Orientador: Patricia Pintor dos Reis / Resumo: Introdução: O tumor carcinoide do pulmão pertence ao tipo neuroendócrino das neoplasias pulmonares. Devido à sua baixa incidência (~2%), pouco se conhece sobre suas alterações moleculares. Os microRNAs (miRNAs) têm importante papel na regulação gênica e têm sido associados ao câncer como biomarcadores diagnósticos, prognósticos e preditivos. Objetivos: Determinar o perfil global de expressão de miRNAs em tumores carcinoides do pulmão e identificar (in silico) vias moleculares envolvendo os miRNAs desregulados e genes-alvo preditos. Material e Métodos: Dois fragmentos de um tumor carcinoide típico e sua metástase correspondente foram obtidos, o RNA extraído de cada amostra e analisado na plataforma TaqMan Low Density Array (TLDA), a qual contém sondas para 384 miRNAs. Os dados foram analisados no Expression Suite software. Adicionalmente, 7 tumores (5 carcinoides típicos e 2 atípicos) foram utilizados para análise de expressão de 2,578 miRNAs na plataforma GeneChip™ miRNA 4.0 e os dados analisados utilizando o Transcriptome Analysis Console software. A análise estatística dos dados foi realizada para identificação dos miRNAs significativamente (p<0,05) alterados. Métodos de análise in silico incluíram a identificação de mRNAs-alvo dos miRNAs e vias moleculares de tumorigênese. Resultados e Discussão: No tumor carcinoide típico e metástase (TLDA), 15 miRNAs estavam com expressão comumente diminuída, os quais regulam genes associados a vias de resposta imune adaptativa. Adiciona... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Lung carcinoid tumors are a type of neuroendocrine lung neoplasia. Due to its low incidence (~2%), little is known about the molecular alterations associated with these tumors. microRNAs (miRNAs) have an important role in gene regulation and have been associated with cancer as diagnostic, prognostic and predictive biomarkers. Objectives: To determine the global expression miRNA profiles of lung carcinoid tumors and to identify (in silico) molecular pathways including deregulated miRNAs and predicted target-genes. Material and Methods: Two fragments of a typical carcinoid tumor and its corresponding metastasis were obtained, the RNA extracted and analyzed using the TaqMan Low Density Array (TLDA) platform containing 384 miRNAs. Data were analyzed using Expression Suite software. Additionally, 7 tumors (5 typical and 2 atypical carcinoids) were used for expression analysis of 2,578 miRNAs in the GeneChip™ miRNA 4.0 platform and data were analyzed using the Transcriptome Analysis Console software. Statistical analysis was performed to identify the significantly (p<0,05) deregulated miRNAs. In silico analyses methods included the identification of miRNA target genes (and enriched pathways. Results and Discussion: In the typical carcinoid tumor and metastasis (TLDA data), 15 miRNAs were commonly down-regulated and these modulate genes associated with the adaptive immune system pathway. Additionally, the comparison of typical carcinoids or atypical vs. normal (GeneChi... (Complete abstract click electronic access below) / Mestre

Les stratégies de traitement biologiques ciblant le TNF-α se sont avérées efficaces pour réduire l'inflammation et les symptômes cliniques dans plusieurs maladies inflammatoires chroniques et sont maintenant couramment utilisées pour les patients qui ne répondent pas aux AINS au cours de la spondyloarthrite (SpA). Cependant, 30 à 40% des patients ne répondent pas aux anti-TNF, et il est actuellement impossible de prédire la réponse des patients à ces biomédicaments. Pour améliorer les résultats cliniques, nous avons besoin d’une part d’une meilleure compréhension des mécanismes d’action des anti-TNF sur le système immunitaire, et d’autre part de biomarqueurs permettant de prédire la réponse à ces biomédicaments afin de guider la décision thérapeutique. Mon projet de doctorat a porté sur deux objectifs complémentaires: (i) l'objectif principal était de progresser dans notre compréhension des mécanismes pathogéniques impliqués dans la SpA axiale et de définir de quelle façon les anti-TNF-α affectent les réponses immunitaires des patients, (ii) de développer des biomarqueurs pour prédire la réponse thérapeutique aux inhibiteurs du TNF. En collaboration avec l'équipe du Pr. Dougados à l'Hôpital Cochin, nous avons recruté deux cohortes indépendantes de patients SpA ayant une maladie active et pour lesquels nous avons collecté des échantillons de sang avant l'initiation du traitement par anti-TNF puis 1 semaine et 3 mois après le début du traitement. Les réponses immunitaires de ces patients ont été analysées à l'aide de tests hautement standardisés réalisés ex-vivo sur sang circulant. Ces tests "TruCulture" se présentent sous forme de seringues, dans lesquelles 1 ml de sang total est mis à incuber avec un stimulus spécifique ; 20 stimuli différents ont été testé et validé avant et après traitement dans les deux cohortes de patients. Nous avons observé une réduction très significative de la sécrétion de IL-1ra, IL-1β, IL-8, and