The thymus in mice and humans originates from the third pharyngeal pouch endoderm. This process is divided into early Foxn1-independent stages and later Foxn1-dependent stages. Foxn1 is indispensible for the differentiation of thymic epithelial progenitor cells (TEPCs) as the development of thymus in Foxn1 mutant mice is arrested around E12.5. The transcriptional changes associated with the developmental of the thymus are poorly understood. In particular, the transcriptional regulation of Foxn1 in the developing thymic rudiment has not been definitively identified. Recently, Pax1, Pax9, Tbx1, and E2Fs have been implicated in transcriptional regulation of Foxn1. However, with the exception of E2Fs, evidence regarding their direct involvement in regulating Foxn1 expression is missing. Therefore, the aims of this thesis were to study the transcriptional regulation of Foxn1 through identification of its regulatory regions and studying the transcriptional changes associated with the developing thymus. These aims were addressed through the use of chromatin-immunoprecipitation technique combined with next-generation sequencing and gene expression analyses of the developing TEPCs. The data presented in this thesis identified H3K4me3 and H3K27ac marked Foxn1 promoter and five H3K4me1 and H3K27ac marked putative enhancer regions. The combination of gene expression analyses and transcription factor binding sites within the above regions suggested Ets1, Isl1, Foxc1, Nfia, Nfib, Srf, Foxo1, Nfatc2, Ing4, Foxa2, Hes1, E2Fs, and p53 as candidate transcriptional regulators of Foxn1. Nfatc2 appears also to be a target of Foxn1 that could play an important role in thymus development by regulating a large set of genes. Comparison of wild type and Foxn1 null thymus showed that Foxn1 could act as positive regulator of Pax1 and negative regulator of Gata3 and Eya1, genes important for third pharyngeal pouch development. The comparison of transcriptome of E10.5 and E11.5 third pharyngeal pouch cells and E12.5 TEPCs showed that genes involved in tissue development are downregulated while those involved in antigen presentation, a process important for thymus function, are upregulated during development. These results also demonstrated a decrease in the activity of transcription factor network involving Hox genes and an increase in the activity of a network involving Nfkb, Rela, and Irf genes. Analysis of signalling pathways suggested that the NFκB signalling pathway could be important for thymus development after E12.5 while TGFβ signalling pathway appeared to be detrimental to Foxn1 expression in thymic epithelial cells. Together, I identified several transcription factors that could be involved in transcriptional regulation of Foxn1 in TEPCs, several genes that could be a target of FOXN1, changes in transcription factor network and signalling pathways associated with the developing thymic rudiment.

Orientador: : Liana Maria Cardoso Verinaud / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T07:17:37Z (GMT). No. of bitstreams: 1 Francelin_Carolina_M.pdf: 13380043 bytes, checksum: 2e65773bab75b0bdc836c19740bd7d3e (MD5) Previous issue date: 2011 / Resumo: O timo é um órgão linfóide primário, responsável pela diferenciação, maturação e desenvolvimento dos linfócitos T (ou timócitos, enquanto no timo). Este órgão apresenta complexa arquitetura tímica composta por elementos celulares e solúveis responsáveis pela migração dos precursores de linfócito T do espaço perivascular para a região cortical e subseqüente migração para a região medular. A trajetória do timócitos pelos microambientes tímicos permite que estas células recebam estímulos que os tornam linfócitos maduros, simples positivos TCD4+ ou TCD8+, prontos para migrar para os órgãos linfóides secundários. Durante a maturação, timócitos que são auto-reativos ou anérgicos são eliminados pelo processo de morte celular programada. Entretanto, a desregulação da apoptose nos linfócitos pode acarretar no desenvolvimento de doenças auto-imunes, tumores e imunodeficiências. Estudos em modelos murinos de atrofia tímica relatam que em