The thymus is the major site for T-cell generation and thus is important for the adaptive immune system. Development of a properly selected, functional T-cell repertoire relies on interactions between developing T cells and a series of functionally distinct thymic stroma cell types including the cortical and medullary thymic epithelial cells (TECs). The thymus is one of the first organs to degenerate in normal healthy ageing. Related to this, there is strong interest in developing protocols for improving thymus function in patients by cell replacement or regenerative therapies. Thymic epithelial progenitor cells (TEPCs) represent a potential source of cells for thymus transplantation. However, the only source of these cells for transplantation is currently fetal thymus tissue. If TEPCs could be generated from pluripotent cells, this could provide an alternative source of cells for transplantation. The work described in this thesis therefore had two central aims (i) to test the stability of thymic epithelial progenitor cells in vivo and (ii) to investigate the possibility of generating TEPCs or TECs from mouse embryonic stem (ES) cells. The forkhead transcription factor, Foxn1, is essential for the development of a functionally mature thymic epithelium, but is not necessary for formation of the thymic primordium or for medullary thymic epithelial sub-lineage specification. By reactivating Foxn1 expression postnatally in mice carrying a revertible hypomorphic allele of Foxn1, Foxn1R, I herein demonstrate that TEPCs that can express only low levels of Foxn1 mRNA can persist postnatally in the thymic rudiment in mice until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression. In parallel with this work, I confirmed the possibility of generating Foxn1-expressing cells from mouse ES cells by using a Foxn1 reporter cell line. As the thymic epithelium has a single origin in the third pharyngeal pouch (3pp) endoderm, I then tested whether or not TEPCs and /or TECs were generated during ES cell differentiation via existing protocols for generating anterior definitive endoderm differentiation cells from mouse ES cells. From this work, I showed that genes expressed in the 3pp and/or TEPC,-including Plet-1, Tbx1, Hoxa3 and Pax9, were induced by differentiation of ES cells using these protocols. I further showed that cells expressing both Plet-1, a marker of foregut endoderm and 3pp, and EpCAM, a marker of proliferating epithelial cells, were induced using a novel protocol (2i ADE) for generating ES cells from ADE. However, gene expression analysis and functional testing suggested that the majority of these cells were non-thymus lineage. I subsequently developed a novel protocol which combined this 2i ADE protocol with co-culturing of the differentiating ES cells with fetal thymic lobes, and demonstrated that this further induced 3pp and TEPC related genes. Finally, I modified the culture conditions in this protocol to conditions predicted to better support TEPC/TEC, and showed that in these conditions, the TEPC-specific markers Foxn1 and IL-7 were induced more strongly than in any other conditions tested. The data presented in this thesis therefore represent an advance towards an optimized protocol for successfully generating TEPCs from ES cells in vitro.

O sistema linfoide é composto de órgãos linfoides primários e secundários. O timo é um órgão linfoide primário responsável pela maturação, diferenciação e seleção da linhagem linfocitária do tipo T que é responsavel pela imunidade celular do individuo. Para cumprir estas funções, o timo possui uma disposição peculiar das suas células epiteliais morfologicamente distintas e de suas estruturas vasculares. Seus vasos sanguíneos possuem um papel na oxigenação tecidual e no processo de migração das células precursoras de linfócitos T para o interior do parênquima tímico e por isso apresentam uma arquitetura típica caracterizada por vasos de grande calibre, localizados na junção cortico-medular e uma fina rede de ramos e anastomoses que se estendem para o córtex. Este processo de estruturação e arquitetura vascular ainda possui sua base molecular desconhecida, assim como os mecanismos que provocam a involução do órgão. O VEGF é um fator angiogênico que atua na formação vascular e na modulação de funções relacionadas à vascularização, sendo um importante marcador da angiogênese. A fim de se melhor compreender o comportamento vascular na formação e involução tímica, propôs-se avaliar a expressão gênica e proteica deste fator durante fases de desenvolvimento e involução do órgão, além da quantificação da vascularização do timo pela técnica estereológica, análise do parênquima tímico pela técnica de microscopia eletrônica de varredura e análise dos tipos celulares presentes em cada estágio etário. Para tal utilizou-se amostras de timo de gato em quatro estágios de desenvolvimento fetal (35, 45, 55, 65), e dois estágios pós-natal (6 meses e 1 ano) para