Tout au long de la chaîne alimentaire, des moisissures peuvent se développer et produire des mycotoxines. Ce sont des composés toxiques naturels issus du métabolisme secondaire des moisissures, susceptibles de contaminer l'alimentation animale et humaine, provoquant de nombreuses pathologies (hépatotoxicité, néphrotoxicité, neurotoxicité, mutagénicité, tératogénicité, cancérogénicité,…). La première étape de ce travail était d'évaluer la présence simultanée de l'ochratoxine A (OTA), de la citrinine (CIT), des aflatoxines (AFs), de la zéaralénone (ZEA), de la fumonisine (FB) et des trichothécènes dans des aliments destinés aux humaines (céréales, lait, café, jambon) et aux animaux (croquettes de chat et chien, foins). En général plusieurs mycotoxines coexistaient. Certains échantillons pour les humains dépassaient les limites autorisées en mycotoxines dans l'Union Européenne. Suite à l'étude de simulation d'apport en mycotoxines dans une ration quotidienne, nous avons constaté que les doses journalières admissibles (DJA) peuvent être dépassées. La deuxième phase consistait à étudier l'impact des mycotoxines seules ou en combinaison sur la viabilité cellulaire et la génotoxicité sur des modèles cellulaires (cellules rénales d'opossum (OK), cellules rénales humaines (HK2), cellules humaines de glandes mammaires (MCF7)) et chez des animaux (porc, rat). Nous avons montré que la CIT, la FB1 et la ZEA agissent en synergie sur la génotoxicité de l'OTA. Chez les animaux, nous avons montré qu'à des doses (5 ng d'OTA/kg poids corporel/ jour et de 200ng FB1/kg pc/j) correspondantes aux DJA, il y avait des effets génotoxiques (formation d'adduits à l'ADN). Nous avons mis en évidence l'implication des mycotoxines dans l'alimentation animale sur la baisse de fertilité et la tératogénicité chez les chats, ainsi que sur la mort des chevaux. Au cours de la troisième partie de cette étude, nous avons testé sur des cultures cellulaires (HK2 et MCF7) et in vivo (poulet) l'effet protecteur du glutathion (GSH) et de la sélénométhionine (SeMet) contre l'OTA responsable de cancers de voie urinaire et la ZEA responsable de baisse de fertilité. Le GSH est un puisant antioxydant et le sélénium est un oligoélément indispensable qui intervient comme co-facteur de nombreuses enzymes ayant des propriétés antioxydantes, comme les glutathion peroxydases. D'une manière générale, au niveau des cellules rénales, le GSH seul et la levure correspondante ont un effet bénéfique vis-à-vis de la génotoxicité de l'OTA ; par contre la sélénométhionine et la levure séléniée augmentent la génotoxicité de l'OTA et de la ZEA. Dans les cellules des glandes mammaires, il y a une nette amélioration vis-à-vis de la génotoxicité des deux mycotoxines lorsque les cellules sont exposées à une seule mycotoxine simultanément au GSH, à la sélénométhionine et aux levures enrichies. Chez les poulets, la diminution de la génotoxicité n'est pas exclusivement corrélée à la capacité des parois de levure ou des levures à adsorber l'OTA. Ces dérivés de levure ont gardé la propriété de partiellement métaboliser l'OTA dans l'intestin. Les parois de levures et les levures enrichies en GSH ont un meilleur pouvoir protecteur que celles enrichies en SeMet / Throughout the food chain, mold can grow and produce mycotoxins. These are toxic compounds "natural" from the secondary metabolism of molds that may contaminate the feed and food, causing many diseases (hepatotoxicity, nephrotoxicity, neurotoxicity, mutagenicity, teratogenicity, carcinogenicity, ...). The first stage of this work was to assess the level of multi-contamination by mycotoxins (OTA, CIT, Afs, ZEA, FB, DON) in food (cereals, milk, coffee, ham) and feed (pet food). Some samples analyzed exceeded the limits of mycotoxins in the European Union. Through the simulation study of mycotoxin intake in a daily diet, we found that the acceptable daily intake (ADI) may be exceeded. The second phase was to study the impact of mycotoxins alone or in combination on cell proliferation, genotoxicity in cellular models (OK, HK2, and MCF7) and animal (pig, rat). We have demonstrated genotoxic effects (formation of DNA adducts) at doses (5 ng OTA / kg bw / day and 200 ng FB1/kg bw / day) considered safe (ADI). We have shown that the CIT, FB1 and ZEA act synergistically on the genotoxicity of OTA. We pointed to the involvement of mycotoxins in animal feed on declining fertility and teratogenicity in cats, as well as the death of horses. In the third part of this study, we tested in cell cultures (HK2 and MCF7) and in vivo (chicken) the protective effect of glutathione (GSH) and selenomethionine (SeMet) against OTA responsible for urinary tract cancers and ZEA reducing fertility. GSH is considered as a potent antioxidant and selenium is a trace essential element that acts as a cofactor of enzymes such glutathione peroxidase. In summary, in kidney cells, GSH and GSH enriched yeast decrease OTA genotoxicity whereas SeMet and SeMet enriched yeast increase genotoxicity of OTA and ZEA. In mammary cells, whatever the compounds gentoxicty of OTA and ZEA significantly decrease. Decrease of OTA genotoxicity in chicken kidney cannot be exclusively explained by adsorption of OTA on yeast by products. The yeast products retain their ability to metabolize the OTA. GSH enriched yeast and yeast cell wells are more efficient than SeMet enriched yeast

