A terapia endodôntica visa o selamento biológico do complexo sistema apical, contribuindo para isso, as substâncias usadas no tratamento e a resposta imune do paciente. A LILT tem mostrado atividade antiinflamatória, favorecendo o processo reparacional. Sendo assim, este trabalho objetivou analisar o efeito da LILT na atividade secretória de macrófagos, previamente ativados por IFN-? e LPS de E.coli, e estimulados por substâncias liberadas de três tipos de cimentos endodônticos, um a base de óxido de zinco e eugenol, outro a base de hidróxido de cálcio e um terceiro resinoso. A citotoxicidade dessas substâncias foi avaliada usando a técnica de análise do MTT. Macrófagos ativados foram estimulados por essas substâncias ou não (controle) e então, irradiados ou não (controle) e a secreção de proteínas próinflamatórias (interleucina-1 b, fator de necrose tumoral-a e metaloproteinase da matriz-1) foram analisadas pelo teste ELISA. As irradiações foram realizadas com um laser GaAlAs (780 nm, 70 mW, ponta da fibra de 4 mm2, 1.67 seg, 3 J/cm2). Foram usadas duas aplicações de irradiação com intervalo de 6 h. Os dados obtidos foram tratados por Análise de Variância, quando de distribuição normal, ou teste de Friedman, quando de distribuição não normal, com nível de significância de 5 % (p = 0,05). A viabilidade dos controles e células tratados pelos cimentos endodônticos foi similar. Produção de IL-1 b e TNF-a foram observadas. Houve alta produção de MMP-1. Entretanto, sem diferenças estatísticas entre os grupos experimentais. Os grupos irradiados apresentaram resultados similares aos não irradiados. Substâncias liberadas pelos cimentos endodônticos testados não se mostraram citotóxicas nas condições deste experimento. Essas substâncias, bem como a LILT, no parâmetro utilizado, não causam alteração na atividade de secreção de MMP-1, IL-1 b e TNF-a por macrófagos ativados. / The endodontic therapy seeks the dental root canal biological sealing, depending on substances used in this process and patient’s defense immune factors. LILT has shown an anti-inflammatory activity, improving the periapical repair process. This in vitro study aimed to analyze the effect of LILT at the secretory activity of macrophages previously activated by interferon-gamma and lypopolisaccharide from E.coli, and stimulated by substances leached from three endodontic sealers (zinc oxide-eugenol based, resinous and calcium hydroxide-based). Cytotoxicity of these substances was assessed by the MTT test. Activated macrophages were stimulated by the substances or not (control) and then, irradiated or not (control) and the secretion of pro-inflammatory proteins (interleukin-1 b, tumor necrosis factor-a and matrix metalloproteinase-1) was analyzed by ELISA test. The LILT was performed using a GaAlAs laser (780 nm, 70 mW, focal spot of 4.0 mm2, 1.67 sec, 3 J/cm2). Two irradiations with 6 h-intervals were done. The data was compared by either ANOVA test or Friedman’s test. The cell viabilities of controls and cells treated by the sealers were similar. Production of IL -1 b and TNF-a were observed. There was a high production of MMP-1. However, statistical differences were not observed amongst the groups. The irradiated groups presented results similar to those of non irradiated groups. Substances leached from the endodontic sealers are non cytotoxic at these experiments conditions . These substances, as well as the LILT, at the parameter used, were not able to change the secretion of MMP-1, IL-1 b e TNF-a by activated macrophages.

O parasita Schistosoma mansoni é um dos principais causadores da esquistossomose, doença que acomete 200 milhões de indivíduos no mundo. O parasita possui um complexo ciclo de vida composto de seis estágios evolutivos em dois hospedeiros. S. mansoni possui um sofisticado sistema de interação com seus hospedeiros de modo a escapar da resposta imune e interagir com moléculas produzidas por eles. Alguns trabalhos na literatura descreveram o efeito do TNF-α humano sobre o processo de ovoposição do parasita adulto. Nosso trabalho teve por objetivo analisar o perfil de expressão gênica do S. mansoni ao longo de seus estágios de desenvolvimento, avaliar o efeito do TNF-α humano sobre o perfil de expressão gênica do parasita em dois estágios de desenvolvimento e descrever o gene homólogo ao receptor de TNF-α humano em S. mansoni. Para isso, duas plataformas de microarrays distintas foram utilizadas: uma composta por 4000 sondas de cDNA dupla fita produzida pelo nosso grupo de pesquisa e a outra, composta por 44000 sondas de oligonucleotídeos desenhadas pelo nosso grupo e produzida pela empresa Agilent Technologies. Com estas plataformas foi detectada a expressão de 5798 genes em vermes adultos, sendo que 156 genes apresentavam a transcrição nas fitas senso e anti-senso; 6 destes tiveram confirmadas a transcrição nas duas fitas por transcrição reversa fita específica seguida de PCR em Tempo Real. Adicionalmente foram identificados 229 genes diferencialmente expressos entre vermes adultos machos e fêmeas. A análise de expressão gênica entre 5 estágios de desenvolvimento do parasita mostrou dois tipos de conjuntos de dados: (i) 1423 genes diferencialmente expressos entre dois estágios subseqüentes de desenvolvimento e (ii) 342 genes com expressão enriquecida em um estágio exclusivamente. 