Accurate replication of the genome is essential for reproduction in all cells. However, even under normal conditions, the replication machinery may face a variety of impediments that can prevent it from completing its task. The mechanism by which cells overcome these hurdles is likely to vary depending upon the nature of the obstacle. Both UV irradiation and inactivation of replicative proteins in DnaB can inhibit the progression of the DNA replication machinery. However, the mechanism by which replication recovers following UV irradiation is different from the mechanism of recovery following the inactivation of the replicative proteins. Previous results show that following UVinduced damage in Escherichia coli, the replication fork is maintained and protected from extensive degradation by RecF, RecO, and RecR until replication can resume. By contrast, replication does not recover following inactivation of the replication protein DnaB, and the nascent DNA is extensively degraded irrespective of whether RecF is present. In this study, we verified DNA replication arrest by monitoring the total DNA accumulation and rate of DNA synthesis following UV-induced DNA damage and inactivation of thermosensitive replication alleles, such as dnaB266. We measured the amount of nascent DNA degradation, allowing us to determine how the newly synthesized strand of DNA is affected following replication fork arrest. Our data indicate that following inactivation of DnaB266, the replication fork is not maintained and is subject to extensive degradation. The degradation that occurs after DnaB266 inactivation is partially reduced in the absence of RecF-O-R, RecJ, and ExoI, suggesting that DNA processing by these enzymes occurs after DnaB arrest. In addition, two-dimensional agarose gel analysis revealed that unique structural intermediates accumulated following inactivation of DnaB266. These observations indicate that the recovery of replication when impeded by DNA lesions, such as those produced by UVirradiation, is maintained and processed through mechanisms that do not resemble the events occurring when replication proteins are inactivated.

No description available.

Diversos estudos têm demonstrado que os danos causados pela exposição da pele à radiação ultravioleta (RUV) são relacionados aos fotodanos ao DNA, geração de espécies reativas de oxigênio e ativação de mediadores do processo inflamatório. Há, portanto, um crescente interesse pelo uso de antioxidantes com potencial fotoquimiopreventivo, como o extrato de maçã e a rutina. O modelo mais utilizado para avaliação de agentes fotoquimiovreventivos é a exposição de camundongos sem pelos à RUV. Porém, os esforços para diminuir ou mesmo evitar a utilização de animais em ensaios científicos tem levado a busca por métodos alternativos à experimentação animal. Na primeira etapa desse trabalho visou-se a otimização de parâmetros relativos ao processo de extração do pó da maçã, bem como a caracterização do extrato obtido e da rutina. Assim, as condições de extração da maçã otimizadas foram tempo de extração de 22 h, teor de etanol no solvente de 60 % (p/p) e proporção solvente:planta de 18 (p/p). A concentração dos ativos presentes na maçã levou ao extrato enriquecido com polifenóis da maçã (EEPM), que apresentou elevada atividade antioxidante in vitro e teor de rutina de 6,1 ± 0.3 ?g/g de extrato. Ambos os ativos apresentaram baixa ou nenhuma toxicidade contra os fibroblastos MRC5, bem como protegeram os fibroblatos contra a morte induzida pela RUV e inibiram a formação de peróxidos lipídicos gerados pelas células irradiadas no tratamento com 4000 e 100 ?g/mL de EEPM e rutina, respectivamente. Na segunda etapa desse trabalho visou-se a avaliação do potencial fotoquimiopreventivo do EEPM e da rutina adicionados a uma formulação tópica em modelos de biópsia de pele humana e pele humana reconstruída in vitro e de camundongo sem pelos in vivo. O EEPM (1,25 %) e a rutina (0,75 %) em formulação foram avaliados quanto à retenção cutânea in vitro utilizando célula de difusão vertical de Franz e, embora não tenha sido possível detectar compostos do EEPM, foi demonstrado que 2,04 ± 0,19 ?g/cm2 da rutina ficou retida na biópsia de pele humana. Na avaliação da eficácia fotoquimiopreventiva em modelos de pele humana in vitro o EEPM e a rutina adicionados em formulação foram capazes de evitar/diminuir a formação de sunburn cells, a indução de caspase-3, dímeros de ciclobutanodipirimidina, metaloproteinases e peroxidação lipídica em pele exposta à RUV. Quanto a atividade funcional in vivo, o extrato enriquecido com polifenóis da maçã apresentou leve efeito inibidor do infiltrado inflamatório induzido pela RUV, enquanto que tanto a rutina como o extrato inibiram a depleção dos níveis de GSH endógeno, o que sugere uma potente atividade