MIP-1β en réponse à des stimuli microbiens et à des agonistes des TLR dans les échantillons de sang prélevés 7 jours et/ou 3 mois après le début du traitement. Pour identifier les bases moléculaires de l’action des inhibiteurs du TNF nous avons analysé l'expression des gènes dans ces différentes conditions de stimulation. L'analyse bioinformatique quantitative de l'expression des gènes (QuSAGE) a révélé que les gènes les plus modulés par le traitement anti-TNF étaient NF-KB et les gènes cibles de NF-kB, y compris le TNF lui-même et l’IL1B. Nos données suggèrent que les inhibiteurs du TNF agissent principalement en perturbant une boucle autorégulatrice pilotée par NF-kB. Afin d'identifier les signatures immunologiques de réponse aux anti-TNF avant le début du traitement, nous avons corrélé les réponses immunitaires chez les patients analysés au temps 0 à la réponse thérapeutique aux anti-TNF mesurée à 3 mois. Nos résultats suggèrent que les patients atteints de SpA et exprimant des niveaux inférieurs de PAX5 et des niveaux supérieurs de SPP1 en réponse à la stimulation avec SEB avant l'initiation de la thérapie anti-TNF ont les meilleures réponses thérapeutiques. Notre recherche montre que les tests TruCulture sont un outil efficace pour étudier les fonctions immunitaires chez les patients atteints de SpA et que les effets du traitement anti-TNF peuvent être mesurés lorsque les cellules immunitaires sont stimulées. En terme de recherche translationnelle, nous avons identifié des molécules qui pourront être utilisés comme biomarqueurs pour aider les cliniciens à prédire les réponses thérapeutiques aux traitements anti TNF / The introduction of anti-TNF therapy has proven effective to reduce inflammation and clinical symptoms in several chronic inflammatory diseases. However, 30-40% of patients do not respond to TNF blockers and it is currently not possible to predict responsiveness of patients to anti-TNF therapy. Furthermore, their impact on the immune system is incompletely understood. The goals of my PhD project were (i) to define the impact of anti-TNF therapy on immune responses to microbial challenges and stimuli targeting specific immune pathways in spondyloarthritis (SpA) patients, and (ii) to identify immunological correlates associated with therapeutic responses to TNF-blockers.Using a set of whole-blood, syringe-based assays to perform ex vivo stimulation while preserving physiological cellular interactions (TruCulture assays), we have performed a pilot study in SpA patients and investigated immune responses to 20 different stimuli before and 3 months after initiation of anti-TNF therapy. These findings were validated in a replication cohort, also assessing the effects of anti-TNF agents after only one week of treatment. We observed a highly significant reduction of the secretion of IL-1ra, IL-1β, IL-8 and MIP-1β in response to selected stimuli after 3 months of treatment compared to the baseline. Interestingly, these changes were already detectable after a single injection of an anti-TNF agent. To gain insight into the molecular mechanism of TNF blockers, we profiled gene expression in the stimulation cultures from all patients. Quantitative set analysis for gene expression (QuSAGE) revealed that the gene modules most affected by anti-TNF therapy are NF-kB transcription factors and inhibitors and NF-kB target genes, including TNF itself and IL1B. Our data suggest that TNF-blockers primarily act by disrupting an autoregulatory loop driven by NF-kB. We also tested whether there is a correlation between the responses of immune cells to specific stimuli and the clinical response to TNF-blockers. The decision tree model that we trained and validated suggests that SpA patients who expressed lower levels of PAX5 and higher levels of SPP1 in response to SEB stimulation before initiation of anti-TNF therapy had the best therapeutic responses. Our study shows that TruCulture assays are an efficient and robust tool to monitor immune functions in SpA patients and that the effects of anti-TNF therapy can be measured when immune cells are challenged, but not at steady state. Our data also indicate that analyzing immune responses in patients before therapy is a promising strategy to develop biomarkers for prediction of therapeutic responses to TNF-blockers

