolving inflammation (sunburn) of unprotected skin. UVR activates release of membrane fatty acids and upregulates their metabolism by cyclooxygenases (COX) and lipoxygenases (LOX) to different eicosanoids. While COX-derived prostaglandin (PG)E2 is a potent mediator of sunburn vasodilatation, LOX-derived 15-hydroxyeicosatetraenoic acid (HETE) and its lipoxin metabolites may contribute to sunburn limitation. We explored the relationships between expression of these lipid mediators and the clinical and histological outcomes, comparing responses of individuals prone and more resistant to sunburn. An acute UVR exposure of 12 SED (standard erythema dose) was applied to buttock skin of 32 white Caucasians (n = 16 phototype I/II, n = 16 phototype III/IV), and over the subsequent 72 h assessments were made of skin erythema, immunohistochemical expression of leukocyte markers, COX-2, 12-LOX, 15-LOX and nitric oxide synthase (NOS), and eicosanoid levels by LC/ESI-MS/MS. Evidence of a significant inflammatory response was seen earlier in phototype I/II with regard to expression of erythema (4h, p < 0.001), neutrophil infiltration (24 h, p = 0.01), epidermal COX-2 (24 h, p < 0.05) and 12-LOX (24 h, p < 0.01), and dermal eNOS (24 h, p < 0.05) proteins, although CD3+ lymphocyte infiltration showed an earlier increase in phototype III/IV (24 h, p < 0.05). Although erythema was equivalent at 72 h in both groups, phototype I/II showed higher PGE2 accompanied by elevated 15-HETE, and a strong positive correlation was seen between these mediators (n = 18, r = 0.805, p = 0.0001). Hence anti-inflammatory eicosanoid 15-HETE may temper the pro-inflammatory milieu in sunburn, having greater influence in those prone to sunburn than those more resistant, given the same high UVR exposure conditions. / The Wellcome Trust

No / Background: Skin cancer is a major public health concern, and the majority of cases are caused by solar ultraviolet radiation (UVR) exposure, which suppresses skin immunity. Omega-3 (n−3) PUFAs protect against photoimmunosuppression and skin cancer in mice, but the impact in humans is unknown. Objectives: We hypothesized that EPA-rich n−3 PUFA would abrogate photoimmunosuppression in humans. Therefore, a nutritional study was performed to assess the effect on UVR suppression of cutaneous cell-mediated immunity (CMI) reflected by nickel contact hypersensitivity (CHS). Design: In a double-blind, randomized controlled study, 79 volunteers (nickel-allergic women, 22–60 y old, with phototype I or II) took 5 g n−3 PUFA–containing lipid (70% EPA plus 10% DHA) or a control lipid daily for 3 mo. After supplementation, nickel was applied to 3 skin sites preexposed on 3 consecutive days to 1.9, 3.8, or 7.6 J/cm2 of solar-simulated radiation (SSR) and to 3 unexposed control sites. Nickel CHS responses were quantified after 72 h and the percentage of immunosuppression by SSR was calculated. Erythrocyte [red blood cell (RBC)] EPA was measured by using gas chromatography. Results: SSR dose-related suppression of the nickel CHS response was observed in both groups. Photoimmunosuppression appeared less in the n−3 PUFA group than in the control group (not statistically significant [mean difference (95% CI): 6.9% (−2.1%, 15.9%)]). The difference was greatest at 3.8 J/cm2 SSR [mean difference: 11% (95% CI: 0.5%, 21.4%)]. Postsupplementation RBC EPA was 4-fold higher in the n−3 PUFA group than in the control group (mean difference: 2.69% (95% CI: 2.23%, 3.14%), which confirmed the EPA bioavailability. Conclusion: Oral n−3 PUFAs appear to abrogate photoimmunosuppression in human skin, providing additional support for their chemopreventive role; verification of study findings is required.