fotoquimiopreventiva para estes princípios ativos. Estes resultados são promissores e apontam para o uso do EEPM e da rutina na prevenção/tratamento dos danos induzidos pela RUV na pele. / Several studies have shown that the ultraviolet radiation (UVR) - induced skin damage are related to DNA photolesions, generation of reactive oxygen species and activation of inflammatory mediators. Therefore, there is an increasing interest in the use of antioxidants with photochemoprotective potential, such as apple extract and rutin. The most used model for evaluation of photochemoprotective agents is the exposure of hairless mice to UVR. However, efforts to reduce or even avoid the use of animals in scientific trials has pursued for alternative methods to replace/reduce animal testing. The first step of this work aimed to optimize parameters for the extraction of apple powder and the characterization of the obtained extract and rutin. Thus, the optimized apple extraction conditions were extraction time of 22 h, ethanol content of 60% (w/w) and plant:solvent ratio of 18 (w/w). The apple extract concentration led to an enriched apple polyphenols extract (EEPM), which showed strong in vitro antioxidant activity and rutin content of 6.1 ± 0.3 ?g / g. The actives showed low or no toxicity against MRC5 fibroblasts, protected these cells against UVR - induced death and inhibited lipid peroxidation in irradiated cells (treatment with 4000 and 100 ?g/mL of EEPM and rutin, respectively). The second step of this study aimed to evaluate the photochemoprotective potential of EEPM and rutin added in a topical formulation and assayed in in vitro models of human skin biopsy and human reconstructed skin and in vivo hairless mouse. The EEPM (1.25%) and rutin (0.75%) formulations were evaluated for skin retention in vitro using the Franz diffusion cell and, although it was not possible to detect compounds of EEPM, rutin was retained in the human skin biopsy (2.04 ± 0.19 mg/cm2). As regard the The photochemoprotective efficacy in in vitro models of human skin, EEPM and rutin formulations were able to prevent/reduce the formation of sunburn cells, induction of caspase-3, cyclobutane pyrimidine dimers, matrix metalloproteinases and lipid peroxidation in skin exposed to UVR. As for in vivo functional activity, EEPM showed a slight inhibitory effect of UVR-induced inflammatory infiltrate, while both EEPM and rutin completely inhibited endogenous GSH levels depletion. These results are promising and suggest the use of EEPM and rutin in the prevention/treatment of ultraviolet radiationinduced damage to the skin.

Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

Submitted by Rosina Valeria Lanzellotti Mattiussi Teixeira (rosina.teixeira@unisantos.br) on 2016-08-09T18:20:11Z No. of bitstreams: 1 Gustavo Abrah¿o dos Santos.pdf: 1810524 bytes, checksum: 2eaefbccae262fa0bc6061286f82adc0 (MD5) / Made available in DSpace on 2016-08-09T18:20:11Z (GMT). No. of bitstreams: 1 Gustavo Abrah¿o dos Santos.pdf: 1810524 bytes, checksum: 2eaefbccae262fa0bc6061286f82adc0 (MD5) Previous issue date: 2016-04-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The theme of this work is the worker's collective health, exposed to risks arising from climate change, for work in the open. The study has the purpose of showing the environmental impacts of climate affect the public health worker, often subject to open in the external environment, with solar power. Regarding the questioning, this occurs in relation to a worker who works in the open and in construction, performing their work subjected to solar charge without receiving the interest of the employer to minimize or neutralize the effects of heat and ultraviolet radiation in these lives. Moreover, the questioning is more reluctant when it prevails the pollution in the environment labor law by failing to apply the principles of environmental law, nor the necessary health protection instruments in the work space, when the failure of employers to international standards and national Related to the theme on screen. As a result of the inscribed problem, clarify are the possible diseases that come from the solar charge exposure as well as recognition of the Nexus Technical Epidemiological Pension (NTEP) in the case of skin cancer and worker exposure to ultraviolet radiation, when active in construction civil. Already as a possible solution, the three-dimensionality of the protection of occupational health in the open and in construction is shown; namely: management, technical and legal. The management solution is elucidated by means of the management systems of the International Organization for Standardization (ISO); the technical solution is illustrated from the Program of Environmental Risk Prevention Program (PPRA) and the Conditions and Environment Programme