Le Strontium 90 (90Sr) est un radionucléide d’origine anthropogénique, relâché en grandes quantités dans l’environnement à la suite d’essais nucléaires aériens ou d’accidents d’installations nucléaires. Le 90Sr persiste à long terme dans l’environnement, ce qui conduit à la contamination chronique par ingestion de populations des territoires contaminés. L’induction de tumeurs osseuses liées à la fixation du 90Sr a été largement décrite. Par contre, l’occurrence d’effets non cancéreux est beaucoup moins connue. Nous avons utilisé un modèle murin avec une contamination chronique par ingestion d’eau contenant 20 kBq/l de 90Sr. Une étude de biocinétique a confirmé l’accumulation de 90Sr dans les os, avec un taux d’accumulation plus rapide durant la croissance osseuse. Cette accumulation est plus élevée dans les os des femelles que chez les males. Les doses absorbées au corps entier varient de 0.33 ± 0.06 mGy (naissance) à 10.6 ± 0.1 mGy (20 semaines). La dose au squelette peut aller jusqu’à 55 mGy. L’ingestion de 90Sr induit une modification de l’expression des gènes impliqués induisant à un déséquilibre favorisant la résorption osseuse, mais sans répercussion sur la morphologie de l’os. Aucun effet majeur n’a été observé pour le système hématopoïétique. Par contre, des modifications mineures du système immunitaire ont été observées. Afin d’évaluer la fonctionnalité du système immunitaire, un test de vaccination avec les antigènes TT et KLH a été utilisé. Les résultats montrent chez les animaux contaminés une diminution significative de la production d’immunoglobulines spécifiques, une modification de la balance Th1/Th2 dans la rate et une différenciation lymphoïde B perturbée. Ces résultats permettent de mieux comprendre certaines des conséquences non cancéreuses de l’exposition chronique à faible dose à des radionucléides à demi-vie longue pouvant être rejetés accidentellement. / Strontium 90 (90Sr) is a radionuclide of anthropogenic origin released in large quantities in the environment as a result of nuclear atmospheric tests or accidents at nuclear facilities. 90Sr persists on a long-term basis in the environment, leading to chronic contamination by ingestion of populations living on contaminated territories. The induction of bone tumours associated with the fixation of 90Sr has been widely described. However, the occurrence of non-cancer effects is much less known. We used a mouse model with chronic contamination by ingestion of water containing 20 kBq/l of 90Sr. A biokinetic study confirmed the accumulation of 90Sr in the bones, with an increased rate of accumulation during bone growth. This accumulation was higher in the bones of females than in males. The whole-body absorbed doses ranged from 0.33 ± 0.06 mGy (birth) to 10.6 ± 0.1 mGy (20 weeks). The absorbed dose for the skeleton was up to 55 mGy. Ingestion of 90Sr induced a change in the expression of genes inducing an imbalance in favour of bone resorption, but without effect on bone morphology. No significant effect was observed for the hematopoietic system. On the other hand, minor modifications were observed for the immune system. To evaluate the functionality of the immune system, a vaccination test with TT and KLH antigens was used. Results showed in contaminated animals a significant decrease in the production of specific immunoglobulins, changes in the Th1/Th2 balance in the spleen and a disrupted B lymphocyte differentiation. These results improve the understanding of some of the non-cancerous consequences of chronic exposure at low dose of radionuclides with a long half-life, which can be accidentally released.

Nos últimos anos houve um aumento da expectativa de vida de indivíduos com síndrome de Down, com causas de morte que diferem da população em geral. Alguns estudos mostraram que a resposta imunológica difere ao longo da vida com alterações relacionadas ao envelhecimento. O gene RCAN1 (regulador de calcineurina tipo 1), localizado na região q22.12 no cromossomo 21 é descrito como responsável pelo fenótipo da SD. O gene RCAN1 inibe a ação da calcineurina, responsável pela desfosforilação do fator nuclear de células T ativadas (NFAT), uma etapa essencial para a ativação de genes responsáveis pela expressão de citocinas. A consequência é uma possível diminuição da resposta imune efetora. Em adultos com síndrome de Down as respostas imunes humoral e celular têm sido pouco investigadas. Apesar da superexpressão do gene RCAN1 já ter sido descrita em diversos tecidos, sua expressão em células mononucleares de sangue periférico (PBMC) de adultos não foi ainda avaliada. Os objetivos deste estudo foram avaliar aspectos humorais e celulares da resposta imune, avaliar a expressão quantitativa do gene RCAN1 e relacionar os achados com a produção de citocinas. O grupo de estudo foi composto de adultos com síndrome de Down (SD) com cariótipo de trissomia livre (n=24), um grupo controle (CTR) composto de deficientes mentais com outras etiologias (n=21) e um grupo de indivíduos saudáveis (n=8), como parâmetros para alguns experimentos. Os grupos SD e CTR convivem na Associação de Pais e Amigos dos Excepcionais (APAE-SP). Foram realizados hemogramas e sorologias para detecção de hepatite B, citomegalovírus, mononucleose infecciosa, toxoplasmose, rubéola, sarampo, PCR, fração de complemento C3, C4, antiestreptolisina O e dosagem de imunoglobulinas IgG, IgM e IgA. Células mononucleares foram obtidas por gradiente de Ficoll-Hypaque e submetidas à cultura celular, análise quantitativa de expressão gênica de RCAN1 e avaliação imunofenotípica por citometria de fluxo. Os sobrenadantes da cultura foram coletados para dosagem das citocinas IL-2, IL-4, IL-5, IL-10, TNF e INF. Os resultados deste estudo mostraram que a frequência de sorologias positivas para os vários agentes infecciosos e os demais parâmetros