diversas infecções parasitárias, virais, helmínticas, bacterianas e fúngicas e até mesmo em distúrbio metabólico como a diabetes experimental o timo é um órgão alvo. Nestes modelos, foi observado intenso acometimento tímico com involução do órgão acompanha por desarranjo da arquitetura tímica e depleção de todas as subpopulações de timócitos. Em estudos anteriores, observamos profundas alterações no timo durante a infecção experimental com Plasmodium berghei que incluíam severa atrofia no décimo quarto dia após inoculação do parasita, alterações histológicas, perda da delimitação cortico-medular, redução de todas sub-populações de timócitos e a presença intra-tímica do parasita. No presente trabalho avaliamos se a grande perda de timócitos observada no timo de animais infectados pelo Plasmodium berghei NK65 é devido a um aumento da ocorrência de apoptose, e/ou necrose, ou se é devida à saída prematura destas células para a periferia.A avaliação de morte celular na população de timócitos total revelou aumento no número de células Anexina-V+/PI- em animais infectados quando comparados aos controles.Ainda, a análise da apoptose em timos atróficos, no pico da parasitemia, revelou aumento de núcleos apoptóticos nas regiões cortical e medular tímica bem como aumento no número de linfócitos (CD4+, DP e DN) Anexina-V positivos. Alteração na expressão de genes pró-apoptóticos no timo de animais infectados quando comparado ao controle, sugerindo a participação de vias de ativação extrínsecas especificas da apoptose, também foram observadas. Embora 90% dos timócitos de animais infectados sejam Anexina-V positivos, as alterações na expressão dos elementos de matriz extracelular, relatadas anteriormente pelo nosso grupo, sugere também a exportação precoce destas células para a periferia do sistema imune. Nossos resultados apontam para um aumento de linfócitos imaturos (DP e DN) nos linfonodos mesentéricos de animais infectados, um indicativo de que parte dos timócitos imaturos sai do timo antes de completarem seu amadurecimento. Em conjunto, nossos dados mostram que as alterações estruturais e de componentes solúveis do timo durante a infecção com Plasmodium berghei NK65 podem favorecer tanto a deleção, por apoptose, de timócitos corticais como também a migração precoce de células imaturas para a periferia do sistema imune. O acometimento tímico na atrofia tímica induzida pelo Plasmodium berghei NK65 revela que durante uma infecção o timo sofre influências de diversos fatores que provocam profundas alterações nas subpopulações linfocitárias. Também, é possível que as alterações observadas em nosso modelo prejudique a resposta imune periférica contra o parasita contribuindo para o agravamento da doença e que a presença de timócitos prematuros nos linfonodos favoreça o desenvolvimento de doenças auto-imunes / Abstract: The thymus is the primary lymphoid organ responsible for the differentiation, maturation and development of T lymphocytes (or thymocytes, as in the thymus).This organ presents a complex architecture composed by soluble and cellular elements that support the migration of lymphocyte precursors from space perivascular to the cortical region, and then their migration toward the medullar area. The trajectory of thymocytes by thymic microenvironments allows these cells to receive stimuli that make them mature into CD4+ or CD8+ T cells, which leave the thymus to populate peripheral lymphoid organs. Studies using murine models of thymic atrophy have reported that thymus is a target organ in several parasitic, viral, helminthic, bacterial and fungal infections. In these models, one can observe intense thymic involution accompanying by disruption of thymic architecture and depletion of all thymocyte subpopulations. In a previous study, our laboratory has demonstrated profound changes in the thymus during experimental infection with Plasmodium berghei, the causative agent of Malaria. Such alterations comprised severe atrophy, histopathological changes with loss of cortical-medullary delimitation, and reduction of all thymocytes sub-populations. In addition, the presence