of Work in the Construction Industry; while the legal solution is by Collective Bargaining Convention, as well as legislative changes that protect occupational health. Finally, the methodology used is the historical normative construction of national and international binding and non-binding documents. / O tema desta dissertação é a saúde coletiva do trabalhador, exposto a riscos decorrentes das alterações climáticas, por trabalho a céu aberto. O estudo possui como objetivo demonstrar que os impactos ambientais do clima atingem a saúde coletiva do trabalhador, não raro sujeito a céu aberto, em ambiente externo, com carga solar. No tocante à problematização, esta se dá em relação ao trabalhador que atua a céu aberto e na construção civil, desempenhando o seu labor submetido a carga solar, sem receber o interesse do empregador em minimizar ou neutralizar os efeitos do calor e da radiação ultravioleta nessas vidas. Mais ainda: a problematização é mais relutante quando se prepondera a poluição no direito ambiental do trabalho, ao não se aplicarem os princípios do direito ambiental, tampouco os instrumentos necessários de tutela à saúde no espaço laboral, quando do descumprimento dos empregadores às normativas internacionais e nacionais relacionadas ao tema em tela. Como consequência do problema inscrito, elucidam-se as possíveis doenças advindas da exposição à carga solar, bem como o reconhecimento do Nexo Técnico Epidemiológico Previdenciário (NTEP), no caso de câncer de pele e exposição do trabalhador à radiação ultravioleta, quando atuante na construção civil. Já como possível solução, é apresentada a tridimensionalidade da tutela da saúde ocupacional a céu aberto e na construção civil; a saber: gestão, técnica e legal. A solução de gestão é elucidada por meio dos sistemas de gestão da Organização Internacional para Padronização (ISO); a solução técnica é ilustrada a partir do Programa de Prevenção de Riscos Ambientais (PPRA) e do Programa de Condições e Meio Ambiente do Trabalho na Indústria da Construção Civil; enquanto a solução legal ocorre por Convenções Coletivas de Trabalho, bem como alterações legislativas que tutelem a saúde ocupacional. Enfim, a metodologia utilizada é a construção histórica normativa dos documentos e internacionais e nacionais vinculantes e não vinculantes.

Global amphibian declines have been attributed to numerous and often synergistic causes, such as invasive species, pathogens, and ultraviolet-B (UV-B) radiation. The effects of these stressors are context dependent and can vary with location, species, and populations. As sensitivity to UV-B has shown inconsistencies across amphibian taxa, it can be expected that variation also occurs between populations of a single species. High elevation populations of the long-toed salamander (Ambystoma macrodactylum) face exponentially higher UV-B radiation levels relative to low elevation populations and these levels are predicted to increase in conjunction with continued ozone depletion. We hypothesized that breeding long-toed salamander females at high elevations have modified oviposition behavior to better protect embryos from UV-B induced damage. In addition, we hypothesized that long-toed salamander embryos at high elevation would exhibit elevated photolyase activity, a photo-reactivating enzyme that repairs UV-B radiation-induced damage to DNA. We predicted that this behavioral defense strategy would be employed together with an elevated physiological response as a correlated defense response to increased levels of UV-B radiation in high elevation populations. We surveyed high and low elevation long-toed salamander breeding sites throughout Oregon to quantify oviposition site characteristics and associated UV-B profiles. We simultaneously collected embryos for quantification of photolyase activity in a bacterial transformation assay. We found significant differences in oviposition behavior across elevations, with high elevation breeding females ovipositing in deeper water and using UV-B protective refugia. Oviposition sites at low elevations, however, were most often found in UV-B exposed microhabitats located at the surface of the water. This population difference in oviposition behavior resulted in a standardization of UV-B and temperature conditions for long-toed salamander embryos across elevation. In contrast, we found no population differentiation in photolyase activity between high and low elevation breeding sites. This indicates that behavioral selection for UV-B protected oviposition substrates may either be negating the need for increased photolyase activity in long-toed salamander embryos, or that populations lack the capacity to adapt a heightened physiological response to UV-B at high elevations. Together, these results show how trade-offs in physiology and behavior are a unique adaptation to a significant environmental stressor. Further research into the susceptibility of amphibian species to changing environmental conditions may help to demonstrate the effectiveness of correlated trait responses and plasticity in behavior, and species persistence under changing climate regimes. / Graduation date: 2012

