Etude du rôle de la réponse UV sur le contrôle de la réparation par excision de nucléotides (NER) des dommages à l’ADN : rôle des voies MAPK et de l’ADN polymérase eta

La réponse cellulaire aux ultra-violets (UV), ou réponse UV, est une réponse complexe et spécialisée dans l’adaptation et la tolérance des dommages aux UV. Celle-ci est initiée par un grand nombre d’évènements moléculaires et de signalisation nucléaire mais aussi au niveau de la membrane plasmique ou du cytoplasme. L’importance et l’influence exactes de ces évènements sur la réparation par excision de nucléotides (NER) des dommages UV à l’ADN sont encore mal comprises et doivent encore être méthodiquement démontrées. Dans cette thèse, grâce à l’utilisation d’une méthode sensible d’analyse de la réparation NER basée sur la cytométrie en flux, il est montré, dans un premier temps, que l’activité des voies MAPK (Mitogen-Activated Protein Kinases), qui sont des voies de signalisation de stress UV d’origine cytoplsamique, ne participent pas à l’efficacité de réparation NER des dommages UV dans les cellules humaines. En effet, l’abrogation de la signalisation MAPK, par inhibition pharmacologique, par utilisation de mutants dominant-négatifs ou par inhibition de leur expression endogène, ne révèlent aucun changement de la cinétique de réparation des dommages UV par excision de nucléotides. Cependant, l’utilisation de cette même méthode de réparation, mais cette fois, appliquée pour l’étude de réparation NER en fonction du cycle cellulaire, a permis de mettre en évidence la nécessité fonctionnelle de l’ADN polymérase translésionnelle eta (Pol η) dans la réparation NER des dommages UV, uniquement en phase S. Cette observation fut initialement caractérisée dans les cellules de patients affectés du syndrome variant de xérodermie pigmentaire (XP-V) puis, confirmée ensuite par l’inhibition de l’expression de Pol η endogène ou par la complémentation avec des mutants non-fonctionnels dans les cellules XP-V. Ces résultats indiquent que, contrairement à la réponse UV MAPK cytoplasmique, les évènements nucléaires comme la synthèse translésionnelle, peuvent influencer l’efficacité de réparation NER en phase S. Plus particulièrement, ces données établissent un lien possible entre la réparation NER en phase S et les niveaux de stress réplicatifs, révélé ici par la déficience fonctionnelle Pol η ou ATR. Les observations, présentées dans cette thèse, renforcent un rôle du point de contrôle S aux UV sur l’efficacité de la réparation NER et suggèrent que l’inhibition NER, observée en phase S dans les cellules XP-V, est modulée par le stress réplicatif. Un tel moyen de contrôle pourrait avoir une action plutôt protectrice pendant cette phase critique du cycle cellulaire. Mots clés: UV, translésionnelle, eta, MAPK, NER, CPD, cytométrie, phase-S, tolérance.

Biochemical and functional characterization of the tumor suppressors BRCA1 and BAP1

L’ubiquitination est une modification post-traductionnelle qui joue un rôle majeur dans la régulation d’une multitude de processus cellulaires. Dans cette thèse, je discuterai de la caractérisation de deux protéines, BRCA1 et BAP1, soit deux suppresseurs de tumeurs fonctionnellement reliés. BRCA1, une ubiquitine ligase qui catalyse la liaison de l’ubiquitine à une protéine cible, est mutée dans les cancers du sein et de l'ovaire. Il est bien établi que cette protéine aide à maintenir la stabilité génomique suite à un bris double brin de l’ADN (BDB), et ce, à l’aide d’un mécanisme de réparation bien caractérisé appelé recombinaison homologue. Cependant, les mécanismes de régulation de BRCA1 suite à des stresses génotoxiques n’impliquant pas directement un BDB ne sont pas pleinement élucidés. Nous avons démontré que BRCA1 est régulée par dégradation protéasomale suite à une exposition des cellules à deux agents génotoxiques reconnus pour ne pas directement générer des BDBs, soit les rayons UV, qui provoquent la distorsion de l’hélice d’ADN, et le méthyle méthanesulfonate (MMS), qui entraîne l’alkylation de l’ADN. La dégradation de BRCA1 est réversible et indépendante des kinases associées à la voie des PI3 kinase, soit ATM, ATR et DNA-PK, protéines qui sont rapidement activées par les dommages à l’ADN. Nous proposons que la dégradation de BRCA1 prévienne son recrutement intempestif, ainsi que celui des facteurs qui lui sont associés, à des sites de dommages d’ADN qui ne sont pas des BDBs, et que cette régulation coordonne la réparation de l’ADN. L’enzyme de déubiquitination BAP1 a initialement été identifiée comme une protéine capable d’interagir avec BRCA1 et de réguler sa fonction. Elle est également connue pour sa capacité à se lier avec les protéines du groupe Polycomb, ASXL1 et ASXL2. Cependant, l’importance de ces interactions n’a toujours pas été établie. Nous avons démontré que BAP1 forme deux complexes protéiques mutuellement exclusifs avec ASXL1 et ASXL2. Ces interactions sont critiques pour la liaison de BAP1 à l’ubiquitine ainsi que pour la stimulation de son activité enzymatique envers l’histone H2A. Nous avons également identifié des mutations de BAP1 dérivées de cancers qui empêchent à la fois son interaction avec ASXL1 et AXSL2, et son activité de déubiquitinase, ce qui fournit un lien mécanistique direct entre la déubiquitination de H2A et la tumorigenèse. Élucider les mécanismes de régulation de BRCA1 et BAP1 menera à une meilleure compréhension de leurs rôles de suppresseurs de tumeurs, permettant ainsi d’établir de nouvelles stratégies de diagnostic et traitement du cancer.