of parasites inside the thymus was detected for the first time. In another study using the same experimental model, we have demonstrated that Plasmodium-infected animals present changes in the expression of extracellular matrix elements and chemokine protein families, which are important molecules for the dynamic thymocyte migration within the thymus. In this study we evaluate whether the acute thymic atrophy observed in Plasmodium berghei-infected animals is correlated with increased apoptotic levels of thymocytes, or with their premature emigration to the periphery. Our data showed profound histological alterations, which included a very large number of cells showing nuclear condensation and karyorrhectic changes surrounded by histiocytes suggesting increased levels of apoptosis. This was confirmed by immunohistochemistry and flow cytometry techniques. Also, increased expression of proapoptotic genes was observed in the thymus of infected animals when compared with controls, suggesting the involvement of the extrinsic, or receptor-iniciated, pathway of apoptosis. Although the rate of apoptosis in the thymus from infected animals is much higher than the controls, the hypothesis that a premature emigration of thymic cells to the peripheral lymphoid organs may also account for the severe atrophy observed during Plasmodium infection could not be ruled out. In fact, we have found a consistent fraction of immature cells, mainly double-negative and double-positive thymocytes, in mesenteric lymph nodes of infected animals. Although this result serves as an evidence of premature emigration of non-mature thymocytes, the precise mechanism by which this process occurs is not clear until now. No significant differences were found in the spleen. Taken together, our data show that thymic alterations observed during Plasmodium berghei infection can favour both the deletion of thymocyte subpopulation by apoptosis and the early emigration of immature cells to the periphery of the system immune. As a consequence, an altered peripheral immune response to the parasite can be expected. Our findings provide relevant information concerning the immune involvement in malaria infection. Moreover, the murine model described here could be useful for mechanist studies of acute thymic atrophy / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

O timo é um órgão essencial para a maturação, diferenciação e seleção de linfócitos T, consequentemente fundamental para o desenvolvimento da imunidade do organismo. Além do papel na manutenção da integridade tecidual, os vasos sanguíneos tímicos desempenham função no processo de migração de células precursoras de linfócitos para o interior do órgão. Sua arquitetura típica é caracterizada por grandes vasos localizados na junção corticomedular e uma fina cadeia de ramificações e anastomoses no interior do córtex, porém são pouco conhecidas as bases moleculares que induzem a formação desta particular vascularização, bem como os exatos mecanismos que desencadeiam a involução do órgão, evento relacionado a alterações nos padrões vasculares. Entretanto, tem sido demonstrado que as interações entre o estroma, que inclui o endotélio vascular, e o compartimento hematopoiético tímico desempenham papéis cruciais na funcionalidade do órgão. Sendo o VEGF um fator angiogênico essencial na formação do leito vascular tecidual e na modulação de funções diretamente relacionadas à vascularização, objetivamos neste estudo avaliar a expressão gênica e protéica deste fator de crescimento durante estágios de desenvolvimento e involução do órgão. Foram utilizadas amostras de timo suíno em 3 diferentes estágios de desenvolvimento fetal (65, 85 e 111 dias) e 2 estágios de vida pós-natal, um compreendendo fase de maturação (5 meses) e outro de involução do órgão (2 anos), compreendendo um total de 25 animais, divididos em 5 grupos (n = 5). A análise da expressão relativa do mRNA do sistema VEGF-A, acessada por meio de PCR em tempo real, apresentou modificações tempo-dependentes significativas (P<0,05) em relação ao controle endógeno GAPDH. Também foram observadas