L'objectif de cette thèse était de développer de nouveaux « donneurs de NO » stables en liant du S-nitrosoglutathion (GSNO) à une structure polymérique. Dans une première étape, les polymères ont été liés au glutathion (GSH) : le chitosan-GSH et l'alginate-GSH ont ainsi été préparés par la « méthode des carbodiimides » et dans une deuxième étape, les polymères finaux [SNOC (S-nitrosoglutathione-oligosaccharide-chitosan) et SNA (S-nitrosoglutathione-alginate)] ont été préparés par nitrosation des deux conjugués précédent. La quantité de NO fixée a été déterminée par les méthodes Griess et Saville. L’aptitude des polymères à libérer du NO et à passer la barrière intestinale [SNOC et SNA] a été évaluée dans une chambre d’Ussing. Nous avons obtenu des polymères avec des quantités variables de NO en fonction de la méthode utilisée (159 µmol de NO/g à 525 µmol de NO/g pour le SNOC ; 174 µmol de NO/g à 468 µmol de NO/g pour le SNA). Le SNOC était stable pendant au moins 6h et le SNA pendant au moins 10h. Enfin, nous avons essayé de mettre au point des microparticules de GSH et GSNO par spray drying avec de l’Eudragit ® FS 30D gastro-résistant. La caractérisation des microparticules a été réalisée par microscopie électronique à balayage (SEM), par diffraction X (PXRD) et par spectroscopie infrarouge (FTIR). Les essais de libération in vitro ont été réalisés dans un tampon (pH 1,2, 3, 6, 6,8 et 7,4). Les microparticules étaient chargées négativement avec une taille moyenne allant de 5 à 7 µm. La formulation était stable à pH acide mais a montré une libération rapide à pH basique ; elle pourrait donc servir de système de délivrance du NO au niveau intestinal. / The aim of the thesis was to develop novel and stable NO-donors by linking S-nitrosoglutathione (GSNO) to a polymer backbone. In the first step, chitosan-GSH and alginate-GSH conjugates were prepared by a carbodiimide reaction and in the second step SNOC (S-nitrosoglutathione-oligosaccharide-chitosan) and SNA (S-nitrosoglutathione-alginate) were prepared by the nitrosation of both conjugates respectively. The amount of NO was determined by Griess and Saville methods. Stability and ex vivo experiments of SNOC and SNA were performed in an Ussing chamber through rat intestine. We obtained polymers with different amount of NO (i.e. 159 µmol of NO/g to 525 µmol of NO/g for SNOC; 174 µmol of NO/g to 468 µmol of NO/g for SNA) depending upon the procedure of nitrosation. SNOC was stable for at least 6h and SNA for at least 10h. Also, we aimed to develop spray dried microparticles of GSH and GSNO based on Eudragit® FS 30D polymer. The microparticles were characterized by scanning electron microscopy (SEM), X-ray diffraction (PXRD), infrared spectroscopy (FTIR) and in vitro release studies were performed in different pH conditions (pH 1.2, 3, 6, 6.8 and 7.4). The microparticles were negatively charged with mean particle size ranging from 5 to 7 µm. The formulation was stable and was resistant to acidic pH but showed rapid release in basic pH; hence, they can be used as colon specific drug delivery systems for the treatment of Crohn’s disease. We think that these formulations could be used in animal models in the treatment of Crohn’s disease.

Made available in DSpace on 2016-08-10T10:38:29Z (GMT). No. of bitstreams: 1 Ariane Bocaletto Frare.pdf: 1200781 bytes, checksum: 8bd7ce42ea14c46d6e84c7f5311b07ca (MD5) Previous issue date: 2011-06-16 / Endometriosis is a benign gynecological pathology dependent on estrogen, which affects 10% of women of reproductive age, it is characterized by the presence of endometrial tissue outside the uterine cavity. Its etiology remains unknown, thus an increasing number of researches have been conducted in the search for associations between endometriosis and alterations or polymorphisms in candidate genes, among these genes are the GSTM1 and GSTT1. This study aimed to verify the possible correlation between endometriosis and polymorphisms of presence / absence of GSTM1 and GSTT1, and the association between GSTM1 and GSTT1 polymorphisms and p53 Arg / Pro. We analyzed the frequency of GSTM1 and GSTT1 polymorphisms in 50 women diagnosed with endometriosis and in control group with 46 women without complaints related to this pathology, the genotypes for these polymorphisms were determined using PCR. We found an increased frequency of absence of GSTM1 (61%) and presence of GSTT1 genes (55%) in the group of women studied, which corroborate with the literature. When we relate the endometriosis with the studied polymorphisms we verified that the endometriosis group had a higher presence of both polymorphisms 50% of GTSM1 and 68% of GSTT1 whereas in the control group there was the opposite, a higher absence of polymorphisms, 74% of GSTM1 and 59% of GSTT1, showing that these polymorphisms has no relation to the proliferation of endometriosis, however the analysis of the pathology with the association of GSTM1, GSTT1 polymorphisms with the p53 codon 72 gene was statistically significant. / A endometriose é uma patologia ginecológica benigna dependente de estrógeno, que afeta 10% das mulheres em idade reprodutiva, é caracterizada pela presença de tecido endometrial fora da cavidade uterina. Sua etiologia permanece desconhecida, com isso um número cada vez maior de pesquisas tem sido realizado na procura de associações entre endometriose e alterações ou polimorfismo em genes candidatos, entre esses genes estão o GSTM1 e o GSTT1. Este estudo teve como objetivo verificar a possível correlação entre a endometriose e os polimorfismos de presença/ausência GSTM1 e GSTT1. Nós analisamos a freqüência dos polimorfismos GSTM1 e GSTT1 em 50 mulheres diagnosticadas com endometriose e no grupo controle, com 46 mulheres sem queixas relacionadas com a patologia, os genótipos para esses polimorfismos foram determinados por PCR. Encontramos maior freqüência de ausência dos genes GSTM1 (61%) e de presença do GSTT1 (55%) no grupo de mulheres estudadas, o que corroboram com a literatura. Quando relacionamos endometriose com os polimorfismos estudados verificamos que o grupo endometriose apresentou maior presença de ambos os polimorfismo 50% GTSM1 e 68% GSTT1, enquanto que no grupo controle verificou-se o contrário, maior ausência dos polimorfismos, 74% do GSTM1 e 59% do GSTT1, mostrando que esses polimorfismos não tem relação com a proliferação da endometriose.