68 destes são transcritos intrônicos que não possuem potencial codificador de proteinas. Um gene ortólogo ao receptor de TNF-α humano (SmTNFR) foi clonado e sequenciado. O transcrito SmTNFR possui 1967pb e codifica uma proteína de 599 aminoácidos. Outros 9 genes codificando elementos conservados da via de sinalização de TNF-α também foram identificados no conjunto de ESTs público, revelando uma via completa de sinalização de TNF-α no parasita. Por fim, identificamos com microarrays o conjunto de genes com expressão alterada pela adição de TNF-α humano em esquistossômulos e vermes adultos. 340 genes tiveram expressão alterada em vermes adultos utilizando a plataforma de cDNA. 411 genes sofreram mudanças de expressão em esquistossômulos e 1093 genes em vermes adultos detectados na plataforma de oligonucleotídeos. Este trabalho representa uma importante contribuição no entendimento da relação parasita-hospedeiro em nível molecular. / The parasite Schistosoma mansoni is one of major causative agents of schistosomiasis, a disease which affects 200 million people in the world. The parasite has a complex life cycle with six developmental stages in two hosts. S. mansoni has a sofisticated system of interaction with the hosts, permitting it to escape the immune response and to interact with molecules produced by the hosts. The effect of human TNF-α on adult parasite egg-laying has been described in the literature. The present work intended to analyse the gene expression profile of S. mansoni among its developmental stages, to evaluate the effect of human TNF-α on gene expression profile in two different developmental stages and to describe a homologous gene to human TNF-α receptor in S. mansoni. Two microarrays platfoms were used: one comprised by 4000 cDNA probes and printed by our research group and another, comprised by 44000 oligonucleotide probes designed by our group and printed by Agilent Technologies Company. With these platforms, we detected the expression of 5798 genes in adult worms, of which 156 showed transcription in sense and anti-sense strands; 6 of them had their expression levels confirmed by strand specific Real Time PCR. 229 genes were identified as differentially expressed between male and female adult worms. Gene expression analysis among 5 parasite developmental stages identified two data sets: (i) 1423 differentially expressed genes between two subsequent developmental stages and (ii) 342 expressed genes enriched in one exclusive stage. 68 of them are intronic transcripts with no protein-coding potential. An ortologue to human TNF-α receptor (SmTNFR) was cloned and sequenced. SmTNFR transcritpt has 1967bp and encodes a 599-amino acid protein. Other 9 genes encoding conserved elements of the TNF-α signaling pathway were identified among the public S. mansoni ESTs dataset, thus revealing a complete TNF-α signaling pathway in the parasite. Finally, we identified with microarrays the set of genes that have an altered gene expression upon exposure of schistosomula and adult worms to human TNF-α. 340 genes were identified with altered expression in adult worms using the cDNA platform. 411 genes changed their expression pattern in schistosomula and 1093 genes in adult worms using the oligonucleotide platform. This work represents an important contribution to the understanding of host-parasite interaction at the molecular level.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-14T18:03:22Z No. of bitstreams: 1 michelefernandesrodrigues.pdf: 2462897 bytes, checksum: fecbbd695cdb846a7633c19e7fab1160 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:08:37Z (GMT) No. of bitstreams: 1 michelefernandesrodrigues.pdf: 2462897 bytes, checksum: fecbbd695cdb846a7633c19e7fab1160 (MD5) / Made available in DSpace on 2016-10-22T13:08:37Z (GMT). No. of bitstreams: 1 michelefernandesrodrigues.pdf: 2462897 bytes, checksum: fecbbd695cdb846a7633c19e7fab1160 (MD5) Previous issue date: 2008-08-06 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Mycobacterium bovis é um membro do complexo Mycobacterium tuberculosis, um grupo de microorganismos com a capacidade de causar tuberculose em humanos. A característica marcante das micobactérias patogênicas é a sua habilidade para sobreviver e replicar dentro do fagossomo do macrófago. A apoptose dos macrófagos infectados pode ser uma alternativa de defesa do hospedeiro, capaz de eliminar o ambiente de suporte para o crescimento da micobactéria. No presente trabalho investigamos, in vivo, a influência da virulência e da carga bacteriana na ocorrência da apoptose de macrófagos durante a fase inicial da infecção pulmonar experimental por M. bovis. Camundongos BALB/c foram infectados intratraquealmente com alta ou baixa carga das cepas virulenta (ATCC 19274) e atenuada (BCG Moreau) de M. bovis. Após 3 e 7 dias de infecção, avaliamos a freqüência da apoptose dos macrófagos alveolares e sua correlação com o crescimento da micobactéria e com os níveis in situ das citocinas TNF-α, IL-10 e IL 12 e da proteína anti-apoptótica Bcl-2. Foi observado um aumento da apoptose de macrófagos após a infecção com ambas as cepas. Entretanto, a cepa virulenta induziu menos apoptose do que a cepa atenuada, sendo que, no 3o dia houve redução da apoptose