diferenças significativas (P0,05) na quantificação vascular por meio de estereologia entre as idades. A imunolocalização da proteína do VEGF e de seus receptores Flt-1 e KDR foi identificada no timo de todos os grupos experimentais e variou entre os grupos pré e pós-natais, com expressão mais rara nestes últimos, o que sugere diferenças temporais na modulação do mRNA no órgão. Assim como as células endoteliais, as células epiteliais tímicas demonstraram modulação pelo sistema VEGF-A, indicando ação parácrina e/ou autócrina deste fator de crescimento no timo. Os valores das variáveis estereológicas Sv[m] e Vv[m] do timo fetal em período próximo ao nascimento mantiveram-se maiores do que no córtex e se correlacionaram positivamente com a expressão do mRNA do KDR e negativamente com a do Flt-1 até a idade adulta. Ao contrário da expressão protéica, a expressão gênica do sistema VEGF-A pode se correlacionar negativamente com aspectos quantitativos da vascularização do timo pós-natal, como observado para o mRNA do VEGF e seu receptor Flt-1 a partir dos 5 meses de idade, quando os valores das variáveis Vv[vasos,timo], Sv[vasos,timo] e Lv[vasos,timo] diminuíram significativamente (P0,05), o que sugere uma resposta de compensação ou manutenção de uma condição hipóxica instalada no órgão. / Crucial for maturation, differentiation and selection of T lymphocytes, the thymus is an essential organ to the development of immunity. In addition to its role in the maintenance of tissue integrity, thymus blood vessels play a role in migration of T-cell precursors into the organ, and show a typical architecture characterized by the presence of large blood vessels at the corticomedullary junction and fine network of branching vessels and anastomosing arcades extending into the cortex. The molecular basis of this typical vasculature and the exact mechanisms that trigger thymus involution are both not well understood. However, it has been shown that the interactions between thymus stroma, including vascular endothelium and the hematopoietic compartment play crucial roles for normal organ function. Thus, because VEGF is an essential angiogenic factor for induction of the vascular bed and modulation of functions directly related with vascularization, the aim of this study was to assess the temporal protein and mRNA expression of VEGF and its receptors Flt-1 and KDR in the thymus during early stages of its development and involution. Samples of thymus from 3 different stages of fetal development (65, 85, and 111 days) and 2 stages of postnatal life of pigs, including one stage of maturation (5 months) and another of involution (2 years) comprising a total of 25 animals that were divided into 5 groups (n = 5). Relative mRNA expression of VEGF-A system, accessed by real-time PCR, showed temporal significant changes (P<0.05) compared to endogenous control GAPDH. Quantification of blood vessels by means of stereology also showed significant differences (P0.05) between ages. Positive immunostaining for VEGF and its receptors Flt-1 and KDR was identified in the thymus of all groups and it varied between the pre and postnatal groups with rare expression in the latter, suggesting temporal differences in the modulation of mRNA. Endothelial and epithelial cells showed modulation by the VEGF-A system, suggesting paracrine or alternatively autocrine activity of this growth factor. The values of the stereological parameters Sv[m] and Vv[m] of the thymus in fetal period around the birth remained higher than in the cortex and upregulated with mRNA expression of KDR and downregulated with mRNA expression of Flt-1 until adulthood. Unlike protein expression, gene expression gene expression of VEGF-A system can be downregulated with quantitative aspects of the postnatal thymus vascularization as observed for the mRNA of VEGF and its receptor Flt-1 from the age of 5 months, when the values of the stereological parameters Vv[vessels,thymus], Sv[vessels,thymus] and Lv[vessels,thymus] decreased significantly (P0.05), which suggests a compensatory or maintenance response to an hypoxic condition in the organ.