Poverty, Inequality and Natural Resources Degradation

The present study is an attempt to understand the link between natural resource degradation and poverty among people dependent on these resources. This is done by examining the impact of depletion of marine resources on the livelihood and socio-economic condition of the small-scale marine fishery community in South Kerala. In Kerala, nearly ten lakh fisherfolk depend on the marine fishery resources for their livelihood. The overall level of education of the small-scale fishing community is lower than that of the State’s rural population. Almost all the households surveyed, is one way or other, depend on fishery resources for livelihood. Low levels percapita income and high levels of inequality imply the existence of a large proportion of poor people in the community who are vulnerable to external shocks. The study reveals that poverty was comparatively higher among households with no fishing assets, with only one earner, with more than two children, and depending entirely on pensions/remittances. The study has not provided any evidence to show that poverty in the community is the result of depletion of marine resources.

Thermal Degradation of Short Nylon -6 Fiber-Reinforced Styrene Butadiene Rubber Composite

The thermal properties of short Nylon-6 fiber-reinforced Styrene butadiene rubber (SBR) composites were studied by Thermogravimetric Analysis (TGA). The effect of epoxy-based bonding agent on thermal degradation of the gum and the composites was also studied. The thermal stability of the SBR was enhanced in the presence of Nylon-6 fibers and the stability of the composites increased in the presence of bonding agent. The epoxy resin did not significantly change the thermal stability of SBR gum vulcanizate. Results of kinetic studies showed that the degradation of SBR and the short nylon fiber-reinforced composites with and without bonding agents followed first-order kinetics.

Thermal degradation of short polyester fiber polyurethane elastomer composite

The thermal degradation of short polyester fiber reinforced polyurethane composites with and without different bonding agents has been studied by thermogravimetric analysis . It was found that degradation of the polyurethane takes place in two steps and that of the composites takes place in three steps. With the incorporation of 30 phr of fiber in the matrix , the onset of degradation was shifted from 230 to 238 ° C. The presence of bonding agents in the virgin elastomer and the composite gave an improved thermal stability . Results of kinetic studies showed that the degradation of polyurethane and the reinforced composites with and without bonding agents follows first -order reaction kinetics

Thermal degradation of short kevlar fibre thermoplastic polyurethane composite

The thermal degradation of short kevlar fibre-thermoplastic polyurethane (TPU) composites has been studied by Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). TGA showed that the thermal degradation of TPU takes place in two steps with peak maxima (T1max and T2ma,) at 383°C and 448°C, respectively. In the presence of 10-40 phr of short kevlar fibres, T1_ and T2max were shifted to lower temperatures. The temperature of onset of degradation was increased from 245 to 255°C at 40 parts per hundred rubber (phr) fibre loading. Kinetic studies showed that the degradation of TPU and kevlar-TPU composite follows first-order reaction kinetics. The DSC study showed that there is an improvement in thermal stability of TPU in the presence of 20 phr of short kevlar fibres.

Photodegradation of Methylorange over Zirconia Doped TiO2 Using Solar Energy

The heterogeneous photocatalytic degradation of methylorange over TiO2 is studied and is found to be cost effective. Effect of Zirconium metal incorporation over titania system is investigated. Photocatalytic degradation of methylorange using solar radiation is found to be highly economical when compared with the processes using artificial UV radiation, which require substantial electrical power input. The characterization of titania as well as modified zirconium metal doped titania systems are done using XRD, FTIR and EDAX measurements. The catalytic activities of different systems are also compared and is tried to correlate with the crystallite size and presence of dopant metal.

