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Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-01-13T11:27:01Z No. of bitstreams: 1 IsraellyVianaDeSena_DISSERT.pdf: 988106 bytes, checksum: fd7fe778e190889d6f1a07cababa96be (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-01-23T11:12:07Z (GMT) No. of bitstreams: 1 IsraellyVianaDeSena_DISSERT.pdf: 988106 bytes, checksum: fd7fe778e190889d6f1a07cababa96be (MD5) / Made available in DSpace on 2017-01-23T11:12:07Z (GMT). No. of bitstreams: 1 IsraellyVianaDeSena_DISSERT.pdf: 988106 bytes, checksum: fd7fe778e190889d6f1a07cababa96be (MD5) Previous issue date: 2015-06-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Neste estudo foi avaliado o papel das esp?cies reativas de oxig?nio na infec??o experimental pelo T. cruzi. Inicialmente, realizamos cultura de macr?fagos na presen?a de formas tripomastigotas do parasito e demonstramos que o T. cruzi induz a produ??o de ROS in vitro. Posteriormente, camundongos albinos Swiss foram infectados com 1 ? 104 formas tripomastigotas sangu?neas da cepa Y e tratados com os inibidores de ROS (N-acetilciste?na), e da ?xido n?trico sintase induz?vel/iNOS (aminoguanidina) ou ambos, e determinado a parasitemia, mortalidade, produ??o de IgG e de citocinas no soro (IL-17, IFN-?, TNF-?) por meio de ELISA; e a express?o de RNAm de citocinas no mioc?rdio dos camundongos por RT-PCR em tempo Real. Animais infectados com a cepa Y que tiveram a inibi??o simult?nea de ROS e NO apresentaram mortalidade inicial precoce (in?cio da mortalidade no 6? D.A.I), foi observado 100% de mortalidade em todos os grupos de animais. Camundongos que tiveram a inibi??o de ROS e NO, apresentaram maior produ??o de IFN-? e TNF-? no soro e tamb?m maior express?o de RNAm destas citocinas no tecido card?aco, quando comparado ao grupo controle. O excesso na produ??o de IFN-? e TNF-? pode conduzir a efeitos delet?rios ao hospedeiro. Quanto ? produ??o de IL-17 e IgG total, n?o houve diferen?a entre os grupos de animais que tiveram ROS ou NO bloqueado. A express?o da iNOS foi menor no grupo de animais tratados com os inibidores de ROS e NO, quando comparado ao controle n?o tratado. Os resultados indicam que as esp?cies reativas de oxig?nio participam da regula??o imunol?gica durante a infec??o experimental pelo T. cruzi, evitando mortalidade precoce ao hospedeiro.

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Oxidative stress (OS) plays a critical role in the pathogenesis of atherosclerosis potentially through interaction with nitric oxide (NO) generated by the inducible nitric oxide synthase (iNOS) pathway. Although considerable literature supports a pro-atherogenic role for iNOS-induced NO, recent evidence suggest an anti-atherogenic property for this enzyme where iNOS-induced NO attenuates atherosclerotic lesions after immune injury, enhancing endothelial integrity, survival, protecting against OS-induced apoptosis and necrosis. We therefore hypothesize that iNOS may have a cardio-protective role in the atherosclerotic vessel and that under conditions of OS, expression and function of this enzyme may be impaired, thus contributing to the deleterious consequences of OS. Experiments have therefore been conducted to establish whether pro-oxidants regulate iNOS expression/function in rat cultured aortic smooth muscle cells (RASMCs). These cells were induced for 24 hours with LPS and IFN-γ to mimic inflammatory conditions. Oxidative stress inducers may modulate iNOS-induced NO production through alteration of the expression and/or function of the inducible L-arginine-NO pathway. We examined the effects of hydrogen peroxide (H2O2), antimycin A and diethyl maleate (DEM) on this pathway in vascular smooth muscle cells. H2O2 had little effect on NO production or L-arginine transport while antimycin A and DEM independently caused a concentration dependent inhibition of both processes. Only DEM induced hemeoxygenase-1 (HO-1) expression, monitored by western blotting as a marker of OS. The effects of statins on NO synthesis and L-arginine transport in the presence and absence of OS were also investigated. The benefits of statins therapy in cardiovascular medicine are ascribed in part to their lipid-lowering effect by inhibiting 3-hydroxy-3-methoxyglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme for cholesterol synthesis. However, statins may possess anti-inflammatory properties and are able to improve endothelial function, stabilize atherosclerotic plaque, and inhibit platelet aggregation, vascular smooth muscle cells proliferation and vessel wall inflammation. These effects may be exerted through novel actions of statins that include interaction with specific signalling pathways in cells which may be associated with the induction of iNOS and/or cationic amino acid transporters (CATs). Thus, we have extended our investigations to include an examination of the effects of statins on both iNOS and CAT function and expression under control conditions and following exposure of cells to OS. Atorvastatin caused a bell shaped response on NO production and iNOS expression and also enhanced L-arginine transport but in a non-concentration dependent manner. Simvastatin only affected NO synthesis without altering transporter activity. Pravastatin was without effect on either system. Further studies demonstrated that that atorvastatin was able to reverse the effects of antimycin A and DEM but only on NO production. These findings confirm that the inducible L-arginine-NO pathway can be downregulated by pro-oxidants. This mechanism may therefore contribute to the deleterious effects observed in disease states associated with OS. Moreover, statins (in particular atorvastatin) appear to be effective in reversing the inhibition of NO production caused by inducers of OS. This, together with the fact that atorvastatin and simvastatin can potentiate iNOS-induced NO production and indeed L-arginine transport (with atorvastatin), highlights a potential novel mechanism through which the cardio-protective actions of these compounds could be mediated.

