Made available in DSpace on 2016-12-08T15:59:05Z (GMT). No. of bitstreams: 1 TESE DAIANA BUNDCHEN.pdf: 729468 bytes, checksum: baf7cf5ef2b6e7a70f9f8546f04ecd74 (MD5) Previous issue date: 2013-12-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The effects of training on the metabolic pathways in chronic heart failure (CHF), specially the intravascular lipid metabolism are largely unexplored and deserve further investigation. Objectives: To analyze the effects of short-term exercise training on the plasma lipids, on lipid transfer to HDL and cytokine levels in CHF patients.Method: We compared plasma lipids, in vitro transfer of four lipids from a radioactively labeled lipid donor nanoemulsion to HDL and cytokine levels (TNF-&#945; and IL-6) in CHF patients, class II or III (NYHA) with (n=9) or without (n=10) statin treatment before and after 12 weeks of exercise training. The aerobic exercise was performed three times a week, during 40 minutes, with heart rate intensity between L1 and L2 of the cardiopulmonary test. Results: Exercise training reduced the LDL-C in the statin-treated group (-16%; p=0.03) and increased HLD-C (+24%; p=0.05) in without statin group. Exercise training elicited a significant increase the transfer of triglycerides from the donor nanoemulsion to HDL in statin-treated group (p=0.03). The transfers of the three other lipids (unesterified and esterified cholesterol and phospolipids) were unchanged. In the without statin-treatment the transfers of all four lipids were not change by training. For cytokines, in those without statintreatment, the TNF-&#945; reduced 28% (p<0,01), while in the group with statin reduced 12% (p=0,07). The seric concentration of the IL-6 reduced 41% (p<0,001) in the group without simvastatin and 45% (p<0,001) in the statin-treatment group. In both groups no correlation was observed between HDL and proinflammatory cytokines. Conclusion: In CHF patients, the short-term training , increased HLD-C in the statin-treated group, showing no functional change in this lipoprotein particles; reduced the LDL-C and increased significantly the lipis transfer only triglycerides in the statin-treated group. The decrease in cytokine levels for both groups indicated the early benefits of exercise. / Os efeitos do treinamento físico sobre as vias metabólicas na insuficiência cardíaca (IC), especialmente o metabolismo lipídico intravascular e sua associação com as citocinas pró-inflamatórias, são em grande parte inexplorados e merecem uma investigação mais aprofundada. Objetivos: analisar os efeitos de um curto período de treinamento físico nos lipídeos plasmáticos, na transferência de lípides para HDL e nos níveis de citocinas pró-inflamatórias em pacientes com IC. Métodos: antes e após 12 semanas de treinamento físico foram avaliados 19 homens com IC, classe funcional II ou III (NYHA), sendo nove sujeitos em uso de sinvastatina e 10 sem uso de sinvastatina. Foi comparado o perfil lipídico, transferência in vitro dos quatro lípides de uma nanoemulsão doadora de lípides marcada radioativamente para a HDL e níveis de citocinas pró-inflamatórias (TNF-&#945; e IL-6). O exercício aeróbio foi realizado três vezes por semana, durante 40 minutos, com intensidade na frequência cardíaca correspondente a faixa entre o primeiro e segundo ponto dos limiares ventilatórios, determinados pelo teste de esforço cardiopulmonar. Resultados: Foi observada redução do LDL-Colesterol (-16%; p=0,03) no grupo tratado com sinvastatina e aumento de HDL-Colesterol (+24%; p=0,05) no grupo sem sinvastatina. No grupo com sinvastatina ocorreu um aumento significativo da transferência de triglicérides da nanoemulsão doadora de lípides para HDL (p=0,03) enquanto a transferência dos outros três lípides (fosfolípides, colesterol livre e colesterol esterificado) não se modificou. No grupo sem sinvastatina a transferência de todos os quatro lípides não se alterou. No grupo sem uso de sinvastatina o TNF-&#945; reduziu 28% (p<0,01), enquanto no grupo com estatina reduziu 12% (p=0,07). A concentração sérica de IL-6 reduziu 41% (p<0,001) no grupo sem sinvastatina e 45% (p<0,001) no grupo em uso de sinvastatina. Em ambos os grupos não foi observado correlação entre HDL e citocinas pró-inflamatórias. Conclusão: Em pacientes com IC, o curto período de treinamento físico aumentou HDL-Colesterol no grupo sem sinvastatina, não apresentando mudança funcional das partículas desta lipoproteína; reduziu o LDL-Colesterol e aumentou significativamente a transferência apenas dos triglicérides no grupo em tratamento com sinvastatina. A diminuição dos níveis de citocinas para ambos os grupos indicou os benefícios precoces do exercício físico.