Yes / This article describes the synthesis and application of lanthanide-based luminophores for visualisation under short wavelength ultraviolet (UV) activation (typically 254 nm). Luminophores are chemical compounds which re-emit light of longer wavelength than typical fluorescence and phosphorescence, following photoexcitation, because of efficient energy transfer from the ligands to the lanthanide ion emission levels. The luminophores described in this article are all derived from europium and terbium metal complexes and cover the complete colour gamut. Selection of appropriate ligands allows for water solubility or water insolubility (if required pigment or disperse “dye” applications). Similar to dyeing or printing processes, the anionic complexes can be applied to polyamide fibres or to cellulosic fibres, whereas dispersions of the non-ionic complexes are suitable for polyester or cellulose acetate fibre application. The water-soluble derivatives are also suitable for ink-jet printing and the water-insoluble derivatives are suitable for lithographic or intaglio paper printing. The novel complexes are excited by radiation below 300 nm and thus cannot be activated by sunlight (the earth's atmosphere prevents light below about 300 nm reaching the surface). Accordingly, the photo-stability of the dyed materials and prints obtained is excellent. These materials could be used in security marking applications, for example bank-notes and passports, where they could replace the current fluorophores that are rendered visible under near UV light (typically 365 nm) illumination but exhibit relatively poor photo-stability. The development of an RGB (red, green and blue) colour palette for inkjet printing based on these innovative luminophores will significantly support the design of digitally printed security features. / The research was part funded by Inovink Ltd and theWorshipful Company of Clothworkers of the City of London.

Résumé : L’exposition aux rayons ultraviolets (UV) du soleil est le principal facteur menant au développement du cancer cutané. Les dimères cyclobutyliques de pyrimidines (DCP) résultant d’une exposition aux UV sont considérés comme les principaux dommages à l’ADN impliqués dans la cancérogenèse cutanée. La fréquence des DCP et leur distribution entre les quatre types de sites dipyrimidiniques ont souvent été étudiés in vitro ou en utilisant des UVC alors que nous ne sommes pas exposés à ce type d’UV. L’utilisation d’écrans solaires permet de prévenir la formation des DCP. Grâce à sa capacité à absorber les UVB, l’acide sulfonique 2-phénylbenzymidazole-5 (PBSA) est utilisé dans des écrans solaires mais des études in vitro ont montré qu’il peut oxyder les guanines lors d’une irradiation aux UVB. Face à ces problèmes, nous avons choisi d’étudier l’effet du PBSA, la fréquence et la distribution des DCP, de même que l’influence de la séquence nucléotidique sur la formation des DCP, dans des conditions plus représentatives de la réalité. Nous avons irradié des fibroblastes humains normaux (in cellulo) et de l’ADN purifié (in vitro) aux UVB et aux UVC. Nous avons comparé la formation des DCP in cellulo et in vitro. Suite à une exposition aux UVB, nous avons trouvé que la distribution des DCP in cellulo est similaire à la distribution des DCP in vitro. Nous avons ensuite comparé l’effet du type d’UV sur la formation des DCP et nous avons trouvé que les TT sont plus souvent endommagés après une irradiation aux UVC alors que les sites potentiellement mutés (contenant des cytosines) sont plus souvent endommagés après une exposition aux UVB, in cellulo. Concernant l’effet de la séquence d’ADN sur la formation des DCP, nous avons trouvé que certains sites dipyrimidiniques sont beaucoup plus fréquemment endommagés que les autres et que la position d’un site dipyrimidinique dans une série de pyrimidines adjacentes est un facteur influençant la formation des DCP. Nous avons étudié l’effet du PBSA in cellulo et in vitro suite à une irradiation aux UVA et aux UVB. Nous avons montré que le PBSA protège efficacement contre la formation de DCP, lors d’une irradiation UVB mais qu’il photosensibilise la formation de guanines oxydées lors d’une irradiation aux UVA et aux UVB. Le PBSA peut donc agir comme une épée à double tranchant et ceci questionne son utilisation dans les écrans solaires. Les travaux réalisés