Investigations on Eco-friendly Natural Insecticides

Instead of developing easily degradable, and low-priced insecticides, we are going after highly sophisticated chemicals. Here, an attempt is being made to develop safer formulations of insecticides of botanical origin. Different parts of the plants were chosen based on their use in countryside and villages The dried plant materials were extracted with petroleum ether, and were applied on Tribolium castaneum. The results were statistically analysed. The active principles from Croton tigilium and Leea sambucina, the most potential plants, were isolated using Column Chromatography, TLC, and Hydrolysis. The isolated principles were analysed spectroscopically ( UV-Vis., IR, NMR, and MS ) to identify their chemical nature. The active principles from Leea and Croton were identified as a cholisterate derivative and a phorbol derivative respectively. In order to ascertain the environmental combatibility of the principles, degradation by soil bacteria was studied. The isolated principles were made into three type of formulations using stabilizers .The formulations were applied on Snake gourd semilooper, Pulse beetle, and mosquito larvae. Also the biocidal activity of the formulations was studied. Both Leea derivative and Croton derivative could be formulated effectively and were effective against a variety of pests. They are eco-friendly, as there is no artificial chemicals involved.

NIR to UV absorption spectra and the optical constants of phthalocyanines in glassy medium

Optical absorption studies of phthalocyanines (Pc-s) in borate glass matrix have been reported for the first time. Measurements have been done corresponding to photon energies between 1.1 and 6.2 eV for free base, manganese, iron, nickel, molybdenum, cobalt and copper phthalocyanines. Several new discrete transitions are observed in the UV–vis region of the spectra in addition to a strong continuum component of absorption in the IR region. Values of some of the important optical constants viz. absorption coefficient (α), molar extinction coefficient (ε), absorption cross-section (σa), band width (Δλ), electric dipole strength (q2) and oscillator strength (f) for the relevant electronic transitions are also presented. All the data reported for Pc-s in the new matrix have been compared with those corresponding to solution, vapor and thin film media.

Modification of Linear Low Density Polyethylene for Improved Photo and Biodegradation

LLDPE was blended with poly (vinyl alcohol) and mechanical, thermal, spectroscopic properties and biodegradability were investigated. The biodegradability of LLDPE/PVA blends has been studied in two environments, viz. (1) a culture medium containing Vibrio sp. and (2) a soil environment over a period of 15 weeks. Nanoanatase having photo catalytic activity was synthesized by hydrothermal method using titanium-iso-propoxide. The synthesized TiO2 was characterized by X-Ray diffraction (XRD), BET studies, FTIR studies and scanning electron microscopy (SEM). The crystallite size of titania was calculated to be ≈ 6nm from the XRD results and the surface area was found to be about 310m2/g by BET method. SEM shows that nanoanatase particles prepared by this method are spherical in shape. Linear low density polyethylene films containing polyvinyl alcohol and a pro-oxidant (TiO2 or cobalt stearate with or without vegetable oil) were prepared. The films were then subjected to natural weathering and UV exposure followed by biodegradation in culture medium as well as in soil environment. The degradation was monitored by mechanical property measurements, thermal studies, rate of weight loss, FTIR and SEM studies. Higher weight loss, texture change and greater increments in carbonyl index values were observed in samples containing cobalt stearate and vegetable oil. The present study demonstrates that the combination of LLDPE/PVA blends with (I) nanoanatase/vegetable oil and (ii) cobalt stearate/vegetable oil leads to extensive photodegradation. These samples show substantial degradation when subsequent exposure to Vibrio sp. is made. Thus a combined photodegradation and biodegradation process is a promising step towards obtaining a biodegradable grade of LLDPE.

Studies on Catalysis by Nano Titania Modified with Metals and Nonmetals

Semiconductor photocatalysis has received much attention during last three decades as a promising solution for both energy generation and environmental problems. Heterogeneous photocatalytic oxidation allows the degradation of organic compounds into carbon dioxide and water in the presence of a semiconductor catalyst and UV light source. The •OH radicals formed during the photocatalytic processes are powerful oxidizing agents and can mineralise a number of organic contaminants. Titanium dioxide (TiO2), due to its chemical stability, non-toxicity and low cost represents one of the most efficient photocatalyst. However, only the ultraviolet fraction of the solar radiation is active in the photoexcitation processes using pure TiO2 and although, TiO2 can treat a wide range of organic pollutants the effectiveness of the process for pollution abatement is still low. A more effective and efficient catalyst therefore must be formulated. Doping of TiO2 was considered with the aim of improving photocatalytic properties. In this study TiO2 catalyst was prepared using the sol-gel method. Metal and nonmetal doped TiO2 catalysts were prepared. The photoactivity of the catalyst was evaluated by the photodegradation of different dyes and pesticides in aqueous solution. High photocatalytic degradation of all the pollutants was observed with doped TiO2. Structural and optical properties of the catalysts were characterized using XRD, BET surface area, UV-Vis. DRS, CHNS analysis, SEM, EDX, TEM, XPS, FTIR and TG. All the catalysts showed the anatase phase. The presence of dopants shifts the absorption of TiO2 into the visible region indicating the possibility of using visible light for photocatalytic processes.