Les champignons saprophytes sont les acteurs principaux du recyclage de la matière organique morte au sein des écosystèmes forestiers. Ces microorganismes possèdent la capacité unique de dégrader la totalité des polymères constitutifs du bois. L'analyse de la structuration des communautés durant les stades initiaux de la colonisation du bois par séquençage à haut débit a révélé que celui-ci influence la distribution et la dynamique des communautés qui lui sont associées. A l'échelle de l'organisme, les différents groupes écologiques de champignons décomposeurs du bois possèdent des systèmes de dégradation extracellulaires reflétant cette complexité chimique. Le séquençage du génome d'un grand nombre de ces organismes a permis l'identification de superfamilles d'enzymes impliquées dans les mécanismes de résistance et de détoxication des composés toxiques exogènes. Parmi elles, la superfamille des glutathion transférases présente une extension de classes spécifiques au sein des champignons décomposeurs du bois. La détermination des propriétés biochimiques et structurales d'une isoforme, issue d'une de ces classes spécifique (les Etherase-like), présente chez Phanerochaete chrysosporium a révélé des caractéristiques particulières. Cette enzyme possède un mode de dimérisation atypique ainsi que la capacité à séquestrer des composés phénoliques toxiques via une propriété ligandine unique. La comparaison des propriétés de plusieurs isoformes de cette classe d'enzymes appartenant aux champignons C. cinereus et P. chrysosporium a démontré que celle-ci exhibe une grande versatilité intra- et interspécifique, de leurs activités enzymatiques et de leur propriété ligandine / Saprophytic fungi are key players of dead organic matter recycling in forest ecosystems. These microorganisms possess the unique ability to degrade the integrality of wood constitutive polymers by secretion of complex oxydative and hydrolytic enzymatic systems. Communities structuration analysis during the initial stages of wood colonisation by high throughput sequencing revealed that the latter beyond being a source of nutrients, influences the distribution and dynamic of communities by its broad chemical variability. At the organism level, the different ecological groups of wood decomposing fungi possess extracellular degradation systems reflecting this chemical complexity. Genome sequencing of these organisms allowed the identification of enzymes superfamilies involved in resistance and detoxification mechanisms towards exogenous toxic compounds. Among them, the glutathione transferases superfamily exhibit extension of specific classes in wood decaying basidiomycetes. Biochemical and structural properties determination of one isoform belonging to one of these specific classes (the Etherase-like), found in Phanerochaete chrysosporium revealed unusual characteristics. This enzyme possesses an atypical dimerization mode as well as the ability to sequestrate toxic phenolic compounds resulting from wood degradation through a unique ligandin property. Properties comparison of several isoforms from this class belonging to C. cinereus and P.chrysosporium demonstrated a huge intra- and interspecific versatility of their enzymatics activities and ligandin property in response to environmental constraints arising from the great chemical heterogeneity of wood composition

Sinorhizobium meliloti est une bactérie du sol Gram- capable d'induire la formation denodosités fixatrices d'azote lors d'une interaction symbiotique avec les plantes de la familledes légumineuses. L'importance de la balance redox au cours de cette interaction a été miseen évidence. Ainsi, des mutants bactériens déficients dans la production du glutathion (GSH),présentent un phénotype altéré d'infection et de fixation de l'azote atmosphérique.Le premier objectif a donc été de déterminer si les phénotypes observés chez les mutants de lavoie de biosynthèse du GSH étaient liés à l'activité des glutarédoxines (GRX). Une analysebioinformatique a révélé la présence de trois gènes codant des GRX chez S. meliloti. Lesmutants, Smgrx1, Smgrx2 et Smgrx3, déficients pour chacune des GRX, ne produisent pasdes phénotypes similaires à ceux observés avec les mutants GSH. Si Smgrx2 présente unphénotype moins marqué, Smgrx1 est plus sévèrement affecté puisqu'il n'est plus capable dese différencier en bactéroïde. L'implication de SmGrx2 dans la régulation du métabolisme dufer et la mise en place des centres Fe-S a, par ailleurs, été mise en évidence.Le second objectif a été de définir s'il existait, chez S. meliloti, une redondance fonctionnelleentre les GRX et les thiorédoxines (TRX). Ainsi, le mutant SmtrxB, dépourvu de thiorédoxineréductase, présente la particularité d'induire la formation d'un plus grand nombre de nodulesque la souche sauvage. Le