imunológicos foram comparáveis nos dois grupos de doentes mentais. A análise imunofenotípica dos indivíduos com SD mostrou aumento de células NK, de linfócitos TCD8+, alteração na relação CD4:CD8 (1:1) e diminuição de linfócitos B (CD19+) quando comparados ao grupo controle (p<0,05). A produção espontânea de INF, TNF e IL-10 foi maior no grupo SD em relação ao grupo CTR (p<0,05). Porém, quanto à expressão do gene RCAN1, não foi observada diferença entre os dois grupos analisados. O estudo do perfil imunológico humoral e celular de adultos com síndrome de Down provenientes da APAE-SP permitiu concluir que não houve diferenças quanto aos aspectos humorais avaliados nos dois grupos (SD e CTR). Quanto aos aspectos celulares, a imunofenotipagem sugere um possível sinal de envelhecimento precoce do sistema imune e a produção de citocinas um predomínio de perfil pró-inflamatório. Contudo, o perfil de citocinas não apresenta relação com o nível de expressão do gene RCAN1. / In recent years there has been an increase in life expectancy of individuals with Down´s syndrome (DS), with death causes differ from the general population. Some studies have shown that the immune response differs throughout life with changes related to aging. The RCAN1 gene (regulator of calcineurin type 1), located in the q22.12 region of chromosome 21 is described as responsible for the phenotype of DS. The gene RCAN1 inhibits the calcineurin activity, responsible for the dephosphorylation of the nuclear factor of activated T cells (NFAT), an essential step for the activation of the genes responsible for cytokines expression. The consequence is a possible reduction of the effector immune response. In adults with DS, the humoral and cellular immune responses have not been throughly investigated. Although the overexpression of the RCAN1 gene has already been described in many tissues, its expression in mononuclear cells of peripheral blood (PBMC) of adults with DS has not been evaluated. The objectives of this study were to evaluate aspects of humoral and cellular immune response, evaluate the quantitative expression of the RCAN1 gene and correlate the findings with the production of cytokines. The study group consisted of adults with Down´s syndrome (DS) with free trisomy karyotype (n = 24), a control group (CTR) composed of the mentally disabled of other etiologies (n = 21) and a group of healthy subjects (n = 8), as parameters for some experiments. The SD and CTR groups are followed in Associação de Pais e Amigos dos Excepcionais (APAE-SP). It was evaluated Hemogram and serology for detection of hepatitis B, cytomegalovirus, infectious mononucleosis, toxoplasmosis, rubella, measles, cRP, complement fraction C3, C4, antistreptolysin O and IgG, IgM and IgA immunoglobulin isotypes. The mononuclear cells were obtained by Ficoll-Hypaque gradient and the cells were cultured without stimuli, analyzed for the quantitative gene expression of RCAN1 and evaluated for immunophenotyping by flow cytometry. The culture supernatants were collected for measurement of cytokines IL-2, IL-4, IL-5, IL- 10, TNF and IFN.The results of this study showed that the frequency of positive tests for various infectious agents and other immunological parameters were comparable in both groups (DS and CTR). Immunophenotyping of individuals with DS showed an increase in NK cells, CD8 + lymphocytes, changes in CD4: CD8 ratio (1:1) and decreased B lymphocytes (CD19 +) when compared to the control group (p <0.05). The DS group had a spontaneous production of IFN, TNF and IL-10 higher than the CTR group (p<0.05). However, there was not any difference in RCAN1 gene expression (mRNA) between the two groups of the mentally disabled. The humoral and cellular immune profile in adults with Down´s syndrome from APAE-SP showed that there was no difference in the humoral aspects assessed in both groups (SD and CTR). For the cellular aspects, the immunophenotyping suggests a possible sign of premature aging of the immune system and the cytokine production show a proinflammatory profile. Nevertheless, this cytokines profile is not associated with level of expression of the RCAN1 gene.

O diabetes mellitus (DM) é uma doença com prevalência e morbidade elevadas em todo o mundo, sendo que o DM1 é responsável por 5-10% dos casos. A vitamina D, hormônio de ação pleiotrópica, pode melhorar o curso do DM1, embora os mecanismos não estejam completamente elucidados. Dessa forma, ampliar o conhecimento sobre a ação desse hormônio pode auxiliar no prognóstico, bem como na compreensão dos possíveis mecanismos envolvidos na prevenção do DM. Neste trabalho, foram avaliados os efeitos fisiológicos da suplementação de vitamina D (800 UI/dia/sete dias; via oral) em camundongos machos (n=31; linhagem C57BL/6) distribuídos em quatro grupos: Controle + Água (CA; n=9); Controle + Vitamina D (CV; n=9); Diabético + Água (DA; n=6) e Diabético + Vitamina D (DV; n=7). Os camundongos tornados diabéticos (aloxana, 60 mg/Kg, intravenosa), quando comparados aos controles, exibiram redução do peso corporal e concentrações