na infecção com alta carga, enquanto que no 7o dia uma maior inibição ocorreu em baixa carga. Essa inibição da apoptose, promovida pela cepa virulenta, foi associada com o aumento da produção de IL-10, redução da produção de TNF-α e aumento da expressão de Bcl-2. Observamos ainda uma menor produção de IL-12 nos pontos de menor apoptose dos macrófagos o que reforça a hipótese de que a apoptose das células infectadas contribui para o desenvolvimento de imunidade específica contra o patógeno. Em conjunto esses dados sugerem que a cepa virulenta de M. bovis é capaz de modular a apoptose em um modo carga dependente de acordo com suas necessidades de crescimento intracelular e disseminação para células adjacentes. / Mycobacterium bovis is a member of the Mycobacterium tuberculosis complex, a group of organisms with the capacity to cause tuberculosis in humans. The hallmark of pathogenic mycobacteria is their ability to survive and replicate inside macrophage phagosome. The apoptosis of macrophages infected can be a host defense alternative capable of removing the environment supporting bacterial growth. In this work we investigate, in vivo, the influences of virulence and bacterial load on the apoptosis of macrophages during the initial phase of experimental pulmonary M. bovis infection. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) and attenuated (BCG Moreau) strains of M. bovis. The frequency of apoptosis and the growth of mycobacteria in macrophages from lung tissue, and the in situ levels of the cytokines TNF-α, IL-10 and IL-12 and of the anti apoptotic protein Bcl-2 were measured at day 3 and day 7 postinfection. An increase of macrophage apoptosis was observed after the infection with both strains, but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day of infection there was a reduction of apoptosis in the infection with high dose, whereas on the 7th day we found more inhibition in the lower dose. This inhibition of apoptosis was associated with the increased production of IL-10, reduced production of TNF-α and increases of Bcl-2. In addition, the production of IL-12 was reduced at the points of lowest apoptosis of macrophages, which suggests that the apoptosis of the infected cells contributes to the development of specific immune response against the pathogen. Our results indicate that the virulent strain of M. bovis is able to modulate apoptosis in a dose dependent fashion in accordance with its necessities for intracellular growth and dissemination to adjacent cells.

O tratamento radioterápico continua aperfeiçoando-se, porém ainda pode estar associado com toxicidade pulmonar. Objetivo: Estudar os efeitos locais e sistêmicos provocados pela radioterapia conformacional torácica adjuvante em mulheres portadoras de câncer de mama, sem fatores de riscos prévios para desenvolvimento de alterações pulmonares. Por meio da tomografia computadorizada de alta resolução; identificar as possíveis alterações radiológicas no parênquima pulmonar. Se presentes, correlacioná-las com parâmetros obtidos da exploração funcional dos pulmões, com os efeitos sistêmicos, pela dosagem de mediadores inflamatórios, IL-1, IL-6 e TNF- , e suas repercussões sobre a qualidade de vida. Material e Métodos: Em 25 pacientes saudáveis foram coletadas amostras de sangue para serem utilizadas apenas como referência de normalidade da IL-1, IL-6 e TNF- . Em decorrência dos rígidos critérios de inclusão e exclusão estabelecidos, das 157 pacientes entrevistadas apenas 24 foram selecionadas para o estudo. A avaliação funcional pulmonar foi abrangente e constou de: medida dos volumes e capacidade dos pulmões, sendo o volume residual obtido pelo método de diluição do hélio em circuito fechado; estudo dos fluxos expiratórios máximos (curva fluxo x volume e curva volume x tempo) e medida da capacidade de difusão pulmonar pela técnica de respiração única do CO. A tomografia computadorizada de alta resolução (16 detectores) foi realizada no pré-planejamento da radioterapia conformacional do tórax, esta com dose total de 45-50 Gy em 25 frações. Para as dosagens de citocinas plasmáticas foram empregadas as técnicas de imunoabsorção enzimáticas (ELISA). Na avaliação da qualidade de vida foi aplicado o questionário Saint George´s Respiratory Questionnary traduzido e adaptado culturalmente ao Brasil. Todos estes procedimentos foram obtidos na fase pré e repedidos 3 meses após a radioterapia. Os resultados das duas fases foram comparados utilizando-se a versão exata do teste Wilcoxon e o teste de Correlação de Spearman, nível de significância p 0,05. Resultados: Entre parâmetros funcionais houve queda significativa apenas na difusão e no fluxo expiratório a 50% da capacidade vital forçada. Mesmo comportamento observou-se para citocina IL-6, já que os mesmos encontravam-se aumentados pré-RT. Não ocorreram mudanças significativas em nenhum dos domínios do questionário de qualidade de vida. As alterações tomográficas ocorreram 60,87% das pacientes na fase pós-radioterapia, em sua maioria de graus leves a moderados e não correlacionaram-se com as alterações observadas em outros parâmetros estudados. Conclusão: O aumento observado na IL-6 durante a fase pré-RT, parece ser um bom índice preditivo de alteração pulmonar. A capacidade de difusão foi a alteração