Uma das formas de se quantificar os possíveis efeitos de metais sobre o estado de saúde de organismo é analisar os biomarcadores do estresse oxidativo. Bioensaios foram realizados com a tilápia do Nilo (Oreochromis niloticus) com concentrações de cádmio (4,25 \'mü\'g/L), cobre (45,0 \'mü\'g/L ) e zinco (260,0 \'mü\'g/L). Os peixes foram expostos aos metais separados (\'CD\', \'CU\' e \'ZN\'), associados dois a dois (\'CD\'/\'CU\', \'CD\'/\'ZN\' e \'CU\'/\'ZN\') e aos três metais juntos (\'CD\'/\'CU\'/\'ZN\'), por doze dias. Foram retiradas amostras de rim, fígado e brânquias para análise de biomarcadores de estresse oxidativo, a superóxido dismutase (SOD), catalase (CAT) e glutationa peroxidase (GPx), o efeito da peroxidação de lipídios (HP), a glutationa S-transferase (GST) e a metalotionéina (MT). Pelos reusltados obtidos constatou-se que, mesmo nas baixas concentrações de exposição aos metais, ocorreram alterações no sitema de defesa antioxidante de O. niloticus. A primeira barreira antioxidante, composta pela SOD, não foi suficiente para barrar os efeitos da exposição aos metais. Em brânquias, onde a ativação desta enzima foi proeminente, formação de HP ocorreu. E, mesmo inicialmente em rim e fígado, tecidos onde ocorreu diminuição da atividade da SOD, ocorreu também a formaçao de HP nas etapas seguintes. A CAT e a GPx também atuaram no sentido de tentar evitar o aparecimento de HP, mas não foram bem sucedidas neste processo. A GST, como enzima da fase II de biotransformação pode ter auxiliado no processo de eliminação dos metais, mas como ocorre de forma tardia em relação à defesa antioxidante, acarretando danos celulares. Em relação às enzimas que compõem o sistema de defesa antioxidante não se verificou também especificidade de tecidos nas respostas. No entanto, a brânquia foi o órgão que teve maior intensidade nas respostas (em termos de ativação das enzimas), nos tratamentos de metais combinados dois a dois, evidenciando que este órgão, por estar em contato com a água e ser sede de processos fisiológicos de homeostase, desempenhou papel importante na tentativa de neutralizar os efeitos dos metais. / One way of quantifying metals effects upon organisms\' health state is to analyse oxidative stress biomarkers. Bioassays were conducted with Nile tilapia (Oreochromis niloticus) with cadmium (4,25 \'mü\'g/L), copper (45,0 \'mü\'g/L ) and zinc (260,0 \'mü\'g/L). Fishes were exposed to metals separatelly (\'CD\', \'CU\' e \'ZN\'), associated two by two (\'CD\'/\'CU\', \'CD\'/\'ZN\' e \'CU\'/\'ZN\') and to three metals together (\'CD\'/\'CU\'/\'ZN\'), for twelve days. Gills, liver and kidney samples were taken to analyse oxidative stress biomarkers, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), the effect of lipid peroxidation (HP), an enzyme of phase II biotransformation, glutathione S-transferase (GST) and metallothionein (MT). From results obtained, it was stablished that, even in low concentrations of metals, O. niloticus antioxidant defense system was modified. The first antioxidant barrier, SOD, wasn\'t able to minimize metal exposition effects. In gills, where activaton of this enzyme was prominent, didn\'t avoi HP formation. And even in liver and kidney, tissues where SOD activity decreased, HP formation also occurred. CAT and GPx also acted trying to avoid HP formation, but weren\'t successfull. GST, like a phase I enzyme, could help eliminating metals, but in a late moment in relation to antioxidant defense, leading to cell damage. For antioxidant enzymes no relation to tissue-specificity response was observed. But gills were the organ which had more intense responses (in terms of enzyme activation), mainly in metals combined two by two, evidencing that gills are important like a first organ in antioxidant defense because it is in direct contact with water and determine the main processes in homeostasis, trying to neutralize metals effects.