système TRX de S. meliloti apparaît donc comme un régulateurnégatif de la nodulation. D'autre part, les nodosités formées par ce mutant SmtrxB, ont uneactivité fixatrice d'azote significativement diminuée. Les rôles des TRX et des GRXapparaissent donc, au moins partiellement, distincts.Les résultats obtenus ici apportent des éléments nouveaux sur l'implication du GSH, des GRXet des TRX dans la mise en place d'une nodosité fonctionnelle, et ouvrent de nouvellesperspectives d'études sur les rôles de ces molécules dans le processus de fixation d'azote. / Sinorhizobium meliloti is a soil bacterium Gram- able to induce the formation of nitrogenfixingnodules during a symbiotic interaction with plants of the legume family. Theimportance of redox balance during this interaction has been demonstrated. In this way,bacterial mutants deficient in the production of glutathione (GSH), exhibit an alteredphenotype of infection and fixation of atmospheric nitrogen.The first objective was therefore to determine whether the phenotypes observed in mutants ofthe GSH biosynthesis pathway were related to the activity of glutaredoxins (GRX). Abioinformatic analysis revealed the presence of three genes encoding GRX in S. meliloti. Themutants, Smgrx1, Smgrx2 and Smgrx3, deficient for each of the GRX, do not producephenotypes similar to those observed with the GSH mutants. If Smgrx2 presents a less severephenotype, Smgrx1 is more severely affected since it is incapable of differentiating intobacteroïd. The involvement of SmGrx2 in the regulation of iron metabolism and theestablishment of Fe-S cluster has also been demonstrated.The second objective was to determine if there was, in S. meliloti, a functional redundancybetween GRX and thioredoxin (TRX). Thus, the SmtrxB mutant, devoid of thioredoxinreductase, has the distinctive feature of inducing the formation of more nodules than the wildtype strain. The TRX system of S. meliloti appears to be a negative regulator of nodulation.On the other hand, the nodules formed by this SmtrxB mutant have a significantly decreasednitrogen-fixing activity. Hence, the roles of TRX and GRX appear to be at least partiallydistinct.The results obtained here provide new evidence on the involvement of GSH, the GRX andTRX in the establishment of a functional nodule, and open new perspectives for studying onthe roles of these molecules in the process of nitrogen fixation.

La surexpression de pompes d'efflux par les cellules cancéreuses permet l'élimination d'agents cytotoxiques, induisant alors une résistance à la chimiothérapie. Trois transporteurs ABC sont principalement impliqués dans cette résistance : P-gp/ABCB1, MRP1-ABCC1 et BCRP/ABCG2. La surexpression des ces transporteurs peut également être le "talon d'Achille" des cellules cancéreuses résistantes en les sensibilisant à certains composés. Ce phénomène, appelé sensibilité collatérale, pourrait constituer un nouvel outil thérapeutique conter les les cancers intrinséquement ou rendus résistants en éliminant sélectivement les cellules cancéreuse résistances. Ainsi, le S-vérapamil provoque la mort sélective par apoptose des cellules surexprimant suite à l'extrusion rapide et massive du glutathion 5GSH) intracellulaire par MRP1. Nous avons démontré que le vérapamil est capable de dépléter s"lectivement de leur contenu en GSH les tumeurs de cancer du poumon H69AR, MRP1 positives et résistantes, dès 3 heures d'exposition aiguë. Le vérapamil étant fortemnt carditoxique, nous avons développé de nouveaux agents de sensibilité collatérale, plus sélectif que le vérapamil, comme le xanthone 9, le flavonoïde 36 et le dimère de flavonoïde 4e. Enfin, grâce à l'étude de chimères MRP1/MRP2, nous avons démontré que la région comprenant les boucles L0 et L1-TM12 pourrait constituter les sites modualteurs et substrat du GSH sur MRP1. / Resistance to chemotherapy is partly due to efflux pumps expressed in the plasma membrane, which prevent the accumulation of anticancer drugs in tumor cells. Three human ABC transporters are particulary involved in this chemoresistance : P-gp/ABCB1, MRP1-ABCC1 and BCRP/ABCG2. The overexpression of these trnasporters can also be an "Achille heel" for resistant cancer cells by sensitizing them to various drugs. This phenomenom, called collateral sensitivity, could constitute a new chemotherapy to eradicate cancers becoming resistant or cancer which ara resistant prioir to any treatment. Thus, S-verapamil triggers selective apoptosis of MRP1 overexpressing correlated to the massive and rapide extrusion of cellular glutathione by MRP1. We showed that verapamil is able to selectivity deprive H69AR MRP1 positive and resistant lung tumors, as soon as 3 hours of acute exposure. Verapamil being highly cardiotoxic, we have developed new collateral sensitivity drugs, more selective than verapamil, such as xanthone 9, flavonoïdd 36 and flavonoïd dimer 4e. Finally, thanks to the characterization of MRP1/MRP2 chimera, we showed that the MRP1 region including the intracellular loop L0 L1-TM12 might constitute the substrate and the modulator binding sites for GSH.