mais evidente e parece ser o índice que melhor reflete as alterações pulmonares que afetam essas pacientes. Diante das discretas alterações tomográficas e funcionais observadas após a RT, é provável que para a redução observada na DLCO concorra uma combinação de fatores ao nível da membrana alvéolo-capilar. No conjunto, as alterações induzidas pela radioterapia conformacional nas pacientes estudadas foram de pequena monta, insuficientes para influenciar aspectos funcionais do pulmão e a qualidade de vida. / Radiotherapy continues to improve itself, but it may still be associated with pulmonary toxicity. Objective: To study the effects caused by local and systemic adjuvant thoracic conformal radiotherapy in women with breast cancer, without risk factors prior to the development of pulmonary alterations. Through high-resolution computed tomography, to identify the possible radiological alterations in the lung parenchyma. If positive identification occurs, correlates it with pulmonary functional parameters, its systemic effects, the dosage of inflammatory mediators IL-1, IL-6 and TNF-, and their impact on quality of life. Material and Methods: Blood samples were collected from 25 healthy patients to be used as a normal reference for IL-1, IL-6 and TNF- mediators. Due to the established strict inclusion and exclusion criteria, only 24 from the initial 157 interviewed patients were selected for this study. The evaluation of pulmonary function was comprehensive and included: Lung volume and capacity measurement through closed loop helium residual volume dilution method;Peak expiratory flow study (flow curve x volume and volume vs. time curve) and CO diffusing capacity measurement through single breath technique. High-resolution computerized tomography (16 detectors) was performed in the pre-planning phase of thoracic conformal radiotherapy where 45-50 Gy total dose was applied in 25 fractions. For cytokines plasma measurement, the enzymatic immunosorbent techniques (ELISA) were used. For quality of life assessment, the Brazil´s validated Saint George\'s Respiratory Questionnary was used. All these procedures were applied in the pre-phase and repeated three months later after radiotherapy sessions. The results of the two phases were compared using the exact version of the Wilcoxon test and Spearman correlation test with p 0.05 significance level. Results: Among the functional parameters, there were a significant decrease in dissemination and expiratory flow at 50% of forced vital capacity. The same behavior was observed for cytokine IL-6, since they were already high at pre-RT. There were no significant changes in any of the aspects of quality of life questionnaire. The tomographic alterations occurred in 60,87% of patients in the post radiotherapy phase, mostly having low to moderate degree and not correlated with the observed changes in other parameters. Conclusion: The IL-6 increase in the pre-RT phase appears to be a reasonable predictive index of pulmonary alterations. The diffusing capacity alterations were the most evident and seem to be the index that best reflects the pulmonary alterations that affect these patients. Given the discrete tomographic and functional abnormalities observed after RT, it is likely that for the observed reduction in DLCO compete a combination of factors occurring at alveolar-capillary membrane level. Overall, the conformal radiotherapy induced changes in the studied patients were not expressive, insufficient to influence the pulmonary functional aspects and the quality of life of the patients.

O sistema NADPH oxidase humano é responsável pela geração de reativos intermediários do oxigênio e defeitos neste sistema resultam na Doença Granulomatosa Crônica (DGC). Nesta tese de doutorado, investigamos o efeito do IFN-g sobre eventos pós-transcricionais em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. Os dados obtidos sugerem que o uso do IFN-g in vitro interfere no processamento da mensagem causando aumento da expressão de transcritos do gene CYBB e NCF1 em células B-EBV de indivíduos sadios e pacientes DGC analisados. Observamos também que o IFN-g dimunui a expressão dos genes THOC4 NONO, SF3A1, SRRM1 e UPF3A e promove aumento de expressão de SRSF10, SNRPA1 e C2 em células B-EBV de paciente X-DGC secundária a defeitos de splicing. Identificamos que o IFN-g e o TNF-α aumentam a expressão das proteínas envolvidas no processo do splicing. Concluímos que o IFN-g aumenta a expressão de genes importantes para uma resposta eficiente do sistema imunológico, incluindo os do sistema NADPH oxidase, além de promover aumento da expressão de genes e de proteínas relacionados ao processo de splicing, que podem estar relacionados aos efeitos benéficos observados no uso do IFN-g em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. / The human phagocyte NADPH oxidase is responsible for the generation of reactive oxygen intermediates and defects in this system result in Chronic Granulomatous Disease (CGD). In this PhD Thesis, we investigated the effect of IFN-g on post-transcriptional events in normal individuals and patients with X-linked CGD, caused by splicing defects. The obtained data suggests that the use of IFN-g in vitro interferes in the message processing causing an increase of expression of CYBB and NCF1 gene transcripts in B-EBV cells of healthy individuals and analyzed CGD patients. We also observed that IFN-g decreases the expression of THOC4, NONO, SF3A1, SRRM1 and UPF3A, and increases the expression of SRSF10, SNRPA1 and C2 genes in cells from X-CGD patients, due to splicing defects. We identified that IFN-g and TNF-α induce expression of proteins involved in the splicing process. We conclude that IFN-g increases the expression of important genes for an effective immune response, including the NADPH oxidase system genes, and promotes augment of gene and protein expression related to the splicing process, which may be related to the beneficial effects related to the use of IFN-g in CGD patient caused by splicing defects.