A pele está em contínua auto-renovação graças a vários nichos de células-tronco presentes neste tecido. Células progenitoras epidérmicas surgem durante o desenvolvimento embrionário e contribuem para a reposição celular da epiderme durante todo o período de vida dos mamíferos. Neste trabalho, buscou-se analisar o papel da depleção de glutationa durante a estratificação da epiderme embrionária e na manutenção da homeostase no tecido adulto. Encontramos evidências de que este tiol tem um importante papel durante a proliferação da epiderme e formação do folículo capilar. As alterações observadas na ausência de GSH foram relacionados com um padrão diferencial de fosforilação dos fatores de transcrição forkhead-homeobox- tipo-O (FOXO). Em resumo, foi estabelecida uma correlação entre o estado de GSH, a fosforilação de FOXO e o desenvolvimento da epiderme. Para melhor estudar a importância do balanço de GSH, na pele do adulto, e seu papel na manutenção deste tecido, camundongos foram tratados com um inibidor da síntese de GSH e, com N-aceti-lcisteína. Foi observado um aumento da fosforilação de Akt, padrões alterados de fosforilação FOXO e aumento da expressão de genes de genes relacionados à diferenciação. Estes resultados mostram que a via Akt/FOXO desempenha um papel importante na manutenção e diferenciação de células-tronco epidermais. O envelhecimento cronológico leva a alterações morfológicas/funcionais que conduzem à diminuição da auto-renovação, o que ocorre concomitantemente com uma diminuição dos níveis de GSH na pele. Utilizamos, também, animais idosos e avaliamos quais mecanismos eram compartilhados pelo envelhecimento e a depleção deste tiol. Observou-se que uma resposta hiperproliferativa ligada à exaustão de células-tronco pode ser o elo entre a depleção de GSH e o envelhecimento. A influência desse processo também foi investigada no compartimento dérmico, através da análise do impacto da depleção de glutationa sobre a osteogênese de células-tronco mesenquimais murinas. Quando induzidas a se diferenciarem em osso (Alizarin-Red+/Von-Kossa-stain +, aumento dos níveis de mRNA para fosfatase alcalina/osteopontina/osterix), o balanço GSH/GSSG e seu sistema antioxidante correlato é diferencialmente regulado em células-tronco mesenquimais derivadas da derme. Sendo regulado de uma forma redox-dependente através da via de MAPKs. A depleção de GSH leva à diminuição nos níveis de osteogênese em favor da adipogênese, levando ao processo comumente associado ao envelhecimento denominado \"adipogenic switc\". Em conclusão, os dados obtidos permitem propor um papel central para a glutationa na manutenção/comprometimento de células-tronco na pele. / The skin is continuously self-renewing thanks to several stem cell niches. Epidermal progenitor cells arise during embryonic development and contribute to the replenishment of the epidermis during the lifetime of mammals. We set out to analyze the glutathione (GSH) antioxidant system during embryonic epidermis stratification and follicle development and the effect of glutathione withdrawal in this process. We found that glutathione plays an important role during epidermis proliferation and hairshaft formation. The changes observed in the absence of GSH were related to a differential phosphorylation pattern of the forkhead-homeobox-type-O (FOXO) transcription factors. In brief, a correlation between GSH status, FOXO phosphorylation and skin development was established. To further study the importance of GSH in adult skin maintenance and understand the effects of ROS in the Akt/FOXO pathway, we treated cells and mice with an inhibitor of GSH synthesis, and with N-acetyl-cysteine. Increased Akt phosphorylation, altered FOXO phosphorylation patterns and increased gene expression of differentiation-related genes were observed. Our results show that the Akt/FOXO pathway plays an important role in maintenance/differentiation of epidermal stem cells. Chronological ageing leads to morphological/functional changes causing a decline in self-renewal, as well as decreased levels of GSH. We also observed that a cell cycle hyperproliferative response was the link between stem cell exaustion in GSH-depletion and ageing. Dermal mesenchymal stem cells (MSCs), are capable of adipo-chondro- and osteogenesis. Little is known about the impact of ROS in MSC differentiation. We induced murine skin MSCs to differentiate into bone (Alizarin-Red/Von-Kossastain+, increased levels of mRNA for alkalinephosphatase/ osteopontin/osterix). In brief, the balance of GSH/GSSG and related antioxidant system is differentially regulated during this process, found to be regulated in a redox-dependent fashion through the MAPK pathway. When depleted, GSH leads to an adipogenic switch in MSC differentiation. In conclusion, our data leads us to propose a central role for glutathione in the maintenance/commitment of stem cells in skin.