Made available in DSpace on 2014-12-17T14:03:37Z (GMT). No. of bitstreams: 1 TicianaMLA_TESE.pdf: 1476361 bytes, checksum: 7074b01e6eb22db7b4cacc005194c334 (MD5) Previous issue date: 2014-04-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Proteinases are enzymes distributed widely founded in several organisms and perform many different functions, from maintaining homeostasis to the worsening of some diseases such as cancer, autoimmune diseases and infections. The proteins responsible of controlling the action of these enzymes are the inhibitors, that are classified based on their target proteases and are founded since simple organisms, such as bacteria, to higher organisms, such as larger plants and mammals. Plant proteinase inhibitors act by reducing or inactivating the activity of target proteases, thus, these proteins have been studied as potential tools in the treatment of diseases related to protease activities. In this context, an inhibitor of chymotrypsin from Erythrina velutina, called EvCI was previously purified and it was observed that this protein plays in vitro anticoagulant activity and anti-inflammatory activity in in vivo model. Aiming to reduce the environmental impact caused by the purification EvCI in high amounts and to facilitate the process of obtaining this protein, the recombinant chymotrypsin inhibitor from Eryhrina velutina was produced after cloning and expression in Escherichia coli. The bacteria were grown in LB medium and after induction of the expression this material was subjected to procedures for cell lysis and the product was applied on Nickel-affinity column. The proteins adsorbed were digested by thrombin and applied on Chymotrypsin-Sepharose affinity column, obtaining the purified inhibitor, named recEvCI. After electrophoresis, the recombinant inhibitor showed an approximately molecular mass of 17 kDa, and reduced the chymotrypsin and elastase activities in vitro. The recombinant inhibitor was sequenced and was found similar amino acids residues when compared to other inhibitors deposited in the database, with some modifications. recEvCI showed high stability under pH variations and reducing conditions, maintaining its activity around 80%. This protein increased the blood coagulation time in vitro by acting on the intrinsic pathway and did not show cytotoxicity against strains of mouse 3T3 fibroblasts and RAW 264.7 macrophages. recEvCI showed microbicide activity related to release of nitric oxide and consequently the activation of macrophages, futhermore having proinflammatory effects assessed by increased release of TNF-&#945;. These results indicate that recEvCI can be biotechnologically used as a new tool in the control of coagulation-related diseases as well as can be an activating agent of the immune system in immunosuppressed individuals / Proteinases s?o enzimas amplamente distribu?das em diferentes organismos e que desempenham as mais diversas fun??es, desde a manuten??o da homeostase at? o agravamento de algumas doen?as como c?ncer, doen?as autoimunes e infec??es. As prote?nas respons?veis pelo controle e atua??o destas enzimas s?o os inibidores, que s?o classificados de acordo com suas proteases alvo e s?o encontrados desde organismos mais simples, como bact?rias, aos mais complexos, como plantas de grande porte e mam?feros. Inibidores de proteinases de plantas agem reduzindo ou inativando a atividade de enzimas alvo, dessa forma, estas prote?nas v?m sendo estudadas como poss?veis ferramentas no tratamento de doen?as relacionadas ?s atividades prote?sicas. Neste contexto, um inibidor de quimotripsina de Erythrina velutina, denominado EvCI, foi previamente purificado e foi observado que esta prote?na desempenha atividades anticoagulante in vitro a anti-inflamat?ria em modelo in vivo. Buscando reduzir o impacto ecol?gico causado pela purifica??o de EvCI em grandes quantidades e facilitar o processo de obten??o desta prote?na, o inibidor de quimotripsina recombinante de Erythrina velutina foi produzido ap?s clonagem e express?o em c?lulas de Escherichia coli. As bact?rias foram crescidas em meio LB e ap?s indu??o da express?o este material foi submetido a procedimentos de lise celular e o produto foi aplicado em uma coluna de afinidade de N?quel. As prote?nas ligadas ? coluna foram digeridas por trombina, aplicadas em uma coluna de afinidade de Quimotripsina-Sepharose obtendo-se o inibidor purificado, denominado recEvCI. Ap?s eletroforese, o inibidor recombinante apresentou uma massa molecular de, aproximadamente, 17 kDA e reduziu a atividade de quimotripsina e elastase in vitro. O inibidor recombinante foi sequenciado e foi detectada a presen?a de amino?cidos iguais a outros inibidores depositados em banco de dados, com algumas modifica??es. recEvCI demonstrou alta estabilidade quando submetido a varia??es de pH e sob condi??es redutoras, mantendo sua atividade inibit?ria em torno de 80%. Esta prote?na aumentou o tempo de coagula??o sangu?nea in vitro por atua??o sobre a via intr?nseca e n?o demostrou citotoxicidade contra as linhagens de fibroblasto de camundongo 3T3 e de macr?fagos RAW 264.7. recEvCI apresentou atividade microbicida relacionada ? libera??o de ?xido n?trico e consequente ativa??o de macr?fagos, al?m de possuir efeito pr?-inflamat?rio avaliado pelo aumento da libera??o de TNF-&#945;. Estes resultados indicam que recEvCI pode ser utilizado biotecnologicamente como uma nova ferramenta no controle de doen?as relacionadas ? coagula??o assim como pode vir a ser um agente ativador do sistema imune em indiv?duos imunossuprimidos