L'arthrose (OA) est une maladie articulaire dégénérative, classée comme la forme la plus fréquente au monde. Elle est caractérisée par la dégénérescence du cartilage articulaire, l’inflammation de la membrane synoviale, et le remodelage de l’os sous-chondral. Ces changements structurels et fonctionnels sont dues à de nombreux facteurs. Les cytokines, les prostaglandines (PG), et les espèces réactives de l'oxygène sont les principaux médiateurs impliqués dans la pathophysiologie de l'OA. L'interleukine-1β (IL-1β) est une cytokine pro-inflammatoire majeure qui joue un rôle crucial dans l'OA. L'IL-1β induit l'expression de la cyclooxygénase-2 (COX-2), la microsomale prostaglandine E synthase-1 (mPGES-1), la synthase inductible de l'oxyde nitrique (iNOS), ainsi que leurs produits la prostaglandine E2 (PGE2) et l'oxyde nitrique (NO). Ce sont des médiateurs essentiels de la réponse inflammatoire au cours de l'OA qui contribuent aux mécanismes des douleurs, de gonflement, et de destruction des tissus articulaires. Les modifications épigénétiques jouent un rôle très important dans la régulation de l’expression de ces gènes pro-inflammatoires. Parmi ces modifications, la méthylation/ déméthylation des histones joue un rôle critique dans la régulation des gènes. La méthylation/ déméthylation des histones est médiée par deux types d'enzymes: les histones méthyltransférases (HMT) et les histones déméthylases (HDM) qui favorisent l’activation et/ou la répression de la transcription. Il est donc nécessaire de comprendre les mécanismes moléculaires qui contrôlent l’expression des gènes de la COX-2, la mPGES-1, et l’iNOS. L'objectif de cette étude est de déterminer si la méthylation/déméthylation des histones contribute à la régulation de l’expression des gènes COX-2, mPGES-1, et iNOS dans des chondrocytes OA humains induits par l'IL-1β. Nous avons montré que la méthylation de la lysine K4 de l'histone H3 (H3K4) par SET-1A contribue à l’activation des gènes COX-2 et iNOS dans les chondrocytes humains OA induite par l'IL-1β. Nous avons également montré que la lysine K9 de l’histone H3 (H3K9) est déméthylée par LSD1, et que cette déméthylation contribue à l’expression de la mPGES-1 induite par IL-1β dans les chondrocytes humains OA. Nous avons aussi trouvé que les niveaux d'expression des enzymes SET-1A et LSD1 sont élevés au niveau du cartilage OA. Nos résultats montrent, pour la première fois, l'implication de la méthylation/ déméthylation des histones dans la régulation de l’expression des gènes COX-2, mPGES-1, et iNOS. Ces données suggèrent que ces mécanismes pourraient être une cible potentielle pour une intervention pharmacologique dans le traitement de la physiopathologie de l'OA. / Osteoarthritis (OA) is a disabling disease classified as the most common form of arthritis worldwide. It is characterized by cartilage degeneration, synovium inflammation, and subchondral bone remodeling resulting in a loss of joint function. These structural and functional changes are due to numerous factors. Cytokines, prostaglandins (PG), and reactive oxygen species are the major mediators implicated in the pathophysiology of OA. Interleukin-1 (IL-1) is a major pro-inflammatory cytokine that plays a crucial role in OA. IL-1 induces the expression of Cyclo-oxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), inducible nitric oxide synthase (iNOS), as well as their products prostaglandin E2 (PGE2) and nitric oxide (NO). These are critical mediators of the inflammatory response during OA causing pain, swelling, and joint tissue destruction. The activation of these pro-inflammatory genes results from different changes at the level of chromatin known as epigenetic modifications. Epigenetic modifications such as DNA methylation and histone modifications play a crucial role in gene expression. Among these modifications, histone methylation/demethylation is the most critical one. Histone methylation/demethylation is mediated by two types of enzymes: histone methyltransferases (HMT) and histone demethylases (HDM) which can either activate or repress transcription. It is therefore necessary to understand the molecular mechanisms which underlie the regulation of COX-2, mPGES-1, and iNOS expression. The objective of this study is to investigate whether histone methylation/demethylation can modulate COX-2, mPGES-1, and iNOS expression in IL-1 induced OA human chondrocytes. We demonstrated that histone H3 lysine K4 (H3K4) methylation by SET-1A contributes to IL-1-induced COX-2 and iNOS expression in human OA Chondrocytes. We showed also that LSD1-mediated demethylation of histone H3 lysine 9 (H3K9) contributes to IL-1β-induced mPGES-1 expression in human OA chondrocytes. We found that levels of SET-1A and LSD1 expression are elevated in OA cartilage as compared with normal cartilage. Our data demonstrates, for the first time, the implication of histone methylation/demethylation in COX-2, mPGES-1, and iNOS regulation suggesting that these mechanisms could be a potential target for pharmacological intervention in the treatment of the pathophysiology of OA.