by Lawrence Chi-ming Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 142-151). / Abstract --- p.i / Acknowledgments --- p.iv / Table of Contents --- p.v / List of Figures --- p.vii / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Polysaccharide-peptide (PSP ) --- p.1 / Chapter 1.2 --- Paracetamol (APAP ) --- p.6 / Chapter 1.2.1 --- Metabolism of APAP --- p.9 / Chapter 1.2.2 --- Mechanisms of APAP toxicity --- p.11 / Chapter 1.2.3 --- Factors influencing the hepatotoxicity of APAP --- p.17 / Chapter 1.3 --- Aim of the present study --- p.23 / Chapter Chapter 2: --- Studies on the effects of PSP on APAP-hepatotoxicity and glutathione levels / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Materials and methods --- p.30 / Chapter 2.3 --- Results / Chapter 2.3.1 --- The effects of PSP on APAP-induced hepatotoxicity --- p.41 / Chapter 2.3.2 --- The acute and sub-chronic effects of PSP on glutathione in rats --- p.45 / Chapter 2.4 --- Discussions --- p.66 / Chapter Chapter 3: --- Studies on the effects of PSP on the covalent binding and metabolism of APAP / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Materials and methods --- p.85 / Chapter 3.3 --- Results / Chapter 3.3.1 --- The effects of PSP on the covalent binding of radiolabelled paracetamol (14C-APAP ) in vitro --- p.102 / Chapter 3.3.2 --- The effects of PSP on the metabolism of APAP --- p.107 / Chapter 3.4 --- Discussions --- p.119 / Chapter Chapter 4: --- Conclusion --- p.137 / References --- p.142

La biologie des réactions redox est particulièrement difficile à étudier de par la compartimentation spatiale mais aussi cinétique des différents systèmes redox cellulaires. Les biosenseurs codés génétiquement, incluant la «redox-sensitive Yellow Fluorescent Protein» (rxYFP) sont une manière de contourner les limitations des méthodes conventionnelles de mesure du couple glutathion/glutathion disulfure (GSH/GSSG). Cette étude présente l’utilisation des biosenseurs rxYFP pour analyser les états redox dans des différents compartiments cellulaires, et leur dynamique en réponse au stress dans les cellules humaines. La rxYFP exprimée soit dans le cytosol, le noyau ou la matrice mitochondriale de cellules HeLa s’est révélée sensible aux changements de l’état redox intracellulaire provoqué par des traitements aussi bien réducteurs qu’oxydants. La rxYFP est capable de détecter des différences de l’état redox, entre les compartiments, mais aussi entre différentes lignées cellulaires. Les senseurs exprimés dans des kératinocytes humain de l’épiderme HEK001 ont réagi au stress induit par les UVA, de façon dose-dépendante, mais pas au stress induit par les UVB. De plus, ces senseurs ont pu détecter les changements redox induits par des faibles doses (30 µM) ainsi que par des doses modérées (100 µM) de peroxyde d’hydrogène (H2O2), de façon dynamique et spécifique des compartiments cellulaires. La rxYFP exprimé dans la matrice mitochondriale a montré une vitesse d’oxydation plus élevée que les senseurs rxYFP exprimés dans le cytosol ou le noyau, ce qui est attribuable à un pH local plus basique. De plus, la déplétion en GSH provoquée par un traitement au buthionine sulphoximine (BSO) a affecté spécifiquement le potentiel redox mitochondrial mais pas cytosolique ni nucléaire. Ces observations soutiennent l’idée que l’état redox du GSH mitochondrial est maintenu et régulé de façon indépendante par rapport à celui du cytosol ou du noyau. Nous avons également montré que dans les cellules humaines, les sondes rxYFP réagissent de façon prédominante avec le GSH/GSSG, puisque la déplétion en GSH ralentit la vitesse d’oxydation de la rxYFP en réponse à un traitement par H2O2. De plus, grâce à l’utilisation des sondes rxYFP et à l’analyse de l’état redox des antioxydants cellulaires, nous démontrons que l’oxydation des thiols se produit après l’activation des caspases au cours de l’apoptose induite par TRAIL. L’ensemble de nos données montrent la robustesse des senseurs rxYFP pour la mesure des changements d’état redox dans les cellules humaines. En complément d’autres senseurs redox ainsi que des méthodes conventionnelles de mesure des états redox, les senseurs rxYFP ciblés aux différents compartiments cellulaires sont un nouvel outil pour étudier l’homéostasie redox dans les cellules de mammifères, et permettent l’étude de l’état redox du glutathion et de la dynamique des changements redox avec une grande précision. / The kinetic and spatial separation of redox systems renders redox biology studies a particularly challenging field. Genetically encoded biosensors including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP) may provide an alternative way to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. This study describes the use of rxYFP sensors for investigating compartment-specific steady redox states and their dynamics in response to stress in human cells. RxYFP expressed either in the cytosol, nucleus or mitochondrial matrix of HeLa cells was responsive to the intracellular redox state changes induced by reducing as well as oxidizing agents. Compartment-targeted rxYFP sensors were able to detect different steady state redox conditions between the cytosol, nucleus and mitochondrial matrix as well as between the cell lines. These sensors expressed in human epidermal keratinocytes HEK001 responded