La apigenina (4', 5, 7 trihidroxiflavona), flavonoide presente en distintos vegetales, tiene numerosas características saludables por las cuales elegimos un derivado hidrosoluble, la apigenina potásica, para el presente estudio.Hemos estudiado su actividad frente a la inflamación aguda, al cáncer de próstata y a los daños causados por las radiaciones, tanto ionizantes como no ionizantes (radiación UV), utilizando diversas técnicas, tanto in vitro como en modelos in vivo. Este flavonoide demostró actividad antiinflamatoria, reduciendo la inflamación hasta un 78%. Asimismo, la apigenina potásica mostró actividad quimiopreventiva frente al cáncer de próstata, al reducir la viabilidad y la migración e inducir apoptosis in vitro, y aumentar la supervivencia de animales con tumores. Además, la apigenina potásica presentó efecto geno- y citoprotector frente a la radiación ionizante (radiación γ y X), con un factor de protección de un 27-35 % en linfocitos humanos, y de un 50-86 % en dos líneas celulares de próstata. Por último, el flavonoide ha demostrado proteger frente al fotoenvejecimiento causado por la radiación UV, reduciendo la displasia epitelial y la elastosis dérmica, dos marcadores de cáncer cutáneo; además, ha sido posible detectar la apigenina en diversos tejidos, entre ellos el cerebro. / Apigenin (4', 5, 7 trihidroxyflavone), a flavonoid present in different plants, shows some healthy characteristics for which a water soluble derivated, potassium apigenin, was chosen for this study.It has been studied the activity of potassium apigenin against acute inflammation, prostate cancer and the effect of ionising and non-ionising (UV) radiations, using different techniques, both in vitro and in vivo models.This flavonoid showed anti-inflammatory effects, inhibiting the inflammation by up to 78%. It also demonstrated chemopreventive activity against prostate cancer, by reducing the cell viability and migration, inducing apoptosis and increasing the animal survival. Moreover, potassium apigenin showed genoprotector and citoprotector effects against ionising radiation (radiation γ and X), with a protection factor of 27-35% in human lymphocytes and of 50-86% in two prostate cell lines. Finally, the flavonoid protected from the photoaging induced by UV radiation, diminishing epithelial dysplasia and dermal elastosis, two markers of skin cancer; furthermore, potassium apigenin was detected in some tissues, brain among them.

Outbreaks of food-born illness associated with the consumption of unpasteurized juice and apple cider have resulted in a rule published by the U.S. Food and Drug Administration (FDA) in order to improve the safety of juice products. The rule (21 CFR120) requires manufacturers of juice products to develop a Hazard Analysis and Critical Control Point (HACCP) plan and to achieve a 5-log reduction in the number of the most resistant pathogens. Ultraviolet (UV) disinfection is one of the promising methods to reach this 5-log reduction of pathogens. The absorption coefficients of juices typically vary from 10 to 40 1/cm and can be even higher depending on brand and processing conditions. Thin film reactors consisting of two concentric cylinders are suitable for inactivating pathogens in juices. When the two concentric cylinders are fixed, the flow pattern in the gap can be laminar Poiseuille flow or turbulent flow depending on flow rates. If the inner cylinder is rotating, and the rotating speed of the inner cylinder exceeds a certain value, the flow pattern can be either laminar or turbulent Taylor-Couette flow. UV disinfection between concentric cylinders in laminar Poiseuille flow, turbulent flow and both laminar and turbulent Taylor-Couette flow was investigated experimentally and numerically. This is the first systematic study done on UV disinfection between concentric cylinders in all three flow patterns. The present work provides new experimental data for pathogen inactivation in each of the three flow patterns. In addition, the present study constitutes the first systematic numerical CFD predictions of expected inactivation levels. Proper operating parameters and optimum gap widths for different flow patterns are suggested. It is concluded that laminar Poiseuille flow provides inferior (small) inactivation levels while laminar Taylor-Couette flow provides superior (large) inactivation levels. The relative inactivation levels are: laminar Poiseuille flow < turbulent flow < laminar Taylor-Couette flow.