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-02-03T13:11:46Z No. of bitstreams: 1 caiocesardesouzaalves.pdf: 1872262 bytes, checksum: f03fadd9407262d508f6744a7f73820f (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-02-03T13:14:01Z (GMT) No. of bitstreams: 1 caiocesardesouzaalves.pdf: 1872262 bytes, checksum: f03fadd9407262d508f6744a7f73820f (MD5) / Made available in DSpace on 2017-02-03T13:14:01Z (GMT). No. of bitstreams: 1 caiocesardesouzaalves.pdf: 1872262 bytes, checksum: f03fadd9407262d508f6744a7f73820f (MD5) Previous issue date: 2007-08-30 / A Paracoccidioidomicose é uma micose sistêmica humana causada pelo fungo dimórfico, Paracoccidioides brasiliensis, que acomete, principalmente, indivíduos adultos do sexo masculino. O presente estudo propôs a padronização do modelo de infecção com P. brasiliensis pela via intratorácica em camundongos BALB/c. Este estudo foi monitorado pela detecção do P. brasiliensis através da contagem de unidades formadoras de colônia e pela presença de DNA do fungo nos pulmões dos animais infectados em diferentes pontos pósinfecção (2o, 7o, 15o, 30o, 45o, 60o e 90o dias) e a taxa de sobrevida dos camundongos. Além disto, foram avaliados alguns parâmetros imunológicos como a produção de óxido nítrico, TNF-alpha, IFN-gama, e IL-10 por células presentes no lavado intratorácico, contagem total e diferencial do número de células do lavado intratorácico, o estudo histopatológico dos pulmões e a detecção de anticorpos específicos anti-P. brasiliensis nos pulmões e no soro. Os resultados mostram um aumento gradual do número de colônias e de DNA de P. brasiliensis nos pulmões. Até o 15o dia após a infecção pode ser observado um aumento na produção de óxido nítrico e IFN-gama pelas células do lavado intratorácico, bem como um aumento do número total de células e da porcentagem de leucócitos mononucleares. A partir do 30o dia após a infecção observa-se um aumento de anticorpos específicos (IgG1) no soro e no pulmão, um aumento da produção de IL-10 e TNF-alpha pelas células do lavado intratorácico e conseqüente diminuição da produção de IFN-gama e óxido nítrico. Além disso, observa-se um aumento da porcentagem de células polimorfonucleares no lavado. No estudo histopatológico pode ser constatado um aumento gradual no tamanho e complexidade dos granulomas presentes nos cortes histológicos. Os camundongos utilizados no estudo de sobrevida começaram a morrer no 60o dia após a infecção. Os resultados mostram uma resposta inicial do hospedeiro com um perfil Th1 mudando durante a infecção para uma resposta Th2 que leva ao óbito dos camundongos BALB/c. / Paracoccidioidomycosis or South American blastomycosis, is a chronic granulomatous human male infection caused by the Paracoccidioides brasiliensis. The present study it considered the standardization of the model of infection with P. brasiliensis for the intrathoracic route in BALB/c mice. This study was monitored by the detection of the P. brasiliensis through the counting of colony forming units and by the presence of DNA of fungi in the lungs of the infected animals in different points (2, 7, 15, 30, 45, 60 and 90 days) and the survival rate of the mice. Moreover, some immune parameters had been evaluated as the nitric oxide production, TNF-alpha, IFN-gamma, and IL-10 for cells in the intrathoracic washed, total and distinguishing counting of cells of the intrathoracic washed, the lung histopathology and the detection of specific antibodies anti-P. brasiliensis in the lungs and serum. The results show a gradual increase of the number of colonies and P. brasiliensis DNA in the lungs. Until 15 day after the infection can be observed an increase in the nitric oxide production and IFN-gamma for the cells of the washed, as well as an increase of the total number of cells and the percentage of mononuclear. From 30 day after the infection observes an increase of specific antibodies (IgG1) in the serum and the lung, an increase of the production of IL-10 and TNF-alpha for the cells of the washed and consequent reduction of the IFN-gamma production and nitric oxide. Moreover, observed an increase of the percentage of cells polimorphonuclear in the washed. In the histopathology it can be evidenced a gradual increase in the size and complexity of granulomas in the cuts. The mice used in the survival study had started to die in 60 day after the infection. The results show an initial reply of the host with a Th1 profile moving during the infection for a Th2 reply that leads to the death of the BALB/c mice.