Orientador: Liana Maria Cardoso Verinaud / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T08:26:55Z (GMT). No. of bitstreams: 1 Thome_Rodolfo_D.pdf: 18503134 bytes, checksum: f4906f6cd7d52ca62d211efbbca1732a (MD5) Previous issue date: 2014 / Resumo: A malária permanece como a maior doença infecciosa do mundo, sendo que metade da população mundial se encontra em risco de ser infectada. A destruição do agente causador, protozoários do gênero Plasmodium, é essencial para a resolução da moléstia e é mediada pela atuação coordenada entre linfócitos T e B. Foi noticiado a presença de auto-anticorpos em pacientes portadores de malária, e em alguns casos o agravamento de doenças autoimunes como o lúpus e, possivelmente, a esclerose múltipla. Estudos conduzidos em nosso laboratório demonstraram que a infecção por P. berghei NK65 em camundongos promoveu atrofia do timo e subsequente migração de células T CD4+CD8+ (DP) para órgãos linfoides secundários. Tendo em vista que o timo é o órgão linfoide primário responsável pela maturação e desenvolvimento de linfócitos T, desempenhando um papel primordial em gerar a tolerância central, o presente estudo teve por objetivo investigar o efeito da infecção por P. berghei sobre a evolução clínica da Encefalomielite Autoimune Experimental (EAE) e também sobre o perfil de ativação de células dendríticas. Os resultados obtidos mostraram que camundongos curados da malária apresentaram quadro clínico exacerbado de EAE quando comparados com animais que não entraram em contato com o plasmódio. A piora no quadro clínico de EAE se associou com a migração precoce de células T-DP para o Sistema Nervoso Central (SNC) e pela produção de citocinas inflamatórias por tais células. Camundongos resistentes a EAE desenvolveram a doença após a infecção por plasmódio, indicando que a atrofia tímica induzida pela infecção é capaz de alterar a susceptibilidade ao desenvolvimento de doenças autoimunes. Por outro lado, o tratamento de células dendríticas (DCs) com extratos derivados do plasmódio modificou seu estado de maturação/ativação para um perfil tolerogênico. A transferência adotiva de células dendríticas moduladas com extratos de plasmódio foi capaz de reduzir a EAE, bem como a neuro-inflamação do SNC pela supressão da resposta imune celular a neuro-antígenos. Tomados em conjunto, os resultados obtidos neste estudo mostram que a infecção por Plasmodium berghei NK65 promove alterações significativas no sistema imune que auxiliam na exacerbação da neuro-inflamação autoimune. Por outro lado, a utilização de extratos do plasmódio pode se tornar uma alternativa inovadora na modulação da inflamação por meio da transferência adotiva de células dendríticas tornadas tolerogênicas com tais extratos / Abstract: Malaria remains as the most frequent infectious disease in the world, where half of the world population is at risk. Destruction of the causative agent, protozoan of the Plasmodium gender, is essential for the resolution of the disease and this is mediated by the coordinated action of both T and B lymphocytes. It has been demonstrated that malaria-bearing patients possess auto-antibodies, e in some cases, the worsening of autoimmune diseases such as lupus and multiple sclerosis. Studies, conducted by our group, have shown that P. berghei NK65 infection promoted thymic atrophy and the subsequent migration of CD4+CD8+ (DP) T cells towards the peripheral immune system. Since the thymus is the primary lymphoid organ responsible for the generation and maturation of T cells, playing a major role in the shaping of T cells repertoire, the present study aimed to investigate the influence of P. berghei infection in the clinical course of Experimental Autoimmune Encephalomyelitis (EAE), the mouse model for multiple sclerosis, and also over the maturation/activation status of dendritic cells. Results showed that malaria-cured mice developed a more severe EAE clinical course compared with control mice. The worsening in EAE score was related to the migration of DP-T cells towards the Central Nervous System (CNS), where these cells produced high amounts of inflammatory cytokines. Interestingly, EAE-resistant BALB/c mice developed the disease after plasmodia infection, indicating that the thymic atrophy induced by the infection is able to alter the susceptibility to autoimmune diseases. On the other hand, treatment of dendritic cells (DCs) with P.berghei extracts (PbX) modified their activation/maturation status towards a tolerogenic profile. The adoptive transfer of DC-PbX was able to suppress the development of EAE, as well as neuro-inflammation, through the reduction in cellular immune responses towards neuro-antigens. Taken together, the results collected in this study show that Plasmodium berghei NK65 infection promotes significant alterations in the immune system that aid the development of autoimmune neuro-inflammation. On the other hand, the use of plasmodia extracts may become an interesting approach to modulate inflammation through the adoptive transfer of tolerogenic dendritic cells / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

(ENG) This thesis summarizes the results of a project dedicated to adaptive immune system of patients with partial DiGeorge syndrome caused by deletion of 22q11.2. The introduction sets the DiGeorge syndrome into a broader context of international pathophysio- clinical classification of primary immunodeficiencies and goes into detail describing its history, causes, clinical phenotype, therapeutic options and changes of the immune system. The attached manuscripts illustrate the premature aging of the T cell population, but also impaired development of B cells with low class-switched memory and high naïve subpopulations, along with high serum levels of BAFF, a B cell survival factor. The surprising lack of T independent marginal zone- like (MZ-like) B cells is reflected in decreased natural anti-α-Gal antibodies. The faulty B cell maturation and imperfect germinal center response is not caused by a deficit of follicular helper T cells, which are in fact increased in DiGeorge syndrome patients, and in most cases doesn't lead to hypogammaglobulinaemia. Despite the high incidence of autoimmune disease, in particular thyroiditis and thrombocytopenia, and a trend towards hypergammaglobulinaemia in adolescence and adulthood, we saw normal proportion of regulatory T cells (Tregs) and normal expression of...