Dayglow Emissions on Mars and Venus

An attempt has been made in this thesis to model some of the emissions observed by SPICAM and SPICAV on Mars and Venus, respectively, viz., CO Cameron band, CO+ 2 ultraviolet doublet, N2 triplet bands, atomic oxygen green (5577 A), red doublet (6300, 6364 A), and ultraviolet (2972 A) emissions. One of major sources of these emissions is photoelectron impact ionization/excitation. In this thesis, an electron degradation model based on Monte Carlo technique has been developed to calculate the production/excitation rates of above mentioned emissions due to electron impact. The limb brightness pro les of emissions are calculated and compared with the observations wherever available. The e ect of various model input parameters on dayglow emissions intensities is also evaluated

Diversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags

The diversity and load of heterotrophic bacteria and fungi associated with the mangrove soil from Suva, Fiji Islands, was determined by using the plate count method. The ability of the bacterial isolates to produce various hydrolytic enzymes such as amylase, gelatinase and lipase were determined using the plate assay. The heterotrophic bacterial load was considerably higher than the fungal load. There was a predominance of the gram positive genus, Bacillus. Other genera encountered included Staphylococcus, Micrococcus, Listeria and Vibrio. Their effectiveness on the degradation of commercial polythene carry bags made of high density polyethylene (HDPE) and low density polyethylene (LDPE) was studied over a period of eight weeks in the laboratory. Biodegradation was measured in terms of mean weight loss, which was nearly 5 % after a period of eight weeks. There was a significant increase in the bacterial load of the soil attached to class 2 (HDPE) polythene. After eight weeks of submergence in mangrove soil, soil attached to class 1 and class 3 polythene mostly had Bacillus (Staphylococcus predominated in class 2 polythene). While most of the isolates were capable of producing hydrolytic enzymes such as amylase and gelatinase, lipolytic activity was low. Class 2 HDPE suffered the greatest biodegradation.

Formation of an unusual copper(II) complex from the degradation of a novel tricopper(II) carbohydrazone complex

An unusual copper(II) complex [Cu(L1a)2Cl2] CH3OH H2O H3O+Cl (1a) was isolated from a solution of a novel tricopper(II) complex [Cu3(HL1)Cl2]Cl3 2H2O (1) in methanol, where L1a is 3-(2-pyridyl)triazolo [1,5-a]-pyridine, and characterized with single crystal X-ray diffraction study. The tricopper(II) complex of potential ligand 1,5-bis(di-2-pyridyl ketone) carbohydrazone (H2L1) was synthesized and physicochemically characterized, while the formation of the complex 1a was followed by time-dependant monitoring of the UV–visible spectra, which reveals degradation of ligand backbone as intensity loss of bands corresponding to O?Cu(II) charge transfer

Enhanced UV emission from ZnO nanoflowers synthesized by the hydrothermal process

ZnO nanoflowers were synthesized by the hydrothermal process at an optimized growth temperature of 200 ◦C and a growth/reaction time of 3 h. As-prepared ZnO nanoflowers were characterized by x-ray diffraction, scanning electron microscopy, UV–visible and Raman spectroscopy. X-ray diffraction and Raman studies reveal that the as-synthesized flower-like ZnO nanostructures are highly crystalline with a hexagonal wurtzite phase preferentially oriented along the (1 0 1 1) plane. The average length (234–347 nm) and diameter (77–106 nm) of the nanorods constituting the flower-like structure are estimated using scanning electron microscopy studies. The band gap of ZnO nanoflowers is estimated as 3.23 eV, the lowering of band gap is attributed to the flower-like surface morphology and microstructure of ZnO. Room temperature photoluminescence spectrum shows a strong UV emission peak at 392 nm, with a suppressed visible emission related to the defect states, indicating the defect free formation of ZnO nanoflowers that can be potentially used for UV light-emitting devices. The suppressed Raman bands at 541 and 583 cm−1 related to defect states in ZnO confirms that the ZnO nanoflowers here obtained have a reduced presence of defects