Phanerochaete chrysosporium est un champignon forestier faisant partie des organismes saprophytes capables de recycler la matière organique morte. Grâce à l'excrétion de nombreuses enzymes de dégradation, en particulier des lignine peroxydases, il est capable de décomposer la matière végétale dont la lignine, un polymère complexe de composés phénoliques très résistant. L'élimination de la lignine permet la libération des autres composants du bois tels que la cellulose et l'hémicellulose qui peuvent être utilisés dans l'industrie papetière ou pour la production de bioéthanol de deuxième génération. La structure des intermédiaires et produits de dégradation de la lignine est souvent proche de celle denombreux polluants, d'où l'intérêt biotechnologique de P. chrysosporium dans les processus de bioremédiation. Cependant, les systèmes de dégradation engendrent des composés plus ou moins toxiques pour le champignon et contre lesquels il doit faire face. C'est pourquoi il possède un système de détoxication impliquant des enzymes telles que les cytochrome P450 monooxygénases ou encore les glutathion transférases (GST). Les Ure2p forment une classe de GST étendue chez Phanerochaete et d'autres basidiomycètes saprophytes. Leur étude par des approches phylogénétiques, biochimiques, structurales et transcriptomiques a permis de mieux comprendre les mécanismes d'évolution que peut subir une classe d'enzymes potentiellement soumises à une forte pression de sélection / Phanerochaete chrysosporium is a forest fungus being part of saprophytic organisms able to recycle dead organic matter. Thanks to the excretion of numerous wood decaying enzymes, and especially lignin peroxidases, this fungus is able to break down plant material including lignin, a complex polymer of phenolic compounds. Lignin removal allows the release of other wood components such as cellulose and hemicellulose, which can be further used in paper industry or to produce second generation bioethanol. The structure of intermediates and products from lignin decomposition is close to that of numerous pollutants making P. chrysosporium biotechnologically interesting for bioremediation purposes. Moreover, the fungus has to deal with more or less toxic compounds created by degradation mechanisms. It thus presents a detoxification pathway involving enzymes including cytochrome P450 monooxygenases and glutathione transferases (GST). Ure2p enzymes belong to an extended GST class in Phanerochaete genus as well as in other saprophytic basidiomycetes. Their study based on phylogenetic, biochemical, structural and transcriptomic approaches provides a better understanding of evolution mechanisms of a class of enzymes potentially subject to strong selection selection pressure

O Adenocarcinoma ductal pancreático (ADP) é o tumor pancreático mais comum e apresenta um dos piores prognósticos. A primeira alteração crítica que desencadeia o processo de progressão tumoral, é a ativação desregulada do gene KRAS, na qual está presente em 90% dos casos. Várias iniciativas terapêuticas buscaram como alvo direto a atividade da oncoproteína RAS, sem no entanto, obter resultados satisfatórios. Desta forma, a investigação de moléculas efetoras downstream às vias reguladas por RAS, poderiam resultar em estratégias mais eficazes. Dentre estas moléculas efetoras estão as MAPKs, que modulam diversos processos celulares essenciais para o desenvolvimento tumoral, onde a cascata RAS/RAF/MEK/ERK representa uma importante via canônica de transdução de sinais. A transdução de sinais desta via pode ser favorecida por proteínas conhecidas como proteínas de arcabouço, como SHOC2, funcionando como uma plataforma para ligação de RAS-RAF-1 e consequentemente potencializando sua ligação. KRAS, têm sido associado à regulação de vias metabólicas importantes, como a glicólise que interferem diretamente na capacidade de proliferação e sobrevivência celular, para o estabelecimento e manutenção da biologia tumoral. Assim, o objetivo deste trabalho foi investigar o papel da proteína SHOC2 na indução do estresse oxidativo e capacidade de sobrevivência de linhagens celulares de ADP. Foram realizados os ensaios de morte celular por apoptose, avaliação da capacidade clonogênica e quantificação dos níveis de glutationa e quantificação da produção de espécies reativas de oxigênio. As linhagens celulares MIA PaCa2 e PANC-1 apresentaram uma redução significativa da capacidade de formação de colônias. A taxa de apoptose induzida pelo tratamento com Gemcitabina não diferiu entre as linhagens modificadas para silenciar a função de SHOC2. No ensaio da quantificação dos níveis de glutationa e na produção de espécies reativas de oxigênio, os resultados não foram concordantes com o esperado. Para análise dos níveis proteicos de p-ERK1/2, podemos observar uma redução na sua expressão, mesmo se mostrando de maneira sutil. Os resultados sugerem que pode haver alguma relação entre o silenciamento de SHOC2, estresse