Chickens in the poultry industry are reared under intensive conditions where they are often exposed to opportunistic pathogens. Escherichia coli strain O78 is responsible for about half of the cases of avian colisepticemia. Potential therapeutic treatments have been proposed to inhibit the hydrolases that controls the endogenous levels of the endocannabinoid, 2-arachidonoylglycerol (2-AG). 2-AG is the full agonist at the CB2 receptors of the endocannabinoid system expressed among leukocytes and it plays a role in mediating the activation of phagocytic macrophages. It is speculated that elevating 2- AG levels could increase macrophage cytokines and promote the recruitment of immune cells at the infected tissues. The purpose of this study was to investigate the immunomodulating effect of the 2-AG hydrolase inhibitor, JZL184 in chickens. The treatments could potentially up-regulate the innate immune responses in chickens during an E. coli infection by conveying a message from the endocannabinoid system to the immune system.

No description available.

La mucoviscidose est la maladie génétique récessive la plus fréquente dans les populations caucasiennes. Elle est dûe à des mutations dans le gène CFTR qui code pour une protéine qui a une fonction canal chlorure et qui est exprimée au pôle apical des épithéliums sécrétoires. La mutation la plus fréquente F508del altère le repliement de la protéine, induisant sa dégradation précoce par le protéasome et son absence à la membrane plasmique. La recherche fondamentale se focalise sur la protéine mutée et cherche à découvrir des molécules capables d’adresser celle-ci au pôle apical des cellules épithéliales où elle pourra remplir sa fonction de canal chlorure. Au sein du laboratoire, nous nous intéressons aux interactions de la protéine CFTR/ F508del-CFTR avec d’autres protéines. Nous avons déjà montré l’existence d’une interaction non voulue entre F508del-CFTR et un filament intermédiaire, la cytokératine 8. En combinant une approche in-silico et du criblage haut débit, nous avons identifié des molécules capables d’interrompre ces interactions. Des tests fonctionnels sur des lignées cellulaires ainsi que sur des modèles murins ont montré une restauration de la fonction canal chlorure après traitements avec ces molécules. Au cours de ma thèse, je me suis intéressée à l’une d’entre elles qui est la molécule « c407 ». L’objectif de ma thèse était de comprendre les mécanismes d’action de cette molécule. J’ai également évalué l’efficacité des traitements actuels dans le contexte des allèles complexes de F508del-CFTR. Dans une seconde partie de ma thèse, j’ai étudié l’effet d’une cytokine (TNFα) sur la protéine mutée F508del-CFTR. De façon inattendue, j’ai observé que le TNFα, à des concentrations physiologiques, corrige le défaut de routage de la protéine F508del-CFTR. Cette observation pourrait expliquer une fonction résiduelle de la F508del-CFTR chez certains patients atteints de mucoviscidose. En conclusion, mes travaux ont permis de préciser les mécanismes d’action d’un correcteur et de découvrir un effet inattendu d’une cytokine pro-inflammatoire. Ces travaux permettent de relier la correction d’un défaut de routage au processus inflammatoire ouvrant ainsi un nouveau champ d’investigation. / Cystic fibrosis is due to the loss of epithelial chloride transport caused by mutations in the CFTR gene, the most frequent mutation being F508del. One of the strategies developed to find new treatment for Cystic fibrosis (CF) is to discover compounds that correct the trafficking of F508del-CFTR to the plasma membrane. Using hypothesis-driven approach and combining modeling of NBD1, molecular docking and functional assays, we identified 4 compounds that correct F508del-CFTR function in cells (including human primary bronchial cells in culture) and F508del mice. New correctors probably act by interrupting the interaction between F508del-CFTR with keratin 8 (Odolczyk et al EMBO Mol Med 2013). During my PhD, I focused on one of those molecules, the "c407" molecule. The aim of my thesis was to investigate the mechanisms of action of this molecule. I have also evaluated the effectiveness of current treatments in the context of complex alleles F508del-CFTR. In the second part of my thesis, I studied the effect of a cytokine (TNFα) on the protein F508del-CFTR. Unexpectedly, I observed that the TNFα at physiological concentrations, corrects the trafficking of F508del-CFTR protein. This observation could explain a residual function of F508del-CFTR in some CF patients. In conclusion, my thesis helped to clarify the mechanisms of action of new correctors of F508del-CFTR.