"A thesis submitted in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy". / Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007. / Bibliography: p. 243-277. / Introduction -- A convenient synthesis of 30HKG -- Facile synthesis of the UV filter compounds 30HKyn and AHBG -- Synthesis, identification and quantification of novel human lens metabolites -- Modification of bovine lens protein with UV filters and related metabolites -- Effect of UV light on UV filter-treated lens proteins -- Conclusions and future directions. / The kynurenine-based UV filters are unstable under physiological conditions and undergo side chain deamination, resulting in α,β-unsaturated carbonyl compounds. These compounds can react with free or protein bound nucleophiles in the lens via Michael addition. The key sites of the UV filters kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) modification in human lenses include cysteine (Cys), and to a lesser extent, lysine (Lys) and histidine (His) residues. Recent in vivo studies have revealed that 3-hydroxykynurenine-O-β-D-glucoside (3OHKG) binds to Cys residues of lens crystallins in older normal human lenses. As a result of this binding, human lens proteins become progressively modified by UV filters in an age-dependent manner, contributing to changes that occur with the development of age-related nuclear (ARN) cataract. Upon exposure to UV light, free UV filters are poor photosensitisers, however the role of protein-bound species is less clear. It has been recently demonstrated that Kyn, when bound to lens proteins, becomes more susceptible to photo-oxidation by UV light. Therefore, the investigation of 3OHKG binding to lens proteins, and the effect of UV light on proteins modified with 3OHKG and 3OHKyn, were major aims of this study. As a result of the role of these compounds as UV filters and their possible involvement in ARN cataract formation, it is crucial to understand the nature, concentration and modes of action of the UV filters and their metabolites present in the human lenses. Therefore, an additional aim was to investigate human lenses for the presence of novel kynurenine-based human lens metabolites and examine their reactivity.--As 3OHKG is not commercially available, to conduct protein binding studies, an initial aim of this study was to synthesise 3OHKG (Chapter 2). Through the expansion and optimisation of a literature procedure, 3OHKG was successfully synthesised using commercially available and inexpensive reagents, and applying green chemistry principles, where toxic and corrosive reagents were replaced with benign reagents and solvent-free and microwave chemistry was used. A detailed investigation of different reaction conditions was also conducted, resulting in either the improvement of reaction yields or reaction time compared to the literature method. Applying the same synthetic strategy, and using key precursors from the synthesis of 3OHKG, the UV filters 3OHKyn and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid-O-β-D-glucoside (AHBG), were also successfully synthesised (Chapter 3). / Chapter 4 describes the investigation of both normal and cataractous human lenses in an attempt to identify novel human lens metabolites derived from deaminated Kyn and 3OHKyn (Chapter 4, Part A). Initially, 4-(2-aminophenyl)-4-oxobutanoic acid (AHA), glutathionyl-kynurenine (GSH-Kyn), kynurenine yellow (Kyn yellow), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid (AHB), glutathionyl-3-hydroxykynurenine (GSH-3OHKyn) and 3-hydroxykynurenine yellow (3OHKyn yellow) were synthesised and human lenses were examined for their presence. AHA and AHB were synthesised from similar precursors to those used in the synthesis of 3OHKG, while the GSH adducts and yellow compounds were synthesised from Kyn and 3OHKyn via base induced deamination. Following isolation and structural elucidation, AHA, AHB and GSH-Kyn were confirmed as novel human lens metabolites. They were quantified in low pmol/mg lens (dry mass) levels in normal and cataractous lenses of all ages, while GSH-3OHKyn, Kyn yellow and 3OHKyn yellow were not detected. In contrast to AHA, the lens metabolites AHB, GSH-Kyn and GSH-3OHKyn were found to be unstable at physiological pH. The spectral properties of these compounds suggest that they may act as UV filters. --Chapter 4 (Part B) also describes the identification and characterisation of a novel human lens UV filter, cysteinyl-3-hydroxykynurenine -O-β-D-glucoside (Cys-3OHKG). An authentic standard was synthesised via Michael addition of cysteine to deaminated 