plasmáticas de glicose mais elevadas durante o período experimental de 10 dias (características do estado insulinopênico). Entretanto, a suplementação com vitamina D não alterou essa condição. Camundongos tornados diabéticos, em relação aos controles, exibiram redução do peso corporal (p<0,05) e concentrações plasmáticas de glicose (p<0,001) mais elevadas durante o período experimental. Animais suplementados com vitamina D apresentaram, em relação aos controles, níveis de 25(OH)D mais elevados (CA vs CV, p<0,001; DA vs DV, p<0,001). Níveis séricos maiores de ureia (CA vs DA, p<0,05; CA vs DV, p<0,01; CV vs DA, p<0,05; CV vs DV, p<0,01) e creatinina (CA vs DA, p<0,001; CA vs DV, p<0,001; CV vs DA, p<0,001; CV vs DV, p<0,001), espessamento da cápsula de Bowman, hipertrofia glomerular e destruição de hepatócitos foram observados em camundongos diabéticos em relação aos controles. Entretanto, a suplementação com vitamina D não alterou estas condições. O grupo DA apresentou menor nível sérico de albumina em relação aos grupos CA (p<0,05) e CV (p<0,05); níveis inferiores de hemoglobina (p<0,05) e hematócrito (p<0,05) em relação ao grupo DV; e menor leucometria (p<0,05) e mononucleares sanguíneos (p<0,05) em relação ao grupo CA. Os resultados sugerem que a vitamina D possa influenciar a resposta imunológica em animais diabéticos, modulando hematócrito, hemoglobina, bem como os níveis séricos de albumina / Diabetes mellitus (DM) is a disease with high prevalence and morbidity worldwide, and the DM1 is responsible for 5-10% of cases. The vitamin D hormone pleiotropic action, can improve the course of T1DM, although the mechanisms are not fully elucidated. Thus, better understanding the action of this hormone can aid in prognosis as well as in understanding the possible mechanisms involved in the prevention of diabetes. We evaluated the physiological effects of vitamin D (800 IU/day/seven days, v.o.) in male mice (n=31, C57BL/6 strain) divided into four groups: Control + Water (CW, n=9); Control Vitamin D + (CV n=9); Diabetic + Water (DW, n=6) Diabetic + Vitamin D (VD, n=7). The mice induced-diabetes by alloxan (60 mg/kg, i.v.), when compared to controls, exhibited reduced body weight and plasma glucose concentrations were higher during the experimental period of 10 days (features insulinopenic state). However, vitamin D supplementation did not alter this condition. Diabetic mice, compared to controls, exhibited reduced body weight (p<0,05) and plasma glucose concentrations (p <0.001) higher during the trial period. Animals supplemented with vitamin D showed higher levels of 25 (OH) D than controls (CW vs CV, p <0,001; DW vs DV, p<0,001). Higher serum urea (CW vs. DW, p <0,05; CW vs DV, p <0,01; CV vs DA, p <0,05; CV vs DV, p <0,01) and creatinine (CW vs. DW, p <0,001; CW vs DV, p <0,001; CV vs DW, p <0,001; CV vs DW, p <0,001), thickening of Bowman\'s capsule, glomerular hypertrophy and destruction of hepatocytes were observed in diabetic mice compared to controls. However, vitamin D supplementation did not alter these conditions. The DW group showed lower serum albumin compared to CW (p<0,05) and CV (p<0,05) groups; lower hemoglobin (p<0,05) and hematocrit (p <0,05) compared to the DV group; and lower leukocyte counts compared to CW (p <0,05) and mononuclear blood (p <0,05) compared to the CW group. The results suggest that Vitamin D may influence the immune response in diabetic animals, modulating hematocrit, hemoglobin and serum albumin

Pteridium aquilinum, conhecida popularmente como &quot;samambaia-do-campo&quot; ou simplesmente &quot;samambaia&quot;, é considerada uma das plantas tóxicas mais importantes no mundo, não só pela sua distribuição cosmopolita e intoxicação de rebanhos em diversas partes do mundo, mas também pelo seu alto potencial carcinogênico observado em animais e seres humanos que se alimentam com esta planta. Por outro lado, não havia dados na literatura a respeito dos possíveis efeitos tóxicos desta planta sobre o sistema imune, o qual se sabe, tem papel fundamental não só para o controle de doenças infecciosas, como também, para impedir a proliferação de células mutantes e, conseqüentemente o desenvolvimento de câncer. Assim, o presente estudo avaliou os efeitos da P. aquilinum sobre as respostas imune inata e adaptativa em camundongos, através dos seguintes protocolos: produção e titulação de anticorpos T - dependente, proliferação de linfócitos T e B, resposta de hipersensibilidade tardia, fenotipagem linfocítica e citotoxicidade de células NK. Além disso, foram feitas a avaliação histológica e a contagem da celularidade dos órgãos linfóides. Resultados mostraram diminuição da resposta de hipersensibilidade tardia (resposta celular) nos grupos tratados com 10 e 30 g/kg de samambaia, redução da citotoxicidade das células NK, redução da polpa branca do baço, diminuição da camada celular do timo e desorganização dos folículos linfóides nos linfonodos mesentéricos e placas de Peyer dos camundongos tratados com a dose de 30 g/kg de samambaia e diminuição na celularidade da medula óssea em