oxidativo e sobrevivência, porém existem outras vias alternativas modulando este processo. / Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic tumor and has one of the worst prognoses. The first critical change that triggers the process of tumor progression is the dysregulated activation of the KRAS gene, in which it is present in 90% of cases. Several therapeutic initiatives aimed directly at the activity of the RAS oncoprotein, without, however, obtaining satisfactory results. Thus, investigating downstream effector molecules on RAS-regulated pathways could result in more effective strategies. Among these effector molecules are MAPKs, which modulate several cellular processes essential for tumor development, where the RAS / RAF / MEK / ERK cascade represents an important canonical pathway for signal transduction. Signal transduction of this pathway may be favored by proteins known as scaffold proteins, such as SHOC2, serving as a platform for RAS-RAF-1 binding and hence potentiating its interaction. KRAS, have been associated with the regulation of important metabolic pathways, such as glycolysis, for the establishment and maintenance of tumor biology. Thus, the objective of this work was to investigate the role of SHOC2 in the induction of oxidative stress and survival capacity of ADP cell lines. Cell death assays were performed by apoptosis, and quantification of glutathione levels and the production of reactive oxygen species were performed. MIA PaCa2 and PANC-1 cell lines showed a reduction in colony formation capacity. Gemcitabine-mediated cell death by apoptosis has not been induced after SHOC2 knockdown. Also, the measurement of reactive oxygen species and quantification of glutathione levels did not reveal any change mediated by SHOC2. The analysis of ERK1 / 2 activation has shown a discrete reduction in its expression. The results suggest that there may be some relationship between SHOC2 silencing, oxidative stress and survival, but there are other alternative pathways modulating this process which needs claryfication.

O experimento foi conduzido na FZEA/USP com objetivo de comparar a utilização de fontes orgânicas de selênio (Se) com o selenito de sódio na dieta de cordeiros, pela análise da concentração de Se nos tecidos, da atividade da enzima glutationa peroxidase no fígado, do balanço metabólico e do cálculo da biodisponibilidade. Foram utilizados 40 cordeiros Suffolk, os quais foram submetidos a três fontes e três níveis de Se suplementar por 84 dias. Os tratamentos foram: tratamento 1: sem suplementação; tratamentos 2, 3 e 4: 0,2; 0,8 e 1,4 mg/kg de Se suplementar na forma de selenito de sódio; tratamentos 5, 6 e 7: 0,2; 0,8 e 1,4 mg/kg de Se suplementar na forma de Se-levedura; tratamentos 8, 9 e 10: 0,2; 0,8 e 1,4 mg/kg de Se suplementar na forma de Se-metionina. Foram colhidas amostras de sangue para dosagem sérica de Se e ao final do experimento, os animais foram abatidos para colheita de amostras de fígado, músculo e rim, para determinação dos teores de Se e da atividade da glutationa peroxidase (fígado). Nos últimos cinco dias de experimento foi realizado um balanço metabólico de Se. A biodisponibilidade foi calculada através da técnica “slope ratio", utilizando como parâmetros a concentração de selênio no fígado, músculo, rim e a atividade da glutationa peroxidase. Não houve efeito da fonte de Se utilizada na ingestão, absorção aparente e retenção de Se, atividade da glutationa peroxidase e nas concentrações de selênio no fígado, rim e soro; porém, as concentrações de selênio no músculo foram maiores nos animais suplementados com fontes orgânicas do que nos outros animais (P<0,0001). A biodisponibilidade de Se no músculo foi maior quando foram utilizadas fontes orgânicas de selênio. O uso de fontes orgânicas de Se promove maior acúmulo de Se no músculo de cordeiros e promoveria maior ingestão de Se por consumidores de carne ovina. / This research was carried out at FZEA/USP to compare the utilization of organic selenium (Se) sources to sodium selenite in lambs’ diet, through analyses of tissues Se concentrations, liver glutathione peroxidase activity, metabolic balance of Se and bioavailability assay. Forty Suffolk lambs were used and submitted to three sources and three levels of supplementary Se for 84 days. Treatments were: treatment 1: no supplement; treatments 2, 3 and 4: 0.2, 0.8 and 1.4 mg/kg of supplementary sodium selenite-selenium; treatments 5, 6 and 7: 0.2, 0.8 and 1.4 mg/kg of supplementary selenoyeast-selenium; treatments 8, 9 and 10: 0.2, 0.8 and 1.4 mg/kg of supplementary selenomethionine-selenium. Blood samples were taken to Se serum dosage and in the end of the experiment the animals were killed and samples of liver, muscle and kidney were taken to Se concentrations dosage and glutathione peroxidase activity (liver). In the last five days of the experiment, a Se metabolic balance was realized. Bioavailability was calculated by “Slope Ration Assay", using liver, muscle and kidney Se concentrations and glutathione peroxidase activity as parameters. Se sources did not affect Se intake, apparent absorption and retention, glutathione peroxidase activity and Se concentrations in liver, kidney and serum; however, selenium concentrations in muscle of animals supplied with organic sources were higher than in other animals (P<0.0001). Se bioavailability in muscle was higher when organic Se sources were used. Using organic Se sources provides higher accumulation of Se in lambs’ muscle which would provide higher Se intake to consumers of sheep meat.