Genomweite Assoziationsstudien haben CLEC16A als ein Suszeptibilitätsgen für Typ 1 Diabetes und weitere Autoimmunerkrankungen identifiziert. Die genaue Funktion von CLEC16A bleibt jedoch ungeklärt. Studien zeigten, dass sowohl das Drosophila Ortholog ema als auch das murine Clec16a eine Rolle in Autophagie spielen. Autophagie trägt zur Beladung der MHC-Klasse-II Moleküle und somit der Antigenpräsentation bei. Darüber hinaus konnten Studien belegen, dass Autophagie zur Antigenpräsentation während der T-Zell Selektion in Thymus-Epithelzellen benötigt wird. Dies schlägt eine mögliche Funktion von CLEC16A in Thymus-Epithelzellen während der T-Zell Selektion vor. Außerdem berichteten Arbeiten, dass CLEC16A als quantitativer Trait Locus für seine Nachbargene fungiert und dass Clec16a KD in Langerhans Inseln im Pankreas die Insulinsekretion und den Glukosestoffwechsel beeinträchtigt. Dieser Arbeit vorausgehend hatten Schuster et al. eine Clec16a KD NOD Maus generiert, welche vor spontanem autoimmunem Diabetes geschützt war. Für diese Arbeit wurde vermutet, dass CLEC16A als Suszeptibilitätsgen für Typ 1 Diabetes den Prozess der Autophagie in Thymus-Epithelzellen beeinträchtigt und somit Antigenpräsentation und das T-Zell Repertoire beeinflusst. Um auf der Vorarbeit von Schuster et al. aufzubauen und diese zu ergänzen, zielte diese Arbeit darauf ab, den Einfluss von CLEC16A auf Thymus-Epithelzellen zu untersuchen. Hierfür wurde ein CLEC16A KD in menschlichen Zellen mittels RNA Interferenz erzeugt und Autophagie durch Immunoblotting untersucht. Zusätzlich wurde die Entzündung im Pankreasgewebe von Clec16a KD NOD Mäusen mittels H.E. Färbung beurteilt und bewertet. Thymus-Transplanationen wurden durchgeführt, um zu sehen, ob der Einfluss von Clec16a KD T-Zell intrinsisch ist. Außerdem wurden intraperitoneale Glukosetoleranztests durchgeführt, um den Blutzuckerstoffwechsel in Clec16a KD Mäusen zu beurteilen. Schließlich wurden mittels qPCR Expressionslevel der benachbarten Gene, wie zum Beispiel Dexi und Socs1, erhoben, um die Eigenschaften von CLEC16A als quantitativer Trait Locus einzuordnen. Gemeinsam mit den Ergebnissen von Schuster et al. kann diese Arbeit aufzeigen, dass Clec16a KD die Ausprägung von Insulitis im Pankreas reduziert und Clec16a KD NOD Mäuse vor spontanem Autoimmundiabetes schützt. Dieser Schutz vor Erkrankung wird durch beeinträchtigte Autophagie in Thymus-Epithelzellen hervorgerufen, welche die T-Zell Selektion beeinflusst und die Reaktivität von T-Zellen reduziert. Der Einfluss des Clec16a KD ist innerhalb des Thymus wirksam. Der Blutzuckerstoffwechsel in Clec16a KD NOD Mäusen bleibt unverändert und kann deshalb als Ursache für den Schutz vor Type 1 Diabetes ausgeschlossen werden. Clec16a und Dexi zeigen ähnliche Expressionslevel auf, dennoch benötigt es weitere detaillierte Studien, um eine Beziehung zwischen den beiden Genen etablieren zu können. Letztlich konnte die Beeinträchtigung von Autophagie in menschlichen CLEC16A KD Zellen nachgewiesen werden, was