dans le cadre de cette thèse améliorent notre compréhension de la cancérogenèse cutanée, montrent l’importance du choix de modèles d’étude pertinents, de même que l’importance d’utiliser une protection solaire efficace contre tous les types de dommages causés par les UV. / Abstract : Exposure to the UV component of sunlight is the principal factor leading to skin cancer development. Cyclobutane pyrimidine dimers (CPD) are considered as the most important DNA damage involved in skin carcinogenesis. CPD frequencies and their distribution between the four types of dipyrimidine sites are mostly investigated in vitro or using UVC, even if we are not exposed to these wavelengths. On the other hand, sunscreens are used to protect against CPD formation. The 2-phenylbenzimidazole-5-sulfonic acid (PBSA) is a sunscreen agent used because it absorbs UVB. However, previous studies have shown that PBSA oxidizes guanines in vitro, during UVB exposure. To address these issues, we chose to study the effect of PBSA, the DCP frequencies and their distribution frequency and distribution of CPD, as well as the influence of DNA sequence on CPD formation, in conditions more representative of reality. We irradiated normal human fibroblasts (in cellulo) and purified DNA (in vitro) with UVB and UVC. We compared the CPD distribution in cellulo and in vitro. Our results show that CPD distribution in cellulo is different to CPD distribution in vitro after UVB exposure. Then, we compared the impact of UV type on CPD formation and we found that TT are more frequently damaged after UVC exposure while potentially mutated dipyrimidine sites (dipyrimidine sites containing cytosine) are more frequently damaged after UVB exposure. Concerning the influence of DNA sequence on CPD formation, we observed that some dipyrimidine sites are extremely frequently damaged compared to others and that the position of a dipyrimidine site within a dipyrimidine run is an important factor influencing the frequency of CPD formation. We studied the effect of PBSA, in cellulo and in vitro, during UVA and UVB exposure. We found that PBSA provides good protection against CPD formation during UVB exposure, in cellulo. However, PBSA photosensitized the formation of oxidized guanines during UVA and UVB exposure. This indicates that PBSA can act as a double-edged sword and question its suitability in sunscreens. The works presented in this thesis provide important elements to understand the skin carcinogenesis process and demonstrate the importance to use an effective protection against UV-induced DNA damage.

La réparation par excision de nucléotides (NER) est une voie critique chez l'homme pour enlever des lésions qui déforment l’hélice d'ADN et qui bloquent à la fois la réplication et la transcription. Parmi ces lésions, il y a les dimères cyclobutyliques de pyrimidines (CPDs) et les adduits pyrimidine (6-4) pyrimidone (6-4PPs) induient par les rayons ultraviolets. L'importance physiologique de la NER est mise en évidence par l’existence de la maladie Xeroderma pigmentosum (XP), causée par des mutations affectant des gènes impliqués dans cette voie de réparation. Les personnes atteintes sont caractérisées par une photosensibilité extrême et une forte prédisposition à développer des tumeurs cutanées (plus de 1000 fois). Les patients atteints du type variant de la maladie Xeroderma pigmentosum (XPV), apparemment compétents en réparation, portent plutôt des mutations dans le gène codant pour l'ADN polymérase η (polη). Polη est une ADN polymérase translésionnelle capable de contourner avec une grande fidélité certaines lésions telles que les CPDs, qui autrement bloquent les polymérases réplicatives. Ainsi, la polη prévient la formation de mutations et permet la reprise de la synthèse d'ADN. L'objectif principal de cette thèse est d'évaluer le rôle potentiel de voies de signalisation majeures dans la régulation de la NER, dont celles régulées par la kinase ATR (Ataxia Télangiectasia and Rad3-related kinase). Suite à l'irradiation UV, ATR est rapidement activée et phosphoryle des centaines de protéines qui régulent les points de contrôle du cycle cellulaire et joue un rôle notoire dans le maintient de la stabilité génomique. Nous avons postulé qu’ATR puisse réguler la NER de manière dépendante du cycle cellulaire. Cependant, tester cette hypothèse représente un grand