L’obésité est une maladie associée à des altérations métaboliques et inflammatoires et constitue un facteur de risque important de développer des comorbidités telles qu’un diabète de type 2. De plus, la prévalence de troubles de l’humeur et de la cognition est élevée chez les sujets obèses. Ces troubles neuropsychiatriques compliquent la prise en charge de l’obésité, contribuent à son aggravation et peuvent à terme favoriser le développement des comorbidités associées. Diminuer le développement de ces troubles pourrait donc permettre d’améliorer la santé et la qualité de vie des individus obèses. Dans ce contexte, l’objectif de cette thèse a été de comprendre les mécanismes neurobiologiques sous-tendant l’apparition de ces troubles neuropsychiatriques, dans le but d’identifier de nouvelles cibles potentielles pour le développement de stratégies préventives et/ou thérapeutiques visant à les réduire. Dans ce but, des modèles animaux d’obésité tels que la souris db/db, qui présentent une obésité sévère associée à des altérations caractéristiques du syndrome métabolique, peuvent être particulièrement utiles.[ ]Dans un premier temps, nous avons montré qu’une restriction calorique ou un traitement anti-inflammatoire diminuait les comportements de type anxieux chez la souris db/db. Cette amélioration était associée à une diminution sélective de l’expression génique du TNF-α dans l’hippocampe, ce qui suggère une contribution de cette cytokine pro-inflammatoire dans les comportements de type anxieux associés à l’obésité. Nous avons ensuite confirmé cette hypothèse en montrant que le blocage sélectif du TNF-α cérébral par administration i.c.v. d’étanercept (un récepteur leurre du TNF-α) diminuait les comportements de type anxieux chez les souris db/db. De façon intéressante, des mesures électrophysiologiques ont permis de montrer que cette amélioration des comportements émotionnels par l’étanercept impliquait la modulation de l’activité spontanée des neurones dans l’hippocampe ventral, région connue pour son rôle dans la régulation des émotions. Dans un second temps, nous avons essayé d’identifier de nouvelles stratégies préventives et/ou thérapeutiques pour améliorer l’humeur et la cognition chez les sujets obèses. Nous avons donc évalué l’effet d’un régime enrichi en acides gras polyinsaturés de type n-3 et antioxydants sur les altérations comportementales des souris db/db. En effet, ces nutriments sont connus pour moduler différents paramètres neurobiologiques impliqués dans la régulation du comportement. Nous avons montré que la consommation chronique de ce régime supprimait les déficits de mémoire spatiale dépendante de l’hippocampe chez les souris db/db dans le test de la piscine de Morris et que cette amélioration cognitive était probablement sous-tendue par des changements de plasticité neuronale. Enfin, nous avons évalué si des manipulations du microbiote intestinal pouvaient représenter une stratégie préventive et/ou thérapeutique pour améliorer les altérations neuropsychiatriques associées à l’obésité. Nous avons donc mesuré l’impact d’une manipulation du microbiote intestinal par des prébiotiques sur les altérations métaboliques et comportementales des souris db/db, mais également sur les systèmes biologiques et neurobiologiques auxquels elles sont associées. Nous avons montré que les améliorations métaboliques induites par l’administration de prébiotiques chez la souris db/db étaient accompagnées d’une diminution de l’inflammation périphérique et centrale. [ ] Pour conclure, ces expériences contribuent à montrer que l’inflammation, en particulier le TNF-α, pourrait être une cible importante pour le développement de traitements visant à améliorer les troubles de l’humeur chez les sujets obèses ; alors que des interventions nutritionnelles avec des nutriments d’intérêt pourrait plutôt aider à protéger des altérations métaboliques et/ou cognitives chez ces patients. / Obesity is a metabolic and inflammatory disorder that represents a major risk factor for the development of comorbidities such as type 2 diabetes. Obese patients also often experience mood and cognitive dysfunctions that represent important risk factors for aggravation of obesity and related outcomes. Reducing the development of such alterations may therefore allow improving health and quality of life of obese subjects. In this context, this thesis aimed to decipher the neurobiological mechanisms underlying such neuropsychiatric alterations, in order to identify new targets for the development of potential preventive and/or therapeutic strategies aiming to reduce these alterations. To do so, rodent models of obesity such as the db/db mice, which display severe obesity associated with classical features of metabolic syndrome, can be particularly useful.[ ] Second, we have investigated whether a nutritional intervention with n-3 polyunsaturated fatty acids (n-3 PUFAs) and antioxidants, which are well-known to display anti-inflammatory and neuroprotective properties, improved obesity-associated neuropsychiatric alterations. In addition, we have measured the consequences of chronic administration of the prebiotic oligofructose on the behavioral alterations displayed by db/db mice since previous studies pointed to the gut microbiota as an important player in the regulation of behavior. Finally, we have investigated the potential underlying mechanisms by measuring the impact of this treatment on the metabolism and systemic inflammation, but also on neurobiological systems known to be involved in the control of food intake and behavior. We first showed that an anti-inflammatory treatment or caloric restriction reduced anxiety-like behaviors, and this was associated with a selective decrease of hippocampal TNF-α mRNA expression, suggesting that this pro-inflammatory cytokine likely contributes to induce anxiety-like behavior associated with obesity. We then nicely confirmed this assumption by showing that selectively blocking brain TNF-α by chronically administrating etanercept i.c.v. (TNF-α decoy receptor) indeed decreased anxiety-like behaviors in obese db/db mice.[ ] Secondly, we tried identifying new preventive and/or therapeutic strategies aiming to improve mood and cognitive alterations associated with obesity. Hence, we measured if an n-3 polyunsaturated fatty acids/antioxidants enriched diet, well-known to modulate different neurobiological mechanisms potentially involved in behavioral alterations displayed by db/db mice, improved their behavioral alterations. We showed that chronic consumption of this diet reversed hippocampus-dependent spatial memory deficits displayed by db/db mice in a water-maze task and that this effect likely involved modulation of neuronal plasticity. Thirdly, we tested whether manipulating the gut microbiota composition may constitute a preventive and/or therapeutic strategy to improve the neuropsychiatric alterations associated with obesity. Hence, we assessed for the first time the effect of microbiota manipulation with a prebiotic on the metabolic and behavioral alterations displayed by db/db mice, but also on their systemic and neurobiological correlates. We showed that improvement of metabolic alterations following prebiotic administration in db/db mice was associated with selective reduction of peripheral and central inflammation, which is however not accompanied by detectable improvement of anxiety-like behavior or spatial memory deficits. To conclude, these experiments contribute to show that inflammation, and especially TNF-α, could be an important target to develop therapeutic treatments for mood alterations associated with obesity, whereas nutritional interventions with selective nutrients of interest may rather help preventing associated metabolic and/or cognitive alterations.