todos os grupos tratados com a samambaia, por 14 dias. Os dados obtidos na presente pesquisa permitem sugerir que a diminuição da resposta imune celular foi decorrente do efeito tóxico da P. aquilinum sobre as células NK e não um efeito tóxico direto sobre os linfócitos Th1. / Pteridium aquilinum, known popularly as \"bracken fern\" is considered one of the more important toxic plants in the world, not only for its cosmopolite distribution and poisoning of flocks in diverse parts of the world, but also for its high potential carcinogenicity observed in animals and human that feed with this plant. On the other hand, there are not data available in the literature regarding the possible toxic effects of this plant on the immune system that is fairly known to be important not also for the control of infectious illnesses but also to hinder the proliferation of mutant cells and, consequently cancer development. Thus, the present study evaluated the effect of the P. aquilinum on the innate and acquired immune responses in mice, through the following protocols: production and titer of T - dependent antibody, proliferation of T and B lymphocytes, delayed-type hypersensitivity, lymphocyte subset analysis and natural killer-cell activity. Moreover, histophatological evaluation and cellularity of the lymphoid organs were performed. Results showed reduction of the delayed-type hypersensitivity (cellular immune response) of the mice treated with 10 and 30 g/kg of bracken fern. Besides, it was observed reduction of the natural killer-cell cytotoxicity, decrease of white pulp of spleen and of the cellular layer of the thymus, disorganization of the lymphoid follicle in the mesenteric lymph nodes and Peyer?s patches of the mice treated with 30 g/kg. It was also observed decrease of bone marrow cellularity of all animals treated with bracken fern up to 14 days. Thus, these data found here permit to suggest that P. aquilinum produced reduction of the cellular immune response by a direct toxic effect on the natural-killer cells and not on the Th1 lymphocytes.

La peau constitue l’une des premières lignes de défense contre les menaces extérieures. Elle présente un système nerveux sensoriel particulièrement développé capable d’interagir fonctionnellement avec son système immunitaire. Cependant ces interactions neuro-immunes sont encore très mal comprises et des analyses plus fines sont nécessaires pour décrypter le réel potentiel de ces neurones à réguler les réponses immunitaires. Nos travaux, présentés ici, se concentrent sur le rôle d'une sous-population de neurones sensoriels innervant la peau identifiée par le marqueur GINIP. La déplétion conditionnelle de ces neurones in vivo (souris GINIP-DTR), à révélé leur rôle central dans le contrôle de l'inflammation et de la réparation des tissus cutanés suite à une exposition aux UV. Les souris dépourvues de neurones GINIP+ présentent une augmentation du nombre de macrophages inflammatoires et des lésions profondes du derme comparées aux souris sauvages. Afin de disséquer les mécanismes moléculaires impliqués, nous nous sommes intéressés à la protéine XXX, un médiateur produit par une sous population de neurones GINIP+, les C-LTMR. In vitro, XXX réduit l’expression de cytokines pro-inflammatoires et favorise la production de médiateurs anti-inflammatoires par les macrophages. In vivo, l’absence de cette molécule (souris XXX-KO) accélère la différentiation des monocytes infiltrant en macrophages résidents, les rendant incapables de résoudre la fibrose du derme induite par les UV. Ces résultats suggèrent que les C-LTMR régulent fonctionnellement des cellules myéloïdes via XXX. / The skin is one of the body’s first lines of defense against external threats. This complex tissue contains a highly developed sensory nervous system and an immune system can cooperate to maintain homeostasis. However, these neuro-immune interactions are still poorly understood and further analyses are necessary to understand their role in skin immune response and tissue repair.The goal of the work presented here is to explore the role of a subset of skin sensory neurons identified by the marker GINIP. In vivo, the conditional depletion of these neurons (GINIP-DTR mice) revealed their central role in the control of inflammation and in the repair of skin exposed to UV (ultrat-violet) irradiation. Compared to wild type controls, mice lacking GINIP+ neurons displayed an increase in inflammatory macrophage number in the dermis associated with deep damage. To decipher the molecular mechanisms involved, we focused on the protein XXX, a mediator produced by a subset of GINIP+ neurons, the C-LTMR. In vitro, XXX reduced the expression of pro-inflammatory cytokines and promoted anti-inflammatory factors by macrophages. In vivo, the lack of this molecule (XXX KO mice) accelerated the differentiation of infiltrating monocytes in dermis resident macrophages, making them unable to resolve the fibrosis induced by UV treatment. These results suggest that C-LTMR regulates the myeloid cell response to UV irradiation via XXX.