défi car, pour des raisons techniques, les méthodes conventionnelles n’ont pas à ce jour été adaptées pour l'évaluation de la cinétique de réparation au cours des différentes phases du cycle cellulaire. Nous avons donc développé une méthode novatrice basée sur la cytométrie en flux permettant de quantifier avec grande précision la cinétique de réparation des 6-4PPs et CPDs dans chacune des phases G0/G1, S et G2/M. Avec cette nouvelle méthode, nous avons pu démontrer que l'inhibition d'ATR ou polη résulte en une très forte inhibition de la NER exclusivement durant la phase S du cycle cellulaire. Ces études ont révélé, pour la première fois, une fonction critique pour ces protéines dans le retrait des lésions qui bloquent la réplication. En outre, nous avons démontré que la synthèse d'ADN est indispensable pour l’inhibition de la réparation en phase-S, reflétant un lien potentiel entre la NER et la réplication. Curieusement, nous avons également montré que parmi six lignées cellulaires tumorales choisies aléatoirement, trois présentent une abrogation totale de la NER uniquement pendant la phase S, ce qui indique que de nombreux cancers humains pourraient être caractérisés par un tel défaut. Nos observations pourraient avoir d'importantes implications pour le traitement du cancer. En effet, le statut de la NER semble constituer un déterminant majeur dans la réponse clinique aux médicaments chimiothérapeutiques tels que le cisplatine, qui inhibent la croissance des cellules cancéreuses via l'induction de lésions à l’ADN. / Nucleotide excision repair (NER) is a critical pathway in humans for repairing highly genotoxic helix-distorting DNA lesions that strongly block both replication and transcription. Among these lesions are ultraviolet-induced 6-4 photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). The physiological importance of NER is highlighted by individuals afflicted with Xeroderma pigmentosum (XP), who carry mutations in NER pathway genes and as such exhibit extreme photosensitivity and remarkable predisposition to cutaneous tumorigenesis (1000-fold increase). On the other hand patients with the variant form of Xeroderma pigmentosum (XPV) are considered proficient in NER, and rather carry germline mutations in the gene encoding DNA polymerase η (polη). Polη is a specialized translesion DNA polymerase able to accurately bypass certain lesions including CPDs which otherwise completely inhibit the progression of normal replicative polymerases, thereby preventing mutations and allowing the resumption of DNA synthesis. The main goal of this thesis was to elucidate the potential role in NER of major DNA damage signalling cascades, including that regulated by the ataxia telangiectasia and rad 3-related kinase (ATR). Following UV irradiation, ATR is rapidly activated and phosphorylates hundreds of proteins that regulate cell cycle checkpoints and maintain genomic stability. We postulated that ATR might regulate NER in a cell cycle-specific manner. However testing this presented a great challenge, as (for technical reasons) traditional NER assays have to date not been adapted for evaluation of repair kinetics during individual phases of the cell cycle. We therefore developed a novel flow cytometry-based assay for sensitive quantification of 6-4PPs and CPDs repair efficiency during each of G0/G1, S, and G2/M. With this new assay, we were able to show that inhibition of either ATR or polη results in strong inhibition of NER capacity exclusively during S phase of the cell cycle. This revealed, for the first time, a critical function for these proteins in removal of replication-blocking DNA adducts. In addition, we demonstrated that active DNA synthesis is required for S phase-specific repair inhibition, reflecting a potential relationship between NER and replication. Intriguingly, we also showed that among six tumor cell lines, three exhibit total abrogation of NER uniquely during S phase, indicating that many human cancers may be characterized by such a defect. Our findings therefore could harbour important implications for cancer treatment. Indeed, NER status of tumors clearly appears to constitute a major determinant in the clinical response to chemotherapeutic drugs such as cisplatin, which inhibit the growth of rapidly proliferating cancer cells through induction of replication-blocking DNA lesions.