La tuberculose est encore aujourd’hui un problème de santé majeur et l’augmentation des cas de tuberculoses résistantes au niveau mondial, associé au dépistage et diagnostic insuffisants, suggère une éradication totale encore lointaine. La compréhension des relations hôte-pathogène est essentielle pour permettre d’établir de nouvelles stratégies thérapeutiques. Ces travaux de thèse ont mis en exergue la contribution limitée de la voie de signalisation IL-36, cytokine de la famille de l’IL-1, au cours de l’infection mycobactérienne. Ces données permettent d’envisager sérieusement l’IL-36 en tant que cible thérapeutique pour le psoriasis sans risquer la réactivation de tuberculose latente chez les patients. De plus, nous avons également démontré le rôle différentiel de la voie de signalisation TNF au sein de populations cellulaires spécifiques lors de l’infection par M. tuberculosis, et nos résultats apportent des connaissances solides pour envisager des stratégies immuno-modulatoires qui pourraient constituer l’avenir des traitements antituberculeux. D’autre part, nous avons caractérisé un modèle murin humanisé pour une étude facilitée des anticorps anti-TNF humains actuels. L’utilisation de ce modèle pourrait permettre, à termes, d’identifier et de valider de nouveaux candidats d’anticorps anti-TNF de deuxième génération. Enfin, nous avons montré que des anticorps neutralisants l’IL-17 ne perturbent pas la réponse immunitaire à la tuberculose, contrairement aux anticorps anti-TNF, et que l’absence de l’IL-17 n’est pas compensée par l’IL-22 puisque des animaux déficients pour ces deux voies de signalisation sont capables de contrôler l’infection. / Tuberculosis remains a major health problem in the world nowadays. The increasing incidence of resistant tuberculosis is associated with a poor diagnosis, reflecting important difficulties for total eradication. Understand host-pathogen interactions is crucial to establish new therapeutic strategies. This work first shows the limited contribution of IL-36 pathway during mycobacterial infection. These results suggest that IL-36 could be targeted for the treatment of psoriasis without a high risk of tuberculosis reactivation. We then demonstrate the differential role of TNF pathway among myeloid or lymphoid cells during M. tuberculosis infection, and our data support the development of immunomodulatory strategies to boost host immune response, thus helping to clear the infection. Moreover, we characterize a humanized murine model allowing the study of new anti-TNF candidates in the context of M. tuberculosis infection. Finally, we show that antibodies targeting IL-17 does not dampen host control of M. tuberculosis infection, unlike anti-TNF, and that IL-22 does not compensate absence of IL-17 for this control.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glutaric aciduria type I (GA-I) is an inborn error of metabolism (EIM) characterized biochemically by accumulation of glutaric acid (GA). The clinical manifestations are mainly neurological and develop during childhood. Among these changes, there are the seizures and cognitive deficits, which may be precipitated by infectious processes. Although growing evidence supports that inflammation and oxidative damage are both involved in learning impairment, it is not known whether inflammatory and oxidative stress markers facilitate GA-induced memory impairment. From this, the main objective of this study was to investigate the performance of rat pups chronically injected with GA and lipopolysaccharide (LPS) in spatial memory test on Barnes maze. To evaluate antioxidant defenses, cytokines levels, Na+, K+-ATPase activity, and hippocampal volume. Furthermore, we also evaluated wheter N-acetylcysteine (NAC) could improve these behavioral, biochemical or structural changes induced by GA and LPS administration. For this, the rat pups were injected with GA (5umol g of body weight-1, subcutaneously; twice per day; from 5th to 28th day of life), and were supplemented with NAC (150 mg/kg/day; intragastric gavage; for the same period). In order to mimic a severe infection state, LPS (2 mg/kg; E.coli 055 B5) or vehicle (saline 0.9%) was injected intraperitoneally, once per day, from 25th to 28th day of life.Oxidative stress biomarkers, antioxidant activity and hippocampal volume were assessed. In this study, GA caused spatial learning deficit in the Barnes maze, and that LPS potentiated the memory impairment induced by GA in rat pups. In addition, GA and LPS increased proinflammatory cytokine levels (TNF- and IL-1), and the co-administration of these compounds potentiated the increase of IL-1 levels but not TNF- levels in the hippocampus of this animals. Although GA and LPS administration increased TBARS (thiobarbituric acid-reactive substance) content, reduced antioxidant defenses and inhibited Na+,K+-ATPase activity (total and subunit α1), GA and LPS co-administration did not have additive effect on oxidative stress markers and Na+, K+ pump. The hippocampal volume did not change after GA or LPS administration. N-acetylcysteine protected against impairment of spatial learning and increase of cytokines