O objetivo deste estudo foi avaliar o estado nutricional relativo ao selênio em indivíduos adultos relacionando-o com polimorfismos no gene da enzima GPx e sua influência sobre a enzima e o balanço redox. Foram selecionados 343 estudantes da Universidade Federal do Piauí entre 20 e 50 anos, de ambos os sexos, selecionados de acordo com critérios de inclusão adotados, como ausência de doenças crônicas não transmissíveis (DCNT) dentre outros. Sangue venoso foi coletado para análise de Se, genotipagem dos SNP da GPx1 (Pro198Leu, -602A/G e Arg5Pro), da atividade das enzimas antioxidantes (GSH-Px e SOD) eritrocitárias, malondialdeído (MDA) e capacidade de absorção de radicais de oxigênio (ORAC) plasmáticas. A análise de Se foi realizada por meio de espectrometria de absorção atômica por geração de hidretos, a genotipagem por PCR em tempo real em Step One Plus, as enzimas em analisador bioquímico automático utilizando kits comerciais, o MDA em cromatografia líquida de alta eficiência e ORAC em um leitor de microplaca. A análise estatística foi realizada por meio do software R 3.0.2. Foram realizados testes de comparação de duas e de mais que três médias entre as variáveis genéticas e os parâmetros de avaliação do Se e do balanço redox. Análises de regressão linear e linear generalizada foram realizadas para identificar a influência das variáveis genéticas, antropométricas, do perfil lipídico e estilo de vida sobre o Se sanguíneo e as variáveis do balanço redox. Os dados foram considerados significativos com p menor que 5%. A idade média dos participantes foi de 24,4±5,0 anos sendo 57,7% do sexo feminino. Entre os participantes, 95,7% eram carreadores do alelo Leu do SNP Pro198Leu e G do -602A/G e não possuíam quantidades mínimas de Se plasmático para otimizar a atividade da GPx. A atividade da GPx foi significativamente mais baixa e de ORAC mais alta nos indivíduos com o genótipo Leu/Leu em relação ao Pro/Pro do SNP Pro198Leu. Os indivíduos com o genótipo Arg/Pro apresentaram atividade da GPx significativamente maior que aqueles com o genótipo Arg/Arg do SNP Arg5Pro. Não houve diferença significativa entre as médias de MDA e SOD e os genótipos dos três SNP. As variáveis genéticas, de avaliação antropométrica, do perfil lipídico e estilo de vida mostraram influência sobre os marcadores do balanço redox, alterando o perfil antioxidante dos participantes. Os indivíduos são deficientes em Se e aqueles com o alelo Leu do SNP Pro198Leu apresentam em seu organismo maior concentração de ORAC, provavelmente para proteção contra radicais livres. O alelo variante do SNP Arg5Pro mostrou-se benéfico para o estado nutricional relativo ao Se. / The aim of this study was to evaluate the nutritional status relative to Se of adult individuals relating it to polymorphisms in the GPx enzyme gene and its influence on the enzyme and the redox balance. We selected 343 students of the Federal University of Piauí between 20 and 50 years, of both genders, selected according to inclusion criteria, such as the absence of chronic noncommunicable diseases (NCD) among others. Venous blood was collected for analysis of Se, genotyping of SNP of GPx1 (Pro198Leu, -602A / G and Arg5Pro), the activity of antioxidant enzymes (GSH-Px and SOD) erythrocyte, malondialdehyde (MDA) and absorption capacity of plasma oxygen radicals (ORAC). The analysis of Se was performed by atomic absorption spectrometry through hydride generation, genotyping by real time PCR in Step One Plus, enzymes in automatic biochemical analyzer by the use of commercial kits, MDA analysis was conducted in high-performance liquid chromatography and ORAC in a microplate reader. Statistical analysis was obtained using the software R version 3.0.2. Comparison tests were performed with two and more than three averages between the genetic variables and the evaluation parameters from Se and redox balance. Linear regression analysis and generalized linear were carried out to identify the influence of genetic variables, anthropometric, lipid profile and lifestyle on the sanguine Se and the variables of the redox balance. Data were considered significant for p less than 5%. The average age of participants was 24.4±5.0 years and 57.7% were female. Among the participants, 95.7% were allele carriers Leu of SNP Pro198Leu and G of -602A/G, and they did not have minimal amounts of Se