Depuis sa découverte, le monoxyde d'azote n'a pas cessé d'intéresser la communauté scientifique. Sa biosynthèse est catalysée par les NO Synthases, qui est une famille de trois isoenzymes. La NOS neuronale et la NOS endothéliale sont constitutives et produisent du NO impliqué dans la neurotransmission et la vasodilatation respectivement. La NOS inductible quant à elle produit du NO impliqué dans la réponse immunitaire innée. Une surproduction de monoxyde d'azote est impliqué dans de nombreuses pathologies comme les maladies neuro-dégénératives ou les maladies chroniques à composantes inflammatoires. L'inhibition de iNOS et nNOS présente donc un grand intérêt thérapeutique. Cependant l'enjeu est de développer des inhibiteurs hautement sélectifs, afin de préserver les fonctions vitales de eNOS.Nous avons choisi de synthétiser des composés constitués d'un analogue de substrat tel que thiocitrulline et S-alkyl-isothiocitrulline auquel est ajouté, par un lien peptidique ou hétérocyclique, un résidu susceptible de se lier dans le canal d'accès du substrat, région moins conservée du site actif pouvant fournir des interactions spécifiques. La synthèse de ces composés a été réalisée sur support solide selon une méthode d'ancrage par la chaîne latérale développée au laboratoire, ou pour certains composés, selon une méthode classique d'ancrage par l'amine alpha, ces deux méthodes offrant un fort potentiel en chimie combinatoire. Les différents composés synthétisés ont été testés sur les trois isoformes recombinantes.Un second travail est brièvement exposé dans ce manuscrit. La synthèse de cyclopeptides basés sur le motif RGD impliqué dans la liaison aux intégrines a été réalisée selon un mode de cyclisation développé au laboratoire faisant intervenir la formation d'un pont guanidine qui sera ensuite diversement substitué. L'étude RMN et l'évaluation de leur activité biologique sur les récepteur opioïdes semble montrer un influence de la substitution du pont sur leur conformation et leur activité. / Since its discovery, Since its discovery, the nitric oxide did not stop interesting the scientific community. Its biosynthesis is catalyzed by NO synthases, a family of three isoenzymes. Neuronal NOS and endothelial NOS are constitutive and produce NO involved in neurotransmission and vasodilation process respectively. Inducible NOS produces NO involved in the innate immune response. An overproduction of nitric oxide is involved in many diseases such as neurodegenerative diseases or chronic diseases with inflammatory components. Thus, its inhibition should be of high therapeutical interest. However, it is necessary to develop highly selective inhibitors to preserve the vital functions of eNOS.We chose to synthesize compounds constituted of one substrate analogue as thiocitrulline or S-alkyl-isothiocitrulline which linked by a peptide bond or heterocycles to a residue able to bind into the substrate access channel, a less conserved region into the active site were specific interactions could be established. The synthesis of these compounds was performed on solid support according to an anchoring method through the side chain developed at the laboratory or, for some compounds, according to a conventional anchoring method through the alpha amine. These approaches are particulary interesting for a combinatory chemistry approach. All compounds were tested on the three recombinant isoforms.A second work is outlined in this manuscript. It consist of synthesizing cyclopeptides based on RGD motif involved in the binding to integrins and enkephalins analogues. The cyclisation method was developed in the lab and involves the formation of a guanidine bridge diversely substituted. NMR studies and biological evaluations on opioid receptor suggests that the diverse bridge substitutions could modulate the conformation and activity of the peptides.