In conclusion, the studies reported in this thesis suggested that the CHO amount, whether provided by a pre-exercise CHO meal or short-time, i.e., 3-day, CHO loading, plays a pivotal role in regulating the immune responses before, during, and after endurance exercise. Although GI and GL independently affect the exercise performance and immune responses, the amount of CHO consumed remains a determining factor. The potential benefits on immune system and endurance performance after the low GI and low GL diet (L-L) should be noted and warrant further investigation. Although the HGI and LGI meals demonstrated similar effects on endurance performance when large amount of CHO-electrolyte solution consumed during the exercise, pre-exercise LGI meal can hasten the IL-6 responses during the recovery. (Abstract shortened by UMI.) / The aim of this thesis was to investigate the influence of pre-exercise carbohydrate (CHO) meal(s) with different glycemic index (GI) and glycemic load (GL) on endurance running performance, physiological, and immune responses. / The first study (Chapter 4) examined the influence of a pre-exercise meal with different GI and GL on subsequent endurance running performance, physiological, and immune responses. Eight endurance-trained male runners completed three trials in a randomized order, separated by at least seven days. These responses were characterized by a lower CHO oxidation with a concomitant higher glycerol and FFA in the H-L trial. Consumption of a pre-exercise high CHO meal, i.e., H-H and L-L, resulted in less perturbation of circulating numbers of leukocytes, neutrophils, and T lymphocyte subsets, decreased elevation of plasma IL-6 concentrations immediately after exercise and during the 2 h recovery period when compared to the H-L trial. These responses were accompanied by an attenuated increase in plasma IL-10 concentrations and plasma cortisol concentrations at the end of 2 h recovery. It was concluded that the amount of CHO consumed 2 h before endurance exercise appears to be the main influencing factor on immune responses irrespective of its GI and GL value. / The second study (Chapter 5) examined the influence of a 3-day CHO loading with different GI and GL meals on the supercompensation status, running performance, physiological and immune responses. Nine endurance-trained male runners were recruited in this study. The procedures basically involved a 3-day CHO loading with different GI and GL meal [CHO intake (% of energy intake), GI, and GL per day are 73%, 80, and 553 for the high-GI and high-GL (H-H); 73%, 42, and 249 for the low-GI and low-GL (L-L); 31%, 78.5, and 227 for the high-GI and low-GL (H-L) respectively] after a glycogen-lowering exercise. Two hours after the breakfast on day 4, participants performed the running protocol as described in the first study. There was no difference in time to complete the 10-km TT between the two trials with high-CHO loading, i.e., H-H and L-L (51.3 +/- 5.3 min vs 48.6 +/- 1.3 min, NS). These results suggested that 3-day CHO loading with low GI and low GL (L-L) is more effective in improving endurance performance when compared to a high GI but low GL diet (H-L). It appears that the amount of CHO consumed during the 3-day CHO loading remains the key influencing factor on immune responses despite of the differences in the GI and GL value. / The third study (Chapter 6) investigated the influence of pre-exercise meal with different GI on subsequent endurance running performance and immune responses when CHO-electrolyte solution was consumed during exercise. Pre-exercise LGI meal attenuated the increases of cortisol when compared with CON and hastened the recovery of the IL-6 value to baseline when compared to that in HGI and CON trials during the first hour of the recovery. The results suggested that beside of CHO quantity (CHO content), the role of CHO quality (GI) in the diet consumed 2 h before exercise should be considered when investigating the influence of CHO supplementation on the exercise-induced transitory immunosuppressive effects. / Chen Yajun. / "August 2006." / Adviser: Stephen Wong Heung-Sang. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1597. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 199-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The innate immune system is the first line of defence against pathogen invasion. The range of diseases that are caused by deficiencies in or deregulation of the innate immune system illustrates the importance of maintaining an effective balance between clearance of infectious agents and minimisation of inflammatory mediated tissue damage. This thesis explores the role of two proteins in the regulation of the innate immune system. Primarily, this work investigates the effect of human β-defensin 3 (hBD3) on the response to self-DNA and pathogenic DNA. HBD3 is an antimicrobial peptide (AMP), which has been shown to have a role in regulating the immune response; increased copy number of the region containing the gene for hBD3, DEFB103, is linked to an increased risk of psoriasis. Additionally, a similar cationic AMP, LL37, has been shown to exacerbate the pathogenesis of psoriasis by forming an immunogenic complex with self-DNA. This lead to the hypothesis that hBD3 may also affect the innate immune response to DNA. Therefore this project investigates what effect hBD3 has on the response of the innate immune system to self and pathogenic DNA. Flt-3 dendritic cells were used to show that whilst hBD3 increased cellular uptake of self-DNA, it did not convert self-DNA into an immune stimulus. However, hBD3 significantly exacerbated the response to bacterial DNA in a TLR9-dependent manner, also by increasing cellular uptake into FLDCs. The finding that hBD3 increased cellular uptake of both self- and pathogenic DNA suggests that at sites of infection or increased cell death, where DNA would be found in the extracellular environment, hBD3 may increase uptake into immune cells and could induce an increased immune response. Since increased hBD3 expression is induced by inflammatory stimuli, this process would cause a positive feedback loop of inflammation during bacterial infections. In conclusion, hBD3’s role in regulating the innate immune response to DNA is at the ligand-receptor level rather than affecting signalling pathways. Furthermore, hBD3 promotes the innate immune response to bacterial DNA by increasing the efficiency of cellular uptake possibly by inducing DNA aggregation. These results implicate a possible role for hBD3 in the earliest stages of psoriatic plaque development, which is often initiated or exacerbated by an infection, and this could be investigated further. Secondly, I investigated the innate immune function of an E3 ubiquitin ligase (E3L) not previously associated with human disease. Mutations in E3L have been identified in three microcephalic primordial dwarfism families; these patients also presented with recurrent respiratory illnesses. E3L has been implicated in the regulation of the innate immune system via interactions with signalling pathways downstream of the receptor, though its role is not clear. We hypothesised that E3L had a dual role both in regulating growth and cell division and in regulating the immune system. Primary patient fibroblasts did not demonstrate an altered cytokine response to bacterial or viral ligands, implying that E3L may have a specific function in immune cells. To investigate this further, and to provide a system to study E3L in vivo, two transgenic mouse lines were designed and engineered, firstly a conditional ‘knock-out’ designed to replicate some of the alternative isoforms of E3L seen in RT-PCRs, and secondly a ‘knock-in’ line to recapitulate the human mutation in exon 7 of E3L, R185X. These mouse lines should offer an insight into the developmental role for E3L, and contribute to establishing a potential role for E3L in the innate immune system. This thesis exemplifies the complexity of the innate immune system and the regulatory pathways that interact to maintain a delicate homeostasis preventing pathogenic inflammation. Understanding these regulatory mechanisms may shed light on the pathogenicity of diseases and identification of potential targets for therapeutics.