plasma to optimize the activity of GPx. The GPx activity was significantly lower and that of ORAC was higher in subjects with the Leu/Leu genotype compared to Pro/Pro of SNP Pro198Leu. Individuals with the Arg/Pro genotype had GPx activity significantly higher than those with genotype Arg/Arg of SNP Arg5Pro. There was no significant difference between the means of MDA and SOD of the genotypes of the three SNP. The genetic variables, anthropometric measurements, lipid profile and lifestyle showed influence on markers of redox balance by changing the antioxidant profile of the participants. Individuals are deficient in Se, and those with the Leu allele of SNP Pro198Leu present in their bodies highest concentration of ORAC, probably to protect against free radicals. The variant allele of the SNP Arg5Pro proved to be beneficial to the nutritional status of the Se.

Foi demonstrado o envolvimento das espécies reativas de oxigênio (ERO) na patogênese de algumas retroviroses, levando muitas vezes à progressão e/ou persistência das mesmas. No entanto, o papel das espécies reativas de oxigênio resultante da modulação do sistema antioxidante, e seu envolvimento na infecção de bovinos naturalmente infectados pelo VLEB, até o presente trabalho, não tinha sido ainda investigado. Desta forma, o presente estudo objetivou-se avaliar o envolvimento do estresse oxidativo na infecção pelo VLEB em bovinos naturalmente infectados, associado ou não ao estabelecimento da linfocitose persistente (LP). Assim, a avaliação do estresse oxidativo se deu pelo teor de malondialdeído, concentração de glutationa reduzida, atividade da glutationa peroxidase e da superóxido dismutase, e pela atividade antioxidante total; e a resposta imune pela quantificação das subpopulações de linfócitos, função fagocítica e produção intracelular de peróxido de hidrogênio de leuccitos polimorfonucleares, morte celular e proliferação linfocitária, além da avaliação hematológica e sorodiagnóstico da leucose enzoótica bovina (LEB). Os resultados do presente estudo apontaram para o envolvimento do estresse oxidativo na infecção pelo vírus da leucose enzoótica bovina, dado pela menor concentração de glutationa peroxidase e a tendência a menor atividade da superxido dismutase eritrocitárias nos animais infectados pelo VLEB, não podendo, contudo, ser associado à fase da infecção. Ademais, os marcadores de estresse oxidativo não apresentaram alterados nem sequer a atividade antioxidante total destes animais. Além disso, o presente trabalho apontou para menor proliferação de linfócitos e redução da apoptose de células CD5+ nos animais infectados pelo VLEB manifestando LP, corroborados pelo significante aumento da população de linfcitos B (CD21+), e dos principais co-marcadores das células infectadas, no caso células CD5+ e CD11b+, nestes animais. No entanto, não observou diferenças significativas na população de linfócitos T destes animais, nem sequer alteração da capacidade fagocítica e produção intracelular de peróxido de hidrogênio pela população de leucócitos polimorfonucleares nos animais infectados. / Abundant observations of multiple pathogenic interactions between reactive oxygen species (ROS) and the pathogenesis of some retroviruses have drawn attention to role of ROS on disease progression and/or persistence of infection. Therefore, the role of the oxidative stress resulted from the modulation of the antioxidant system in dairy cows naturally infected with BLV has not been studied yet. So, the present study tries to investigate the involvement of oxidative stress in dairy cows naturally infected with BLV associated or not with the persistent lymphocytosis (PL). Thus, the oxidative stress was evaluated by the malondialdehyde, reduced glutathione content, and by the activity of glutathione peroxidase and superoxide dismutase, and also total antioxidant activity. The immune response was also evaluated by the quantification of lymphocyte subsets, lymphocyte proliferation, polymorfonuclear leukocytes phagocytosis and intracellular hydrogen peroxide production, and cell death. The results of the present work point out to an involvement of oxidative stress in dairy cows naturally infected with BLV, given by a lower glutathione peroxidase activity and also a tendency towards lower superoxide dismutase activity, but it can be not associated with PL. Futhermore, the oxidative stress markers and also the total antioxidant status was not altered in infected animals. The present study also showed a lower lymphocyte proliferarion and apoptosis rates of CD5+ cells, strengthen by a higher B cells (CD21+) counts and also by a significant augment of CD5+ and CD11b+ positive cells that are often co-expressed by the infected cells. Although, no significant difference was observed in T cells counts. Moreover, the phagocytosis rates and the intracellular hydrogen peroxide production by the polymorphonuclear leukocytes are not altered in BLV infected dairy cows.