3OHKG. Cys-3OHKG was detected in low pmol/mg lens (dry mass) levels in normal lenses only after the 5th decade of life and was absent in cataractous lenses. Cys-3OHKG showed rapid decomposition at physiological pH. / Chapter 5 describes the identification and quantification of amino acids involved in covalent binding of 3OHKG to lens proteins. Model studies with bovine lens proteins and 3OHKG at pH 7.2 and 9.5 were undertaken. The amino acid adducts were identified via total synthesis and spectral analysis, and subsequently quantified upon acid hydrolysis of the modified lens proteins. Under both pH conditions, 3OHKG was found to react with lens proteins predominantly via Cys residues with low levels of binding also detected at Lys residues. Comparative studies with Kyn (pH 9.5) and 3OHKyn (pH 7.2 and 9.5) resulted in modified lens proteins at Cys residues, with only minor modification at Lys residues at pH 9.5. The extent of modification was found to be significantly higher at pH 9.5 in all cases. His adducts were not identified. 3OHKG-, Kyn- and 3OHKyn-modified lens proteins were found to be coloured and fluorescent, resembling those of aged and ARN cataractous lenses. In contrast, AHB and AHA, which can not form α,β-unsaturated carbonyl compounds, resulted in non-covalent modification of lens proteins. AHB may contribute to lens colouration and fluorescence as further reactions of this material yielded species that have similar characteristics to those identified from 3OHKyn modification. These species are postulated to arise via auto-oxidation of the o-aminophenol moiety present in both 3OHKyn and AHB.--In Chapter 6, the potential roles of 3OHKG and 3OHKyn, and the related species AHA and AHB, in generating reactive oxygen species and protein damage following illumination with UV light was examined. The UV filter compounds were examined in both their free and protein-bound forms. Kyn-modified proteins were used as a positive control. Exposure of these compounds to UV light (λ 305-385 nm) has been shown to generate H2O2 and protein-bound peroxides in a time-dependent manner, with shorter wavelengths generating more peroxides. The yields of peroxides were observed to be highly dependent on the nature of the UV filter compound and whether these species were free or protein bound, with much higher levels being detected with the bound species. Thus, protein-bound 3OHKyn yielded higher levels of peroxide than 3OHKG, with these levels, in turn, higher than for the free UV filter compounds. AHB-treated lens proteins resulted in formation of low but statistically significant levels of peroxides, while AHA-treated lens proteins resulted in insignificant peroxide formation. The consequences of these photochemical reactions have been examined by quantifying protein-bound tyrosine oxidation products (3,4-dihydroxyphenylalanine [DOPA], di-tyrosine [di-Tyr]) and protein cross-linking. 3OHKG-modified proteins gave elevated levels of di-Tyr, but not DOPA, whereas 3OHKyn-modified protein gave the inverse. DOPA formation was observed to be independent of illumination and most likely arose via o-aminophenol auto-oxidation. AHB- and AHA-treated lens proteins resulted in statistically insignificant di-Tyr formation, while a light independent increase in DOPA was observed for both samples. Both reducible (disulfide) and non-reducible cross-links were detected in modified proteins following illumination. These linkages were present at lower levels in modified, but non-illuminated proteins, and absent from unmodified protein samples. / This work has provided an optimised synthetic procedure for 3OHKG and other lens metabolites (Chapters 2 and 3). Four novel lens metabolites have been identified and quantified in normal and cataractous human lenses (Chapter 4). Subsequent experiments, described in Chapter 5, identified the major covalent binding sites of 3OHKG to lens proteins, while AHA and AHB showed non-covalent binding. Further work described in Chapter 6 showed that protein-bound 3OHKG, Kyn and 3OHKyn were better photosensitisers of oxidative damage than in their unbound state. Together, this research has provided strong evidence that post-translational modifications of lens proteins by kynurenine-based metabolites and their interaction with UV light appear, at least in part, responsible for the age-dependent colouration of human lenses and an elevated level of oxidative stress in older lenses. These processes may contribute to the progression of ARN cataract. / Mode of access: World Wide Web. / xxxix, 308 p. ill. (some col.)