Le mélanome malin est l’un des cancers les plus mortels dont l’incidence continue à augmenter chaque année avec peu de traitement efficace à long terme. Il est causé et initié principalement par l’exposition excessive aux rayons ultraviolets engendrant des photoproduits hautement génotoxiques. Il est bien connu que la cascade de signalisation PI3K/Akt joue un rôle crucial dans la régulation des processus qui sont généralement dérégulés durant le développement tumoral comme la prolifération, le contrôle du cycle cellulaire et l’apoptose. Néanmoins, l’implication de cette voie moléculaire dans la réponse aux dommages à l’ADN est peu caractérisée. Chez les mammifères, trois isoformes de la protéine kinase Akt ont été identifiées: Akt1, Akt2 et Akt3. Bien qu’elles soient très homologues en termes de séquence, plusieurs études ont montré que ces isoformes ont des fonctions biologiques distinctes, et nous suggérons qu’elles puissent contribuer différemment à la régulation de la réponse génotoxique. Les objectifs de ce projet étaient de: (i) évaluer l’activation d’Akt dans les cellules de mélanomes (ii) déterminer l’impact de l’inhibition de cette activité sur la régulation de la réponse cellulaire aux UV (iii) vérifier si la perte d’expression de l’un ou de l’autre des isoformes d’Akt peut réguler la réponse aux UV. Nous avons démontré qu’Akt est transitoirement hyperactivée par phosphorylation suite aux irradiations UV dans les lignées cellulaires de mélanomes. Afin de déterminer l'importance de cette activation dans la réponse cellulaire aux UV, notre approche était de diminuer (i) la phosphorylation d’Akt par l’usage d’inhibiteurs pharmacologiques ou (ii) l’expression de chaque isoforme d’Akt par l’approche des ARN interférents. Nous avons montré que l’inhibition de la phosphorylation d’Akt amène à l’augmentation du taux de l’apoptose induit par les UV d’une manière isoforme spécifique, alors qu’elle n’a aucun effet sur la régulation de la voie de réparation par excision de nucléotides (NER), qui est la seule voie humaine pour éliminer les dommages à l’ADN induits par les UV. En somme, notre étude constitue un nouvel aspect qui permet de mieux comprendre les mécanismes moléculaires du développement de mélanomes malins suites aux irradiations ultraviolettes. / Malignant melanoma is one of the deadliest cancers whose incidence continues to rise each year with a few effective long-term treatments. It is caused and initiated mainly by excessive exposure to ultraviolet radiation generating highly genotoxic DNA photoproducts. It is well known that the PI3K/Akt signaling cascade plays a crucial role in the regulation of processes commonly deregulated in tumor development such as proliferation, cell cycle control and apoptosis. Nevertheless, the nuclear involvement of this molecular pathway in the genotoxic response is poorly characterized. In mammals, three Akt kinase isoforms have been identified: Akt1, Akt2 and Akt3. Although these exhibit a high degree of homology, several studies have shown that they have distinct biological functions; therefore, we suggest that these isoforms may contribute differently to the regulation of genotoxic response. The objectives of this project were to: (i) evaluate Akt activation in UV-irradiated melanoma cells, (ii) determine the effect of the Akt phosphorylation inhibition on the regulation of the cellular response to UV, (iii) evaluate whether the loss of the expression of one or more of Akt isoforms can regulate the cellular response to UV. We demonstrated that Akt undergoes transient hyperactivation after UV treatment in melanoma cell lines. To determine the importance of this activation, our approach was to reduce (i) the phosphorylation of Akt by the use of pharmacological inhibitors or (ii) the expression of each individual Akt isoform using RNA interference. We have shown that inhibition of Akt phosphorylation leads to increased rates of UV-induced apoptosis in an isoform specific manner, while exerting no effect on regulation of nucleotide excision repair (NER), the only human pathway for eliminating UV-induced DNA damage. In summary, our study provides a better understanding of the molecular mechanisms of malignant melanoma development in response to UV.