Made available in DSpace on 2016-04-28T20:17:27Z (GMT). No. of bitstreams: 1 2007 - Antonia Regina Sessa da Silva.pdf: 384094 bytes, checksum: 91d2147f8402249dead112e338e89b66 (MD5) Previous issue date: 2007-03-30 / Brazil has currently the second bigger flock of equines of the world and the brazilian equines breeding plays an important role in the development of the sector of agribusiness. Among the infectious disease that affect the national equines breeding, the Equine infectious anemia (EIAV) has been shown of difficult control. The Equine infectious anemia is caused by a retrovirus, it has a worldwide distribution, and it is recognized as the most important disease of equines. The most common clinical signals in the acute phase, are anemia followed of jaundice in the mucosae, ventral oedema, mioglobinury, caquexy and, mainly, intermittent fever. Once it has no treatment, Ministry of Agriculture, Cattle and Supplying determines, through legal mechanisms, a politics of obligatory examination in credentialed laboratories, for the transport and commercialization of equines in the country. The examination is based on the test of Coggins, an immunodiffusion in agar gel (IDGA) of the serum of the animal tested against antigen of the EIAV. As the infection is lifetime, positive animals are euthanasiated by Veterinarians of the Ministry of Agriculture. Considering that precision of the results is critical, once that false positives animals could uselessly be sacrificed, and false negatives will be preserved as infection source, diverse problems may occur, appeared the interest of a systematic analysis of the reproducibility of kits and on possible sources of error. For this purpose, positive and negative sera, referred by the repetition of the tests for Coggins and ELISA, had been evaluated by IDGA comparatively with three kits of different manufacturers. In the experiments of reproducibility, it observed a great distinction in the quality of the precipitation line promoted for the positive sera in the tests. This phenomenon may be justified by the use of the conditions techniques legal, determined for would carry of the Ministry of Agriculture that is distinct of the recommendation of the analyzed manufacturers of two of kits. Exactly thus, the evaluation of the reproducibility of the results of kits with the referred sera showed high correlation. / O Brasil possui atualmente o segundo maior rebanho de eq??deos do mundo e a Eq?ideocultura Brasileira desempenha um importante papel no desenvolvimento do setor de agroneg?cios. Entre as doen?as infecciosas que afetam a eq?inocultura nacional, a Anemia Infecciosa dos Eq?inos (AIE) tem se mostrado de dif?cil controle. A AIE ? causada por um retrov?rus, apresenta uma distribui??o mundial, e ? reconhecida como a mais importante doen?a dos eq?inos. Entre os sinais cl?nicos mais comuns, na fase aguda da doen?a, encontram-se a anemia seguida de icter?cia nas mucosas, edema ventral, mioglobin?ria, caquexia e, principalmente, febre intermitente. Como n?o h? tratamento, Minist?rio da Agricultura, Pecu?ria e Abastecimento (MAPA) determina, atrav?s de mecanismo legais, uma pol?tica de exame obrigat?rio em laborat?rios credenciados, para o transporte e comercializa??o de eq??deos no Pa?s. O exame ? baseado na prova de Coggins, uma imunodifus?o em ?gar gel do soro do animal testado contra ant?geno do v?rus da AIE. Como a infec??o ? vital?cia, animais soropositivos s?o eutanasiados por M?dicos Veterin?rios do MAPA. Considerando que a precis?o dos resultados ? cr?tica, posto que animais falso positivos podem ser sacrificados inutilmente, e falso negativos preservados como fonte de infec??o, surgiu o interesse de um an?lise sistem?tica da reprodutibilidade dos kits e poss?veis fontes de erro no diagn?stico. Para esta finalidade, soros positivos e negativos, referenciados pela repeti??o dos testes de Coggins e ELISA, foram avaliados pela prova de IDGA, comparativamente com tr?s kits de diferentes fabricantes. Nos experimentos de reprodutibilidade, observou-se uma grande distin??o na qualidade da linha de precipita??o promovida pelos soros positivos nos testes nos diferentes kits. Este fen?meno pode ser justificado pela utiliza??o das condi??es t?cnicas legais, determinadas por portaria do MAPA, que s?o distintas das recomenda??es dos fabricantes de dois dos kits analisados. Mesmo assim, a avalia??o da reprodutibilidade dos resultados dos kits com os soros referenciados mostrou elevada correla??o.

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<p>The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E₂ (PGE₂) formation may impair islet function.</p><p>In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. <i>In vitro</i>, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. <i>In vivo</i>, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin.</p><p>Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The <i>in vitro</i> data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE₂ production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.</p>