La mucoviscidose est la maladie génétique récessive la plus fréquente dans les populations caucasiennes. Elle est dûe à des mutations dans le gène CFTR qui code pour une protéine qui a une fonction canal chlorure et qui est exprimée au pôle apical des épithéliums sécrétoires. La mutation la plus fréquente F508del altère le repliement de la protéine, induisant sa dégradation précoce par le protéasome et son absence à la membrane plasmique. La recherche fondamentale se focalise sur la protéine mutée et cherche à découvrir des molécules capables d’adresser celle-ci au pôle apical des cellules épithéliales où elle pourra remplir sa fonction de canal chlorure. Au sein du laboratoire, nous nous intéressons aux interactions de la protéine CFTR/ F508del-CFTR avec d’autres protéines. Nous avons déjà montré l’existence d’une interaction non voulue entre F508del-CFTR et un filament intermédiaire, la cytokératine 8. En combinant une approche in-silico et du criblage haut débit, nous avons identifié des molécules capables d’interrompre ces interactions. Des tests fonctionnels sur des lignées cellulaires ainsi que sur des modèles murins ont montré une restauration de la fonction canal chlorure après traitements avec ces molécules. Au cours de ma thèse, je me suis intéressée à l’une d’entre elles qui est la molécule « c407 ». L’objectif de ma thèse était de comprendre les mécanismes d’action de cette molécule. J’ai également évalué l’efficacité des traitements actuels dans le contexte des allèles complexes de F508del-CFTR. Dans une seconde partie de ma thèse, j’ai étudié l’effet d’une cytokine (TNFα) sur la protéine mutée F508del-CFTR. De façon inattendue, j’ai observé que le TNFα, à des concentrations physiologiques, corrige le défaut de routage de la protéine F508del-CFTR. Cette observation pourrait expliquer une fonction résiduelle de la F508del-CFTR chez certains patients atteints de mucoviscidose. En conclusion, mes travaux ont permis de préciser les mécanismes d’action d’un correcteur et de découvrir un effet inattendu d’une cytokine pro-inflammatoire. Ces travaux permettent de relier la correction d’un défaut de routage au processus inflammatoire ouvrant ainsi un nouveau champ d’investigation. / Cystic fibrosis is due to the loss of epithelial chloride transport caused by mutations in the CFTR gene, the most frequent mutation being F508del. One of the strategies developed to find new treatment for Cystic fibrosis (CF) is to discover compounds that correct the trafficking of F508del-CFTR to the plasma membrane. Using hypothesis-driven approach and combining modeling of NBD1, molecular docking and functional assays, we identified 4 compounds that correct F508del-CFTR function in cells (including human primary bronchial cells in culture) and F508del mice. New correctors probably act by interrupting the interaction between F508del-CFTR with keratin 8 (Odolczyk et al EMBO Mol Med 2013). During my PhD, I focused on one of those molecules, the "c407" molecule. The aim of my thesis was to investigate the mechanisms of action of this molecule. I have also evaluated the effectiveness of current treatments in the context of complex alleles F508del-CFTR. In the second part of my thesis, I studied the effect of a cytokine (TNFα) on the protein F508del-CFTR. Unexpectedly, I observed that the TNFα at physiological concentrations, corrects the trafficking of F508del-CFTR protein. This observation could explain a residual function of F508del-CFTR in some CF patients. In conclusion, my thesis helped to clarify the mechanisms of action of new correctors of F508del-CFTR.