Suite à la signature du Protocole de Montréal en 1987, la concentration atmosphérique des substances destructrices d’ozone (ODS) est en baisse. La couche d’ozone montre des signes de récupération (Morgenstern et al. 2008a). Toutefois, l’émission des gaz à effet de serre (GHG) est en augmentation et devrait affecter au cours du 21ème siècle la distribution et les niveaux d’ozone dans l’atmosphère terrestre. En particulier, la modélisation du climat futur montre des signes d’accélération de la circulation de Brewer-Dobson transportant l’ozone de l’équateur vers les pôles. L’ozone est un constituant chimique important de l’atmosphère. Bien que nocif dans la troposphère, il est essentiel à la vie sur Terre grâce à sa capacité d’absorption d’une grande partie du rayonnement ultraviolet (UV) provenant du Soleil. Des modifications dans sa variabilité temporelle ou géographique impliqueraient des changements d’intensité du rayonnement UV à la surface de la Terre (Hegglin et al. (2009), Bais et al. (2011)). Le rayonnement UV à la surface affecte toute la biosphère. Les interactions entre rayonnement UV et écosystèmes terrestres et aquatiques sont nombreuses. Ces interactions ont des effets sur les cycles biogéochimiques et engendrent des rétroactions positives et négatives sur le climat (Erickson III et al., 2015a). La capture du CO2 atmosphérique par photosynthèse des plantes terrestres en est un exemple (Zepp et al., 2007a). Dans l’océan la pompe biologique du CO2 par la photosynthèse du phytoplancton est aussi directement affecté par la variabilité du rayonnement UV (Hader et al., 2007a). Pour l’homme le rayonnement UV est nécessaire car il participe à la photosynthèse de la vitamine D (Holick et al., 1980), mais une surexposition à des niveaux d’intensité élevés du rayonnement UV est la cause principale du développement de cancer de la peau (Matsumura and Ananthaswamy, 2004). L’objectif de ce travail de thèse est d’analyser l’évolution possible du rayonnement UV au cours du 21ème siècle, en particulier aux tropiques sud, dans le cadre des modifications climatiques attendues. Une première partie de ce travail consiste à modéliser le rayonnement UV en ciel clair dans les tropiques grâce au modèle TUV (Madronich et al., 1998) et à comparer les résultats aux mesures sols réalisées à la Réunion. Cette première partie permet l’utilisation future du modèle aux tropiques avec un bon niveau de confiance. La sensibilité du modèle de transfert radiatif en fonction de différents paramètres d’entrée est analysée (section efficace d’absorption de l’ozone,spectre extraterrestriel du soleil, ...). Les sorties du modèle sont ensuite validées à partir de mesures UV spectral au sol obtenues grâce à un spectromètre BENTHAM DM300n. Un filtrage ciel-clair des données au sol est opéré à partir de mesures de flux et de l’algorithme de Long and Ackerman (2000). Les projections climatiques des indices UV (Mc Kinlay and Diffey, 1987) sont réalisées par la suite. Pour cela, on utilise les sorties de plusieurs modèles de Chimie-Climat participant à l’exercice d’inter-comparaison CCMI (Chemistry Climate Model Initiative), couplées aux modèle TUV, validé en première partie dans les tropiques. L’exercice CCMI consiste à projeter le climat et la chimie Terrestre jusqu’en 2100 selon différents scénarios. Ces sorties décrivant la chimie et physique de l’atmosphère servent d’entrée au modèle de transfert radiatif, on obtient alors le rayonnement UV jusqu’en 2100 pour différents scénarios. Une première analyse comparative de l’UV obtenue pour quatre scénarios d’émissions (RCP2.6/4./6.0/8.5, Meinshausen et al., 2011) est effectuée. La fin du travail consiste à étudier l’impact des ODS, GHG et aérosols sur l’évolution du rayonnement UV au cours du 21ème siècle, avec un focus particulier sur les tropiques de l’hémisphère sud. / Following the 1987 Montreal Protocol, atmospheric concentrations of ozone-depleting substances are decreasing. The ozone layer shows signs of recovery. Nonetheless, greenhouse gases emissions (GHG) are rising et should affect the ozone distribution in the atmosphere. Ozone is an important due to his ability to absorb ultraviolet (UV) radiation. The goal of this work is to analyse the possible evolution of UV radiation through the 21st century, particularly in the tropics, for possible climate modification. The first part of this work is to UV in clear-sky in the tropics with the TUV (Madronich et al., 1998) model and to compare against ground-based observations made on Reunion Island. This validation allows the utilisation of TUV in the tropics with a good confidence level. The sensitivity of the model is analysed for multiple parameters. Modelling output is validated against spectral ground-based measurement. Climate Projection of UVI (Mc Kinlay and Diffey, 1987) are then realized with the use of output from model participating in the CCMI ( Model Initiative) exercise and the TUV model. CCMI output describes the chemistry and physics of the atmosphere through the 21st century for four climate scenarios (RCP2.6/4.5/6.0/8.5), they are used as input for the TUV model in order to obtain UV radiation. ODS, GHG and aerosols impact on UVI evolution is analysed.