bedeutet, dass die Funktion von CLEC16A evolutionär konserviert ist und ein möglicher Zusammenhang zwischen CLEC16A Polymorphismen und einem erhöhten Risiko für Typ 1 Diabetes im Menschen besteht. / Genome-wide association studies revealed CLEC16A as a candidate gene for Type 1 Diabetes and multiple other autoimmune disorders. The function of CLEC16A remains unknown. However, previous work showed that the CLEC16A ortholog ema and the murine Clec16a were both implicated in autophagy, a process partially required for MHC class II loading and antigen presentation. Furthermore, studies could show that autophagy was required in thymic epithelial cells for antigen presentation during T cell selection, suggesting a possible role of CLEC16A in T cell selection in the thymus. Additionally, it was postulated that CLEC16A may function as an expression quantitative trait locus for its neighboring genes and that Clec16a KD was involved in pancreatic islet function and impaired insulin secretion and glucose homeostasis. Prior to this work, Schuster et al. had created a Clec16a KD NOD mouse, which was protected from spontaneous autoimmune diabetes. For this work it was hypothesized that CLEC16A variation serves as a Type 1 Diabetes risk gene by affecting autophagy in thymic epithelial cells, which modulates antigen presentation and shapes the T cell repertoire. To expand and complement previous findings by Schuster et al., this thesis aimed to investigate how CLEC16A modifies the function of thymic epithelial cells. For this purpose, CLEC16A KD was induced in human cells via RNA interference and autophagy was studied through immunoblotting. Additionally, inflammation of pancreatic tissue in Clec16a KD NOD mice was scored using H.E. stained pancreatic sections. Thymic transplantation experiments were conducted to test whether the effects of Clec16a KD were T cell intrinsic. Also, intraperitoneal glucose tolerance tests were performed to study glucose homeostasis in Clec16a KD NOD animals. Finally, using qPCR, gene expression levels of neighboring genes such as Dexi and Socs1 were measured to study Clec16a as an expression quantitative trait locus. In combination with the findings of Schuster et al., this thesis demonstrates that Clec16a KD reduces the severity of insulitis and protects from onset of spontaneous diabetes in the NOD mouse. Disease protection is conveyed by impaired autophagy in TEC, which leads to altered T cell selection and hyporeactive CD4+ T cells. The effects of Clec16a KD in the NOD mouse are thymus intrinsic. Glucose homeostasis remains unchanged in the Clec16a KD NOD mouse and plays no role in disease protection. Clec16a and Dexi presented similar expression levels, but further studies are required to investigate a clear link between these two genes. Finally, impaired autophagy could be replicated in human CLEC16A KD cells, which demonstrates a conserved function of CLEC16A and suggests a possible link between CLEC16A variation and risk of autoimmune disease in human.