L’athérosclérose est une maladie inflammatoire, caractérisée par une accumulation lipidique et cellulaire, dont le macrophage est l’acteur principal. Dans l’athérosclérose, le macrophage sécrète des cytokines qui favorisent le chimiotactisme et donc la migration et l’internalisation des cellules immunitaires, accentuant le processus pro-inflammatoire et accélérant l’évolution des plaques athéromateuses et leur instabilité jusqu’à éventuellement la rupture et/ou la formation de thrombus. Il est donc important de développer une technique d’imagerie cellulaire permettant de visualiser les macrophages et leur migration dans divers contextes pathologiques pour mieux comprendre leur implication, permettre le suivi thérapeutique et probablement leur utilisation dans la thérapie vectorisée. Le but de ce travail vise principalement à marquer aux nanoparticules de fer des macrophages activés de manière classiques (macrophages dits « M1 ») pour l’évaluation du statut inflammatoire dans un premier temps au sein des plaques d’athérome, et la visualisation de la dynamique de leur recrutement par IRM. Dans un deuxième temps, la méthode est exploitée pour montrer in vivo l’effet d’un traitement permettant de diminuer l’inflammation dans la plaque chez des souris invalidées pour le gène de l’apolipoprotéine E (ApoE-/-). Enfin, l’utilisation du protocole de marquage des macrophages M1 et leur suivi par IRM sont appliqués au diagnostic par imagerie d’une inflammation transitoire et circonscrite au poumon, induite par les lipopolysaccharides (LPS). En conclusion, le marquage des monocytes dirigés vers la voie M1 pour leur détection par IRM représente une méthode solide pour étudier différents phénomènes immunologiques en particulier le processus inflammatoire, et peut être un outil ou une approche de vectorisation permettant le suivi thérapeutique, cellulaire ou génique / Atherosclerosis is an inflammatory disease characterized by vessel wall lipids and cells accumulation, for which the macrophage acts as a central actor. In atherosclerosis the macrophage secretes cytokines that promote chemotaxis and thus migration and internalization of immune cells. This phenomenon emphasizes pro-inflammatory processes and accelerates the development of atherosclerotic plaque unstability to potentially its rupture with or without thrombus formation. It is therefore important to develop a technique for cell imaging to visualize macrophages and their migration in various pathological contexts to better understand their involvement, to allow therapeutic drug monitoring and probably their use in vectorized therapy. The aim of this work is mainly to label activated macrophages in classical way (macrophages called "M1") by iron nanoparticles, first to evaluate the inflammatory status within atherosclerotic plaques, and visualize their dynamic recruitment by MRI. In a second step, the method is used to show in vivo the effect of a treatment to reduce inflammation in the plaque, in mice invalidated for the gene for apolipoprotein E (ApoE-/ -). Finally, the protocol for M1 macrophages labelling and MRI tracking, was used for diagnostic imaging of a transient inflammation confined to the lung induced by lipopolysaccharide (LPS). In conclusion, the labelling of monocytes headed towards M1 polarization for their detection by MRI is a robust method to study various immunological phenomena in particular the inflammatory process, and can be an approach to vectorization and monitoring for gene or cell therapy

La mucoviscidose est la maladie génétique récessive la plus fréquente dans les populations caucasiennes. Elle est dûe à des mutations dans le gène CFTR qui code pour une protéine qui a une fonction canal chlorure et qui est exprimée au pôle apical des épithéliums sécrétoires. La mutation la plus fréquente F508del altère le repliement de la protéine, induisant sa dégradation précoce par le protéasome et son absence à la membrane plasmique. La recherche fondamentale se focalise sur la protéine mutée et cherche à découvrir des molécules capables d’adresser celle-ci au pôle apical des cellules épithéliales où elle pourra remplir sa fonction de canal chlorure. Au sein du laboratoire, nous nous intéressons aux interactions de la protéine CFTR/ F508del-CFTR avec d’autres protéines. Nous avons déjà montré l’existence d’une interaction non voulue entre F508del-CFTR et un filament intermédiaire, la cytokératine 8. En combinant une approche in-silico et du criblage haut débit, nous avons identifié des molécules capables d’interrompre ces interactions. Des tests fonctionnels sur des lignées cellulaires ainsi que sur des modèles murins ont montré une restauration de la fonction canal chlorure après traitements