INTRODUÇÃO E OBJETIVOS: A a-Glutathiona-S-transferase (aGST) vem sendo proposta como um marcador sensível e não-invasivo de lesão hepática em pacientes com Hepatite pelo vírus C. Avaliamos neste trabalho como a (aGST) se correlaciona com características bioquímicas e histológicas em pacientes com HCV. MATERIAL E MÉTODOS: Realizamos a determinação da concentração plasmática das aGST, alanina aminotransferase (ALT), aspartato aminotransferase (AST) e gama-glutamyltransferase (gGT) em 114 pacientes com HCV, dos quais 97 foram submetidos à biópsia hepática em até 6 meses da realização dos testes bioquímicos. Avaliamos também os níveis de aGST em 66 doadores de sangue sadios, que serviram como controles. Comparamos os níveis de aGST com as demais provas bioquímicas e a histologia hepática. RESULTADOS: A aGST estava elevada em 85.96% dos pacientes com HCV e mostrou associação com a elevação das aminotransferases (p<0.01). O valor de 4mg/dL mostrou as melhores sensibilidade (85,96%) e especificidade (92,42%) para determinar a normalidade do teste aGST. aGST ³ 8mg/dL mostrou a melhor especificidade para determinar lesão histológica hepática mais agressiva e o melhor valor preditivo positivo e razão de verossimilhança positiva para inflamação portal e atividade parenquimatosa mais agressiva nos pacientes com HCV. CONCLUSÕES: A aGST está relacionada à lesão hepática na infecção pelo HCV (valor de corte = 4mg/dL) e lesões histológicas hepáticas mais agressivas (valor de corte = 8mg/dL). Neste contexto, a aGST poderia ser utilizada em pacientes com Hepatite pelo HCV com ALT elevada, como um indicador complementar de lesão histopatológica mais agressiva. Entretanto, seu valor preditivo positivo não é suficientemente elevado para evitar a necessidade de realizar a biópsia hepática, mesmo quando está acima de 8mg/dL. / BACKGROUND/AIMS: a-Glutathione-S-transferase (aGST) has been proposed as a sensitive non-invasive indicator of hepatocellular injury due to Hepatitis C virus (HCV) infection. In this work, we evaluate how alpha-GST concentration correlates with biochemical and histological features in HCV patients. METHODS: We assayed plasma aGST, alanine aminotransferase (ALT), spartate aminotransferase (AST) and gama-glutamyl-transferase (gGT) in 114 HCV+ patients, among whom liver biopsy was performed in 97, within 6 months of the biochemical evaluation. We also assessed aGST levels in 66 health blood donors, aimed to serve as control. We compared aGST levels with other biochemical tests and liver histology. RESULTS: In 85.96% of HCV patients aGST was elevated and showed association with serum aminotransferases (p<0.01). The value of 4mg/dL (or lower) showed the best sensitivity (85.96%) and specificity (92.42%) to determine normality on the aGST test. aGST levels 8mg/dL and higher showed the best specificity to determine the presence of more aggressive liver histological damage among HCV patients and the best predictive positive value and positive likelihood for more aggressive portal inflammation and lobular activity. CONCLUSION: aGST is related to HCV infection (cut-off = 4mg/dL) and more aggressive liver histological damage (cut-off = 8mg/dL). In this sense, aGST could be used in HCV patients with altered ALT levels as an indicator of more aggressive hystopathological damage. However, its positive predictive value (PPV) is not high enough to preclude the decision of performing hepatic biopsy, even when it is above 8mg/dL.