to stress induced by UVA radiation in a dose-dependent manner but not to UVB radiation. Furthermore, rxYFP sensors were able to sense dynamic and compartment-specific redox changes caused by low dose (30 µM) and moderate dose (100 M) hydrogen peroxide (H2O2). Mitochondrial matrix-targeted rxYFP displayed a greater dynamics of oxidation in response to a H2O2 challenge than the cytosol- and nucleus-targeted sensors, largely due to a more alkaline local pH environment. Similarly, the depletion of glutathione induced by buthionine sulphoximine (BSO) affected selectively mitochondrial redox potential without inducing changes in cytosol and nucleus. Furthermore, using rxYFP probes and cellular antioxidants redox state analysis, we show that oxidation of thiols occurs after activation of caspases during TRAIL-induced apoptosis. These observations support the view that mitochondrial glutathione redox state is maintained and regulated independently from that of the cytosol and nucleus. We also showed that in human cells the rxYFP probes react predominantly with glutathione since the glutathione depletion slows down the dynamics of rxYFP oxidation in response to H2O2. Taken together, our data show the robustness of the rxYFP sensors to measure compartmental redox changes in human cells. Complementary to existing redox sensors and conventional redox measurements, compartment-targeted rxYFP sensors provide a novel tool for examining mammalian cell redox homeostasis, permitting high resolution readout of steady glutathione state and dynamics of redox changes.

Le climat alpin à plus de 2400 mètres d’altitude montre des fortes variations de température, des intensités lumineuses très élevées (3000 µmol photons m-2 s-1) qui sont connues pour générer un état de réduction importante de la chaine de transport des électrons photosynthétique. Le bon fonctionnement du processus photosynthétique est primordial pour les quelques espèces de plantes vasculaires qui sont présentes à l’étage alpin et qui doivent terminer leur cycle de vie lors d’une très courte période de végétation.Soldanella alpina et Ranunculus glacialis sont deux espèces inféodées aux étages alpin et nival. Dans leur site naturel de croissance nous avons mesuré des températures faibles (0.7°C) et fortes (37°C) sous des lumières supérieures à 2500 µmol photons m-2 s-1. Chez les espèces non-alpines ces conditions induisent la photoinhibition du PSII, ce qui est évité chez S. alpina et R. glacialis, par des mécanismes très différents. Les systèmes antioxydants et le quenching non photochimique sont particulièrement importants chez S. alpina. Chez Ranunculus glacialis, la photorespiration reste très importante et un contenu élevé en PTOX est décrit. Le rôle des antioxydants et de la PTOX dans la photoprotection des deux espèces ont été étudiés. Dans une partie de thèse, nous avons montré qu’une diminution de la capacité antioxydante par une diminution de la concentration en glutathion n’affecte pas la tolérance vis-à-vis de la photoinhibition à basse température. Dans une deuxième partie les résultats supposent qu’une surexpression de la PTOX chez le tabac augmente la photoinhibition à lumière forte par production des espèces réactives d’oxygène. En utilisant différentes conditions environnementales de croissance pour Ranunculus glacialis, nous avons pu montrer que l’expression de la PTOX est induite par des fortes lumières et non par des basses températures. Grâce à une approche associant mesures d’échanges gazeux et mesures de la fluorescence de la chlorophylle, nous avons montré qu’un flux d’électrons conséquent vers l’oxygène, indépendant de la photorespiration, corrélait avec la présence de la PTOX mais que l’activité de la PTOX sous des conditions qui permettent l’assimilation du CO2 et la photorespiration n’est pas maximale. Grâce à des mesures de fluorescence chlorophyllienne en présence de différents inhibiteurs photosynthétiques, nous avons pu montrer que l’importance de ce flux d’électrons vers l’oxygène corrèle avec la quantité de PTOX présente dans les feuilles, dans des conditions réductrices. Ces résultats nous ont amenés à conclure que chez Ranunculus glacialis, la PTOX peut prendre en charge un flux significatif d’électrons, éviter ainsi l’apparition d’un état réduit de la chaine de transfert photosynthétique, et protéger la plante vis-à-vis de la photoinhibition en agissant comme une valve de sécurité. Ces travaux permettent d’apporter des précisions sur un modèle original de photoprotection, qui a été l’objet de nombreuses controverses. / The alpine climate above 2400 meters altitude shows large variations in temperature and very important light intensity (3000 µmol photons m-2 s-1), which are known to generate a state of significant reduction in the photosynthetic electron transport chain. The proper functioning of the photosynthetic process is essential for vascular plants species that are present in this alpine environment and must complete their life cycle within a very short growing season.Soldanella alpina and Ranunculus glacialis are two species restricted to alpine and snow floors. In their natural growth environment we measured very low (0.7 ° C) and high temperature (37 ° C) under lights above 2500 µmol photons m-2 s-1. Among non-alpine species such conditions induce photoinhibition of