levels induced by GA and LPS. The NAC also protected against deleterious effects induced by GA and LPS, as characterized by inhibition of Na+,K+-ATPase activity (total and subunit α1)and increase of TBARS content, as well as the reduction of antioxidant defenses(non protein thiols and glutathione content, superoxide dismutase and catalase activities).These results suggest that inflammatory and oxidative markers may underlie at least in part of the neuropathology of GA-I in this model. Pharmacological protection with NAC during encephalopatic crises could be considered as an adjuvant therapy to prevent hippocampal dysfunction and the progression of disease in children with GA-I. / A acidemia glutrárica do tipo I (AG-I) é um erro inato do metabolismo (EIM) caracterizada bioquimicamente pelo pelo acúmulo de ácido glutárico (AG). As manifestações clínicas são predominantemente neurológicas, e desenvolvem-se principalmente na infância. Entre essas alterações, as quais são precipitadas por processos infecciosos, pode-se citar o déficit cognitivo. Embora estudos recentes sugerem que a inflamação e o estresse oxidativo estão envolvidos no déficit cognitivo, não se sabe se os marcadores inflamatórios e oxidativos facilitam o prejuízo de memória após a administração de AG. A partir disso, o objetivo desta dissertação foi investigar o desempenho de ratos jovens injetados cronicamente com AG e lipopolissacarídeo (LPS) no teste de memória espacial no labirinto de Barnes. Além disso, foi avaliado os níveis das defesas antioxidantes, níveis de citocinas, atividade da enzima Na+, K+-ATPase e volume hipocampal. Como a N-acetilcisteína (NAC) possui propriedades antioxidantes e antiinflamatórias, foi testado se esse composto poderia melhorar as alterações comportamentais, bioquímicas e estruturais induzidas pela administração de AG e LPS. Para isso, os ratos jovens foram injetados com AG (5 μmol/g do peso corporal-1; subcutaneamente; duas vezes por dia; do 5º ao 28º dia de vida), e foram suplementados com NAC (150 mg/kg/dia; por gavagem; pelo mesmo período). A fim de mimetizar um estado infeccioso, LPS (2 mg/Kg: E. coli 055 B5) ou veículo (salina 0.9%) foi injetado intraperitonealmente uma vez por dia, do 25º ao 28º dia de vida. Nesse estudo, AG causou déficit de aprenizagem espacial no labirinto de Barnes, e o LPS potencializou esse prejuízo de memória induzido pelo AG nos ratos jovens. Em adição, a administração de AG e LPS aumentou os níveis de citocinas pró-inflamatórias (TNF- and IL-1), e a associação desses compostos potencializou o aumento dos níveis de IL-1, mas não de TNF-α no hipocampo dos animais. Embora a associação de AG e LPS tenha causado o aumento o conteúdo TBARS (espécies reativas ao ácido tiobarbitúrico), a redução das defesas antioxidantes e inibição da atividade da Na+,K+-ATPase (total e subunidade α1), a associação de AG e LPS não teve efeito aditivo nos marcadores de estresse oxidativo e na atividade da bomba de Na+ e K+. O volume hipocampal não foi alterado após a administração do AG e LPS. A N-acetilcisteína protegeu contra o prejuízo de aprendizagem espacial e aumento de citocinas inflamatórias induzido pelo AG e LPS. A NAC também protegeu contra os efeitos deletérios induzidos pelo AG e LPS, caracterizado pela inibição da atividade da Na+,K+-ATPase (total e subunidade α1) e aumento do conteúdo de TBARS, bem como a redução das defesas antioxidantes (tiós não-proteicos, conteúdo de glutationa, avitidade da superóxido dismutase e catalase). Esses resultados sugerem que marcadores inflamatórios e oxidativos podem estar envolvidos, em parte, na neuropatologia da AG-I neste modelo. Dessa forma, a proteção farmacológica com a NAC durante crises encefalopáticas pode ser considerada como uma terapia adjuvante para prevenir a disfunção hipocampal e a progressão da doença em crianças com AG-I.

B-lymphocytes are lymphoid cells, which are a part of the adaptive/innate immune system and generate antibodies. Recently, many studies have supported hypothesis that different rather minor B-lymphocyte subpopulations may play a direct and indirect role in immunopathogenesis in human pathologies such as Crohn's disease (CD). The aim of current study was therefore to investigate distribution of frequencies of B lymphocyte subpopulations (from transient to mature effector B cell stages) in peripheral blood of healthy subjects (CO), patients with Crohn's disease (CD) and ulcerative colitis (UC). Thus, using 11-colour flow cytometry we have analysed 30 blood samples of individuals, including 14 healthy controls, 11 patients with Crohn's disease and 5 with UC. In 6 patients with CD we have had an opportunity to analyze blood samples collected 2 hours after an administration of anti-TNF therapy. Higher frequencies of memory B-lymphocytes (CD19+ CD27+ , CD19+ CD20+ CD27+ and CD19+ CD20+ CD27+ IgM+) were found in patients with CD as compared to COs. (20.06±13.58%; 17.61±13.48%; 88.60±20.56% vs. 11.75±26.47%; 11.25±26.50%; and 66.82±22.60%), in case of CD19+CD20-CD27-IgM+ B-lymphocytes the difference was statistically significant (57.15±17.21% in CD vs. 19.59±31.79% in CO; p=0.0341), which is in accordance...