Orientador: Iracilda Zeppone Carlos / Banca: Iracilda Zeppone Carlos / Banca: Luis Carlos Spolidorio / Banca: Márcia Antoniazi Michelin / Resumo: No Brasil, o câncer é a segunda principal causa de morte, sendo o câncer de mama o segundo mais frequente entre as mulheres. Um tumor sólido é composto pelo tecido neoplásico, células estromais e o sistema imune. Todas as células envolvidas produzem mediadores de forma local e sistêmica que influenciam no desenvolvimento do tumor. Os macrófagos ativados se polarizam e são capazes de ativar uma resposta imune celular, induzindo as células T para um padrão Th1, eficaz na eliminação de tumores; ou ativar uma resposta imune humoral, induzindo Th2, ineficiente contra o câncer. Na teoria, uma imunoterapia eficiente poderia alterar a ativação das células envolvidas e gerar uma inflamação voltada para um padrão de resposta celular, que gerasse a destruição das células cancerígenas. Dentre as possíveis imunoterapias, encontra-se o consumo de alimentos funcionais. A soja vem sendo estudada como um dos alimentos capazes de prevenir o câncer de mama a longo prazo. Além disso, as bactérias probióticas, parecem ser capazes de prevenir o câncer. Neste trabalho, procuramos observar o potencial do produto de soja fermentado por Enterococcus faecium CRL 183 e Lactobacillus helveticus ssp jugurti 416 enquanto alimento funcional capaz de modular o sistema imune e exercer influência no desenvolvimento de câncer de mama experimental murino. Os animais consumiram diariamente produto não fermentado de soja (PNFS), produto fermentado de soja acrescido (PFSI) ou não (PFS) de isoflavonas por 40 dias, sendo o inóculo tumoral realizado no décimo dia. PNFS, PFS e PFSI elevam a produção de TNF-α, e PFS e PFSI eleva a atividade da enzima arginase de macrófagos peritoneais. PFS gera uma redução na produção de IFN-γ e PNFS, PFS e PFSI reduzem os níveis de IL-5 produzidos por linfócitos esplênicos. O volume tumoral é menor em PFS e PFSI, e o número de células apoptóticas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In Brazil, cancer is the second main cause of death, and breast cancer is the second more frequent neoplasia in women. A solid tumor contains neoplastic tissue, stromal cells and immune cells. All cells present in this microenvironment may produce mediators locally and systemically which can influence tumor development. Activated macrophages polarize and are capable of activating a cellular response, inducing T cells into a Th1 cytokine pattern, which is effective against tumor; or activate a humoral response, leading T cells into Th2, unable to fight cancer properly. In theory, a effective immunotherapy could activate involved cells and trigger a acute inflammation, with a cellular pattern, capable of destroying cancer cells. One of the possible therapies is the use of functional food. Soy has been studied as one of the food capable of preventing breast cancer in a long term diet. Besides, probiotic bacteria seems to be able to prevent cancer as well. In the present work, we intended to observe the potential of a soy product fermented using Enterococcus faecium CRL 183 and Lactobacillus helveticus ssp jugurti 416 as a functional food capable of modulating immune system and influencing on experimental murine mamary tumor growth. Animals were fed daily non-fermented soy product (PNFS), fermented sou product (PFS) or fermented soy product plus isoflavones (PFSI) during 40 days, and tumor implant was performed on 10th day. PNFS, PFS and PFSI showed a higher production of TNF-α, while PFS and PFSI also showed a higher arginase activity by peritoneal macrophages. PFS reduced IFN-γ production, while PNFS, PFS and PFSI reduced IL-5 levels on splenic lymphocytes culture. Final tumor volume is reduced in PFS and PFSI, while number of apoptotic cells is elevated in these two groups. It appears that the product may influence on tumor development in a good way, but it is not clear whether immune system is important. / Mestre