PSII, which is avoided in S. alpina and R. glacialis, by very different mechanisms. Antioxidant systems and non-photochemical quenching are particularly important in S. alpina. In Ranunculus glacialis, photorespiration remains very important and a high content of PTOX is described. The roles of antioxidants and PTOX in photoprotection of both species were studied.In one part of the thesis, we showed that a decrease in antioxidant capacity by reducing the concentration of glutathione does not affect tolerance to low-temperature photoinhibition. In the second part the results imply that overexpression of PTOX in tobacco enhances photoinhibition by strong light to produce reactive oxygen species.Using different environmental conditions for Ranunculus glacialis growth, we showed that expression of the PTOX is induced by strong light, but not by low temperatures. With an approach combining gas exchange measurements and chlorophyll fluorescence measurements, we showed that an electron flow to oxygen, independent of photorespiration, correlated with the presence of PTOX. Through measures of chlorophyll fluorescence in the presence of various inhibitors photosynthetic, we could show that the importance of this electron flow to oxygen correlates with the amount of PTOX in the leaves, under reducing conditions. These results led us to conclude that in Ranunculus glacialis, the PTOX may support a significant flow of electrons, thus avoiding the appearance of a reduced state of the photosynthetic chain transfer, and protect the plant from photoinhibition, acting as a safety valve. These studies are discussed to help clarify a new pathway of photoprotection, which was the subject of much controversy.

Os carrapatos Rhipicephalus (Boophilus) microplus e Haemaphysalis longicornis são ectoparasitas hematófagos que infestam bovinos. O uso de vacinas anticarrapato tem mostrado ser uma estratégia alternativa promissora para o controle desses parasitos. As glutationa-S-transferases (GSTs) pertencem a uma família de enzimas multifuncionais presentes em organismos animais e vegetais, e, entre suas funções, pode-se destacar o transporte intracelular, a participação em processos digestivos, a síntese de prostaglandinas, a detoxificação de substâncias tóxicas e a proteção contra o estresse oxidativo. Em trabalhos anteriores, verificou-se em camundongos que a GST de H. longicornis (rGST-Hl) é mais imunogênica que a GST de R. microplus (rGST-Bm). O objetivo do presente estudo foi investigar a proteção cruzada conferida pela imunização de bovinos com a rGST-Hl frente a uma infestação de R. microplus. Por Western blot, verificou-se que os bovinos vacinados com a rGST-Hl apresentaram um aumento nos níveis de anticorpos anti-rGST-Hl, os quais foram capazes de reconhecer a GST-Bm nativa de extratos de larva, ovário e glândula salivar. Posteriormente, os bovinos foram infestados com 20.000 larvas de R. microplus. O percentual de proteção conferido foi calculado em função da variação entre os grupos tratados e controle, em relação ao número e peso de carrapatos que terminaram a alimentação, a capacidade de postura e a fertilidade dos ovos. O grupo dos bovinos vacinados com rGST-Hl mostrou uma redução de 53% e 52% no número e no peso de teleóginas, respectivamente. A postura de ovos e a eclodibilidade apresentaram uma redução no grupo vacinado de 0,6% e 8%, respectivamente. A eficácia total contra a infestação de R. microplus foi de 57%, quando comparado ao grupo controle. / The ticks Rhipicephalus (Boophilus) microplus and Haemaphysalis longicornis are hematophagous ectoparasites that infest bovines. Vaccine has been considered one of the most promising methods for the control of these parasites. Glutathione Stransferases (GSTs) enzymes are present in animal and vegetal organisms and are important in intracellular transport, digestive process, synthesis of prostaglandins, detoxification and protection against oxidative stress. In a previous study with mice we showed that H. longicornis GST (rGST-Hl) is more immunogenic than R. microplus GST (rGST-Bm). The objective of the present study was to investigate the crossprotection against R. microplus infestation in bovine vaccinated with rGST-Hl. By Western blot, the immunized bovines showed an increase in antibodies anti-rGST-Hl. The native GST-Bm in larvae, ovary and salivary gland were recognized by sera of bovines immunized with rGST-Hl, indicating that the antibodies induced by rGST-Hl immunization recognize native R. microplus GSTs. The bovines were challenged with 20,000 larvae of R. microplus. Protection was calculated by variation in number and weight of fully engorged ticks, egg laying capacity and egg fertility. The bovines vaccinated with rGST-Hl showed a reduction of 53% and 52% in number and weight of engorged ticks, respectively. Egg laying capacity and egg fertility of ticks from vaccinated bovines were respectively 0.6% and 8% lower, as compared to ticks from control animals. The overall efficacy ratio against R. microplus was 57.03%, as compared with the control group.