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Les travaux présentés dans cette thèse ont été entrepris dans le but de mieux caractériser des événements influençant la signalisation du récepteur de surface des lymphocytes T (TCR) au cours de la sélection thymique, étape importante dans la maturation des lymphocytes T. Le premier aspect étudié a été la modulation de l'activation de la MAPK (mitogen-activated protein kinase) ERK par le TCR, puisqu'il semble que l'intensité et la durée de l'activation de cette protéine contrôle l'issue de la sélection thymique soit la survie ou la mort de la cellule activée. Nous avons mis en évidence une interrelation entre l'activation de ERK et celle d'une autre protéine de la famille des MAPKs, p38. En utilisant un inhibiteur spécifique de l'activité p38 et également par la transduction d'une protéine dominante négative de p38 fusionnée au peptide TAT, permettant au complexe TAT-p38 d'entrer rapidement dans les cellules, nous avons observé que l'activation maximale de ERK par le TCR est dépendante de celle de p38. En deuxième lieu nous avons observé que l'adénosine inhibe l'activation du TCR en interagissant avec ses récepteurs de surface. Nous avons tenté d'identifier le ou les récepteurs d'adénosine responsable(s) du phénomène. Notre étude a déterminé que l'activation de ERK par le TCR était diminuée en présence d'adénosine et d'inhibiteur d'adénosine déaminase (de façon à empêcher la dégradation d'adénosine dans le milieu). L'utilisation d'agonistes et d'antagonistes des différents récepteurs d'adénosine connus (A1, A2A, A2B et A3) chez des souris normales ou déficientes pour le récepteur A2A a permis d'impliquer le récepteur A2A et de suggérer l'implication du récepteur A2B dans l'inhibition de la signalisation du TCR par l'adénosine. En conclusion, nos travaux ont apportés une meilleure connaissance des processus de régulation de l'activation de ERK par le TCR, une protéine déterminante dans le résultat final de la sélection thymique. De plus, il a été démontré que l'inhibition de la signalisation du TCR par l'adénosine est attribuable, du moins en partie, au récepteur A2A.

Diethylstilbestrol (DES) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been identified as immunotoxicants causing thymic atrophy, thymocyte hypocellularity, phenotypic changes detected by CD4 and CD8 surface antigens, and progenitor T-cell targeting in the fetal mouse. We hypothesized that gestational exposure to these two compounds may lead to comparable histologic and gene expression alterations in the fetal mouse thymus and liver. Treatment of pregnant C57Bl/6 mice with doses of 5 or 10 ug/kg TCDD or 48 ug/kg DES by oral gavage on gestation days (gd) 14 and 16 severely depressed day 18 thymic cellularity. Histologic evaluation of day 18 fetal thymuses showed disruption of normal cortico-medullary architecture after TCDD or DES. Decreased thymocyte density was noted primarily in cortical zones where pyknotic cells were increased by either TCDD or DES treatment. Using day 18 thymocyte suspensions and flow cytometry, 7-AAD showed decreases in viable thymocytes from TCDD- or DES-treated fetal mice, and concomitant increases in thymocytes in early apoptosis. When thymocytes were co-identified with CD4 and CD8 cell surface antigen expression, enhanced apoptosis occurred in CD4+CD8+ phenotype after TCDD treatment. After DES exposure, increased apoptosis occurred in CD4-CD8- and CD4-CD8+thymocytes. Both TCDD and DES increased liver to body weight ratios and decreased ratios of hematopoietic to hepatic cells present. Cytomegaly was seen in hepatocytes of TCDD and DES treated animals, and these cells had more variable features, such as increased cytoplasmic basophilia and more prominent nucleoli. Real time quantitative PCR demonstrated that DES decreased c-jun, Bcl-2, and PKCalpha mRNA expression. These results suggest a shift away from proliferative activity and may reflect alterations noted predominantly in the hematopoietic population. TCDD increased c-jun mRNA expression with modest decreases in PKCalpha, and marked decreases in p53 also noted. Decreases in p53 suggest a pro-proliferative status of hepatic cells, while decreases in PKCalpha may indicate decreases in phosphorylation of substrates required for normal cell cycle progression. The increased c-jun suggests that this gene may play a role in the hepatocyte hyperplasia, as well as the diminution of hematopoiesis. / Ph. D.