avec ces molécules. Au cours de ma thèse, je me suis intéressée à l’une d’entre elles qui est la molécule « c407 ». L’objectif de ma thèse était de comprendre les mécanismes d’action de cette molécule. J’ai également évalué l’efficacité des traitements actuels dans le contexte des allèles complexes de F508del-CFTR. Dans une seconde partie de ma thèse, j’ai étudié l’effet d’une cytokine (TNFα) sur la protéine mutée F508del-CFTR. De façon inattendue, j’ai observé que le TNFα, à des concentrations physiologiques, corrige le défaut de routage de la protéine F508del-CFTR. Cette observation pourrait expliquer une fonction résiduelle de la F508del-CFTR chez certains patients atteints de mucoviscidose. En conclusion, mes travaux ont permis de préciser les mécanismes d’action d’un correcteur et de découvrir un effet inattendu d’une cytokine pro-inflammatoire. Ces travaux permettent de relier la correction d’un défaut de routage au processus inflammatoire ouvrant ainsi un nouveau champ d’investigation. / Cystic fibrosis is due to the loss of epithelial chloride transport caused by mutations in the CFTR gene, the most frequent mutation being F508del. One of the strategies developed to find new treatment for Cystic fibrosis (CF) is to discover compounds that correct the trafficking of F508del-CFTR to the plasma membrane. Using hypothesis-driven approach and combining modeling of NBD1, molecular docking and functional assays, we identified 4 compounds that correct F508del-CFTR function in cells (including human primary bronchial cells in culture) and F508del mice. New correctors probably act by interrupting the interaction between F508del-CFTR with keratin 8 (Odolczyk et al EMBO Mol Med 2013). During my PhD, I focused on one of those molecules, the "c407" molecule. The aim of my thesis was to investigate the mechanisms of action of this molecule. I have also evaluated the effectiveness of current treatments in the context of complex alleles F508del-CFTR. In the second part of my thesis, I studied the effect of a cytokine (TNFα) on the protein F508del-CFTR. Unexpectedly, I observed that the TNFα at physiological concentrations, corrects the trafficking of F508del-CFTR protein. This observation could explain a residual function of F508del-CFTR in some CF patients. In conclusion, my thesis helped to clarify the mechanisms of action of new correctors of F508del-CFTR.

Nos últimos anos, inúmeros avanços têm sido alcançados no que diz respeito aos mecanismos moleculares que participam na indução e manutenção da dor crônica, incluindo ativação glial. Já foi demonstrado que alguns receptores de reconhecimento padrão (PRRs), como os receptores do tipo Toll (TLRs) participam desse processo e, que em modelos de inflamação/infecção do Sistema Nervoso Central, os TLRs e os receptores do tipo NOD (NLRs) cooperam na ativação das células da glia, o que nos levou a hipotetizar que os receptores NOD1 e NOD2 também possam desempenhar um papel importante no processo de dor crônica. O NOD2 é responsável pela detecção intracelular do muramil dipeptídeo (MDP) enquanto que NOD1 reconhece o ácido diaminopimélico (iE-DAP), ambos padrões moleculares associados a patógenos (PAMPs) encontrados no peptideoglicano de bactérias. Após o reconhecimento, NLRs recrutam diretamente RIPK2 (proteína 2 de interação com o receptor RICK), uma serina-treonina quinase importante na ativação do fator nuclear kB (NF-kB). Assim, o objetivo do presente estudo foi avaliar a participação de NOD1 e NOD2, via RIPK2, no desenvolvimento da hipersensibilidade mecânica neuropática focando principalmente nos mecanismos espinais envolvidos. Dessa maneira, foi observado que os animais NOD1-/-, NOD2-/- e RIPK2-/- apresentaram redução significativa da hipersensibilidade nociceptiva mecânica quando comparado aos animais selvagens após indução de neuropatia periférica pelo modelo experimental de lesão limitada do nervo isquiático (SNI, Spared Nerve Injury). Ao contrário, a hipersensibilidade inflamatória induzida pelo adjuvante completo de Freud (CFA) não foi reduzida nesses animais. A redução da dor neuropática em NOD1-/-, NOD2-/- e RIPK2-/- foi associada a uma diminuição da expressão de IBA-1, GFAP, IL-1, TNF- e P2X4 na medula espinal quando comparado ao grupo WT. In vitro, foi observado que culturas primárias de micróglia não induziram liberação de IL-1, TNF-, IL-6 em resposta ao MDP (10g/mL) e iE-DAP (100ng/mL). No entanto, quando o MDP foi administrado juntamente com uma baixa concentração de lipopolissacarídeo (LPS) (0,1ng/mL), apresentou uma forte produção destas citocinas. Além disso, também foi demonstrado que células periféricas que infiltram na medula espinal podem expressar NOD1 e NOD2 e portanto serem capazes de