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2017-03-31T13:38:17Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação franciene villanova.pdf: 1735262 bytes, checksum: f9a950bdf67d2961e687eac6378b79bd (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-13T19:48:56Z (GMT) No. of bitstreams: 2 Dissertação franciene villanova.pdf: 1735262 bytes, checksum: f9a950bdf67d2961e687eac6378b79bd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-13T19:48:56Z (GMT). No. of bitstreams: 2 Dissertação franciene villanova.pdf: 1735262 bytes, checksum: f9a950bdf67d2961e687eac6378b79bd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O arroz é o principal alimento para mais da metade da população mundial, principalmente nos países em desenvolvimento. O processo de parboilização consiste em três etapas adicionais ao processo convencional de industrialização do arroz, realizadas antes do descascamento: (1) encharcamento, (2) autoclavagem e (3) secagem. Embora a parboilização promova benefícios nutricionais e tecnológicos ao arroz, comparado ao arroz branco polido, o produto parboilizado é mais escuro e amarelado, o que afeta negativamente a aceitabilidade no mercado. Neste contexto, objetivou-se com o estudo avaliar a capacidade do ácido gálico, da glicina, da glutationa reduzida e da L-cisteína na inibição de reações de escurecimento em arroz parboilizado. A cor dos grãos, a composição proximal, as propriedades de pasta da farinha, a massa molecular e a complexação de proteínas, o percentual de proteína solúvel, o percentual de grãos quebrados, manchados e danificados, o tempo de cocção, o perfil texturométrico, o teor de lisina livre, o teor de mono e dissacarídeos, e o teor de 5-hidroximetil-2-furaldeído livre (HMF) foram determinados. A utilização de glicina e as concentrações de 1,0 e 2,0% de glutationa reduzida foram capazes de conferir uma coloração mais branca aos grãos parboilizados, no entanto, esses tratamentos aumentaram o percentual de grãos quebrados, a solubilidade proteica e o conteúdo de lisina livre. Além disso, estes tratamentos provocaram redução no percentual de grãos manchados e nos níveis de HMF. O tempo de cocção do arroz parboilizado tratado com glicina, independente da concentração, foi semelhante ao tempo de cocção do arroz parboilizado sem adição de inibidores. No entanto, a glutationa reduzida foi capaz de diminuir o tempo de cocção em comparação ao arroz parboilizado convencionalmente, e aumentar o percentual de grãos danificados, exceto na concentração de 2,0%. Os teores de glicose no arroz parboilizado com glicina e glutationa reduzida aumentaram, entretanto, não foram observadas alterações nos níveis de frutose e de sacarose. Desta forma, o uso de glicina é recomendável para retardar o escurecimento dos grãos e melhorar propriedades tecnológicas, no entanto, deve ser considerada em estudos futuros a avaliação sensorial destes grãos. / Rice is a staple food for over the half of the world's population, mainly in developing countries. The parboiling process consists of three additional staps to the conventional rice industrialization process, which are done prior dehusking: (1) soaking, (2) pressure steaming, and (3) drying. Although parboiling improves the nutritional and technological properties of rice, compared to polished white rice, the parboiled product is dark, yellowish, which negatively affects their consumers’ acceptability. The objective of the present study was to evaluate the ability of gallic acid, glycine, reduced glutathione and L-cysteine to inhibit browning reactions during the parboiling process of the rice. The color, the proximate composition, the flour pasting properties, the molecular mass and complexation of proteins, the percentage of soluble protein, the percentage of broken, stained and damaged grains, the cooking time, the texturometric profile, the free lysine content, the mono- and disaccharides contents, and the free 5-hydroxymethyl-2-furaldehyde (HMF) content were determined. The use of glycine and higher concentrations of reduced glutathione at 1.0 and 2.0% levels were able to provide a whiter color to parboiled grains. However, these treatments increased the percentage of broken grains, thr protein extractibility, and the free lysine content. Furthermore, 1.0 and 2.0% levels of glycine and reduced glutathione treatments caused a reduction in the percentage of stained grains and HMF levels. The cooking time of parboiled rice treated with glycine, independent of concentration, was similar to the cooking time of parboiled rice without anti-browning. However, reduced glutathione decreased the cooking time of rice compared to rice parboiled without anti-browning of ati-browning agent, and increased the percentage of damaged grains, except at the 2.0% concentration. Glucose content in parboiled rice with glycine and reduced glutathione increased. However, no changes were observed in fructose and sucrose levels. Thus, the use of glycine is recommended to retard the browning of the grains and improve technological properties. However, the sensorial evaluation of these grains should be considered in future studies.