induzir hipersensibilidade mecânica e ativação microglial após a indução de neuropatia. Dessa maneira, os resultados sugerem que NOD1 e NOD2, via RIPK2, contribuem para a gênese da dor neuropática, possivelmente mediando a liberação de citocinas pró-nociceptivas e a ativação de células gliais. Além disso, os resultados apontam ação potencial de NOD2 com TLR4 no intuito de estimular a ativação glial. Estes mecanismos representam uma nova abordagem para elucidar os mecanismos envolvidos na fisiopatologia da dor crônica e um possível alvo para o desenvolvimento de drogas para o tratamento da dor neuropática. / In the last years, many advances have been made related to the molecular mechanisms involved in the induction and maintenance of chronic pain, including glial activation. It has been shown that some pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) are involved in this process, and that in inflammation/infection models of the CNS, the TLRs and Nod-like receptors (NLRs) cooperate in activation of glial cells, which led us to hypothesize that NOD1 and NOD2 receptors may also play an important role in chronic pain process. NOD2 are responsible by intracellular detection of muramyl dipeptide (MDP) and NOD1 detects meso-diaminopimelic acid (iE-DAP), pathogen-associated molecular patterns (PAMPs) found in the peptidoglycan from bacteria. Upon recognition, NLRs recruit directly RIPK2, an adaptor protein, important in NLRs-mediated NFB activation. In the present study, we aimed to evaluate the participation of NOD1 and NOD2, via RIPK2, in the development of neuropathic mechanical hypersensitivity focusing mainly on spinal mechanisms involved. The results demonstrate that NOD1-/-, NOD2-/-, RIPK2-/- showed a significant reduction in mechanical hypersensitivity when compared to WT mice, after submitted to an experimental model of neuropathic pain Spared Nerve Injury (SNI). Interestingly, CFA-induced chronic inflammatory hypersensitivity was not decreased in these mice. The reduction in neuropathic pain in NOD1-/-, NOD2-/- and RIPK2-/- mice was associated with a decrease in the expression of IBA-1, GFAP, IL-1, TNF- and P2X4 in spinal cord when compared with WT. In vitro, it was observed that primary cultures of microglia did not produce IL-1, TNF-, IL-6 in response to MDP (3g/mL) or iE-DAP (100ng/mL). However, MDP, together with an ineffective concentration of LPS (0.1ng/mL), produced a robust production of these cytokines. Moreover, it was also demonstrated that peripheral cells infiltrating the spinal cord could express NOD1 and NOD2 and thus, be able to induce mechanical hypersensitivity and microglial activation after induction of peripheral neuropathy. The results suggest that NOD1 and NOD2, via RIPK2, contribute to the genesis of neuropathic pain, possibly by mediating the release of pronociceptive cytokines and increased glial cells activation. Moreover, the results indicate potential action of NOD2 with TLR4 in attempt to stimulate glial cells activation. These mechanisms represent a novel approach for elucidating the pathophysiology of chronic pain, and a target for the development of drugs for the treatment of neuropathic pain.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Lectins are proteins that bind carbohydrates specifically and reversibly. The legume lectins represent the best studied and established group, from the point of view of physical-chemical and biological and structural, where a well-studied group of these proteins involves lectin obtained from members of the subtribe Diocleinae. Lectins Diocleinae have a high degree of structural similarity, but the same was not true regarding biological activities. This variability, as a rule, is in the detail that can be analyzed in structures based studies. In this context, multiple cardiovascular disease and inflammatory processes, particularly chronic and recurrent nature, arouse the interest of the scientific community because they require a wider range of drugs for therapeutic alternatives. In this sense, they become important research seeking new compounds with vasorelaxant and anti-inflammatory action. The present paper describes the partial structural and biological characterization of two lectins present in Canavalia virosa and Canavalia oxyphylla seeds, belonging to the family Leguminosae, subfamily Papilionoideae, Phaseoleae tribe, subtribe Diocleinae. The lectin from Canavalia virosa seeds (ConV) was purified in a single step by affinity chromatography on mannose-Sepharose 4B column. ConV strongly agglutinated rabbit erythrocytes and was inhibited by monosaccharide (D-mannose, D-glucose and &#945;-methyl-D-manosÃdeo) and glycoproteins (fetuin and ovalbumin). SDS-PAGE revealed three bands, corresponding to three chains (&#945;, &#946;, &#947; and) confirmed by ESI mass spectrometry with masses of 25.480  2 Da, 12.864  1 Da and 12,633  1 Da, respectively. The hemagglutination activity of ConV is great in pH 7.0 to 9.0, stable at a temperature of 80 C, and is not affected by EDTA. ConV showed no toxicity against Artemia sp. and relaxed the endothelized rat aorta, with the participation of the lectin domain. In our tests, the lectin immobilized on CNBr-Sepharose was able to bind 0.8 mg of ovalbumin by chromatography, allowing the use of ConV as a tool to capture and purification of glycoproteins. Moreover, the lectin from Canavalia oxyphylla (CoxyL) was purified in a single step by affinity chromatography on Sephadex G-50 column. SDS-PAGE showed that pure lectin consists of a major band of 30 kDa (&#945; chain) and two minor components (&#946; and &#947; chains) of 16 and 13 kDa, respectively. These data were further confirmed by mass spectrometry via electrospray ionization. Compared to the average molecular weight of the &#945; chains, the partial amino acid sequence obtained corresponds to about 45% of the total sequence CoxyL. CoxyL showed hemagglutinating activity was specifically inhibited by monosaccharide (D-glucose, D-mannose and methyl-&#945;-D-manosÃdeo) and glycoproteins (fetuin and ovalbumin). Furthermore, CoxyL proved to be heat stable at 60 C, and its activity is optimal at pH 7.0. CoxyL caused toxicity in Artemia sp. and induced paw edema in rats. / Lectinas sÃo proteÃnas que se ligam a carboidratos de forma especÃfica e reversÃvel. As lectinas de leguminosas representam o grupo mais bem estudado e estabelecido, tanto do ponto de vista de caracterizaÃÃo fÃsico-quÃmica e biolÃgica como estrutural, onde um grupo bem estudado destas proteÃnas envolve lectinas obtidas de membros da subtribo Diocleinae. As lectinas de Diocleinae apresentam um alto grau de similaridade estrutural, porÃm o mesmo nÃo se observa quanto Ãs atividades biolÃgicas. Esta variabilidade, via de regra, està em detalhes que podem ser analisados em estudos baseados em estruturas. Neste contexto, mÃltiplos processos patolÃgicos cardiovasculares e inflamatÃrios, principalmente de natureza crÃnica e recorrente, despertam o interesse da comunidade cientÃfica por requererem uma maior variedade de fÃrmacos para alternativas terapÃuticas. Neste sentido, tornam-se importantes pesquisas que busquem novos compostos, com aÃÃo vasorrelaxante e anti-inflamatÃria. Assim, o presente trabalho descreve a caracterizaÃÃo estrutural parcial e biolÃgica de duas lectinas presentes em sementes de Canavalia virosa e Canavalia oxyphylla, pertencentes à famÃlia Leguminosae, subfamÃlia Papilionoideae, tribo Phaseoleae, subtribo Diocleinae. A lectina de sementes de Canavalia virosa (ConV) foi purificada em uma Ãnica etapa atravÃs de cromatografia de afinidade em coluna Sepharose-mannose 4B. ConV aglutinou fortemente eritrÃcitos de coelho e foi inibida por monossacarÃdeos (D-manose, D-glicose e &#945;-metil-D-manosÃdeo) e glicoproteÃnas (ovalbumina e fetuÃna). SDS-PAGE revelou trÃs bandas, correspondentes a trÃs cadeias (&#945;, &#946;, e &#947;) confirmadas por espectrometria de massas ESI com massas de 25,480Â2 Da, 12,864Â1 Da e 12,633Â1 Da, respectivamente. A atividade hemaglutinante da ConV à Ãtima nos pH 7.0 a 9.0, estÃvel a uma temperatura de 80 ÂC, e nÃo à afetada pelo EDTA. ConV nÃo demonstrou toxicidade contra nÃuplios de Artemia sp. e relaxou a aorta endotelizada de ratos, com a participaÃÃo do domÃnio da lectina. Em nossos testes, a lectina imobilizada em CNBr-Sepharose foi capaz de se ligar a 0,8 mg de ovalbumina por cromatografia, permitindo o uso de ConV como uma ferramenta para a captura e purificaÃÃo de glicoproteÃnas. Por outro lado, a lectina de Canavalia oxyphylla (CoxyL) foi purificada em um Ãnico passo atravÃs de cromatografia de afinidade em coluna Sephadex G-50.SDS-PAGE mostrou que a lectina pura consiste de uma principal banda de 30 kDa (cadeia &#945;) e dois componentes menores (cadeias &#946; e &#947;) de 16 e 13 kDa, respectivamente. Estes dados foram adicionalmente confirmados por espectrometria de massas por ionizaÃÃo por eletropulverizaÃÃo. Em comparaÃÃo com a massa molecular mÃdia das cadeias &#945;, a sequÃncia parcial de aminoÃcidos obtida corresponde a aproximadamente 45% da sequÃncia total de CoxyL. CoxyL apresentou atividade hemaglutinante que foi especificamente inibida por monossacarÃdeos (D-glicose, D-manose, e &#945;-metil-D-manosÃdeo) e glicoproteÃnas (ovalbumina e fetuÃna). AlÃm disso, CoxyL mostrou ser termoestÃvel a 60 C, e sua atividade à Ãtima no pH 7,0. CoxyL causou toxicidade em nÃuplios de Artemia sp. e induziu edema de pata em ratos.