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / O oxigênio é fundamental para os vertebrados. No entanto, variações dos níveis de oxigênio na água podem provocar estresse oxidante em peixes porque privação de oxigênio seguida de reoxigenação forma espécies reativas de oxigênio (ERO) em células. Níveis intracelulares de ERO aumentados favorecem que moléculas de proteínas, fosfolipídios e ácidos nucleicos sofram alterações, vindo a prejudicar muitas funções celulares. No Pantanal, habitat do pacu, o nível de oxigênio varia circadianamente na água das lagoas rasas que acabam isoladas dos rios na seca. O pacu evoluiu sob a pressão contínua da exposição aos efeitos prejudiciais das ERO causados pelos pulsos de inundação. A melatonina, uma indolamina produzida na glândula pineal, influencia os níveis de atividade de enzimas antioxidantes que reduzem ERO, além de ser capaz de doar elétrons ou captar radicais livres de forma não enzimática. Os níveis de melatonina no pacu são mais altos no verão e menores no inverno. Isoenzimas de glutationa S-transferases que conjugam o tripetídeo glutationa com o 4-hidroxinonenal, aldeído derivado da peroxidação de ácidos graxos por ERO, são importantes para evitar alteração funcional de proteínas por ligação do 4-hidroxinonenal à sua estrutura. Neste trabalho procuramos relação entre estresse oxidante, níveis de atividades de glutationa S-transferase e melatonina, para estabelecer se a melatonina ajudaria pacus a superar os efeitos deletérios das espécies reativas de oxigênio. Ensaiamos atividades de isoenzimas de glutationa S-transferases no citosol de fígado de pacus mantidos em normoxia, hipoxia, reoxigenação e hiperoxia no inverno e no verão. Medimos o efeito da melatonina in vitro e in vivo sobre as atividades de isoenzimas de glutationa S-transferase. Medimos os efeitos do estresse oxidante sobre a ligação do 4-hidroxinonenal com proteínas nos fígados de pacus tratados com melatonina. Somente as isoenzimas que conjugam 4-hidroxinonenal com glutationa mostraram menor atividade no inverno em relação ao verão; outras isoenzimas de glutationa S-transferases não alteram suas atividades sazonalmente. In vitro a melatonina não alterou a atividade de isoenzimas de glutationa S-transferase que conjugam o 4-hidroxinonenal, mas inibiu outras isoenzimas de glutationa S-transferase. In vivo a melatonina aumentou a atividade encontrada no inverno das isoenzimas que conjugam o 4-hidroxinonenal para os níveis do verão. A ligação de 4-hidroxinonenal com proteínas foi menor em pacus inoculados com melatonina. Nossos resultados mostram que a melatonina pode influenciar os efeitos de ERO em fígado de pacus. Ficou claro que a melatonina do plasma mantém os níveis de atividade conjugadora de 4-hidroxinonenal do fígado em pacus e que a baixa produção de melatonina no inverno não é adequada para a conjugação do 4-hidroxinonenal em fígado de pacus. / Oxygen is vital for vertebrates. However, changes in the levels of dissolved oxygen in water might cause oxidative stress in fishes because the shortage of oxygen followed by reoxygenation originates reactive oxygen species (ROS) inside cells. Higher intracellular levels of ROS favor alterations of proteins, phospholipids and nucleic acid molecules, which result in impairment of many cell functions. In Pantanal, the pacus habitat, circadian variation of the oxygen levels occurs in water of the shallow lagoons that ended up isolated from the rivers along the dry season. Pacu has evolved under the pressure of continuous exposition to harmful effects of ROS caused by the annual inundation pulses. Melatonin, an indolamine produced by the pineal gland, influences the levels of activity of antioxidant enzymes that reduce ROS, and is capable of donating electrons or scavenge free radicals nonenzymatically. Pacus melatonin levels are higher during summer than in winter. Glutathione S-transferases isoenzymes that catalyze the conjugation of the tripeptide glutathione with 4-hydroxynonenal, an aldehyde derived from peroxidation of fat acids by ROS, are important to avoid functional alterations of proteins consequential to the binding of 4-hydroxynonenal to their structures. In this work, we searched for facts that linked oxidative stress, levels of activity of glutathione S-transferase and melatonin, in order to establish whether melatonin could help pacus to overcome the pernicious effects of reactive oxygen species. We carried out assays of glutathione S-transferases in liver cytosol of pacus kept under normoxia, hypoxia, reoxygenation and hyperoxia, in the summer and in the winter. We measured the effect of melatonin in vitro and in vivo on isoenzymes of glutathione S-transferases. We measured the effects of oxidative stress on the binding of 4-hydroxynonenal to proteins in liver of pacu treated with melatonin. Only isoenzymes that conjugate 4-hydroxynonenal with glutathione showed less activity during the winter in comparison to the summer; other isoenzymes did not have their activities changed seasonally. In vitro, melatonin did not change the activity of glutathione S-transferases isoenzymes that conjugate 4-hydroxynonenal, but inhibited other isoenzymes of glutathione S-transferase. In vivo, melatonin enhanced the liver activity of the glutathione S-transferase that conjugate 4-hydroxynonenal found in winter up to the levels found in summer. The binding of 4-hydroxynonenal to proteins was lower in liver cytosol from pacus injected with melatonin. Our findings show that melatonin can influence the effects of ROS in liver of pacu. It became evident that plasma melatonin maintains the liver levels of the conjugating activity of 4-hydroxynonenal and that the lower production of melatonin during winter is not adequate to the conjugation of 4-hydroxynonenal.