a realização da imuno-histoquímica, PCR em tempo real e MEV,e para a técnica estereológica 2 estágios pós-natal (6 meses e 1 ano). Na microscopia eletrônica de varredura foram observados os timócitos de diferentes tamanhos, em estágios de maturação distintos. As proteinas do VEGF-A e dos receptores Fit-1 e KDR foram identificadas no timo de gatos em todas as fases do desenvolvimento foram localizadas no citoplasma de células epiteliais e no interior dos corpúsculos tímicos. A expressão do mRNA no período de 1 ano de idade a expressão do mRNA do VEGF e seus receptores tem um aumento significativo, coincidindo com a diminuição do Nvasc e do Nv(vasc) podendo causar um estado de hipóxia no órgão levando a um aumento compensatório de sistema VEGF. A curva de crescimento vascular obedece a um padrão de desenvolvimento e involuçãio do órgão. / The lymphoid system is composed by primary and secondary lymphoid organs. The thymus is a primary lymphoid organ responsible for maturation, differentiation and selection of the lymphoid T cell lineage that is responsible for cellular immunity. To accomplish these functions has a peculiar arrangement with morphologically distinct epithelial cells and vascular structures. The blood vessels have a role in tissue oxygentation and the migration of T cells into the thymic parenchyma, therefore they presents large vessels in cortico-medullary junction and a fine network branches to the cortex. This process has its molecular basis unknown as well as the involution process of the thymus. VEGF is an angiogenic factor that plays a role in the formation and modulation of vascular functions, being an important marker of angiogenesis. We proposed to evaluate the gene and protein of VEGF during the thymus development and involution, stereological quantification and scanning electronic microscopy. Samples of cat´s thymus from 35, 45, 55, 65 days of development and 6 months and 1 year of age. In scanning electronic microscopy different stages maturation thymocytes were observed. Protein expression of VEGF and its receptors were identified in all development stages in epithelial cells, endothelial cells and thymic corpuscles. The VEGF mRNA expression and its receptors in 1 year old animals was significantly increased, coinciding with the decreasing Nvasc and the Nv(vasc) causing a hypoxic condition in the thymus resulting in a compensatory increase of VEGF system. The vascular growth curve follows a pattern of development and involution of the organ.

L’induction de tolérance est le principal objectif de la recherche en transplantation car elle permettrait de prévenir le rejet et d’éliminer la morbidité secondaire à l’immunosuppression prolongée. Dans ce cadre, le thymus pourrait être un organe crucial car il permet d’établir la taille et la diversité de la population lymphocytaire T et est responsable de la génération d’une tolérance immunitaire en façonnant le répertoire T par le processus de sélection négative. En plus des lymphocytes T, les cellules dendritiques (CDs), épithéliales et les lymphocytes B jouent un rôle important dans le thymus, en tant que cellules présentatrices d’antigènes (CPAs). Ainsi, le thymus est un organe hautement dynamique, à l’interface entre système immunitaire central et périphérique. Alors qu’il débute son involution avant l’âge adulte, une fonction thymique persiste toute la vie, avec un grand degré de variabilité interindividuelle.Dans le cadre de la transplantation cardiaque, les patients sont souvent soumis à un traitement lymphodéplétant par sérum anti-lymphocytaire (SAL), afin de réduire le risque de rejet, mais avec un potentiel risque iatrogénique pour le thymus. De plus, ces patients subissent une chirurgie au cours de laquelle les reliquats thymiques pourraient être partiellement réséqués.Sachant que la fonction thymique varie entre les individus et qu’une régénération thymique est possible dans les suites d’une lymphodéplétion, nous avons émis l’hypothèse que la transplantation cardiaque serait un contexte associé à une réactivation de la fonction thymique et qu’une relation pourrait exister entre cette fonction et la survenue d’un rejet.Tout d’abord, j’ai analysé l’influence des thérapies d’induction sur la cinétique de repopulation lymphocytaire. J’ai étudié les sous-populations lymphocytaires avant et peu après la transplantation chez des patients recevant une induction par basiliximab (antagoniste du récepteur de l’IL-2) ou SAL et ai montré que le SAL induisait une sénescence immunologique précoce. Cette première partie a permis la caractérisation des modifications immunologiques induites par le SAL avant d’analyser les populations lymphocytaires chez les patients transplantés traités par le SAL en fonction de la survenue d’un rejet. Dans ce cadre,j’ai ensuite démontré qu’une thymopoïèse plus intense était observée chez les patients développant un rejet humoral (RH), par l’étude des cercles d’excision du récepteur des cellules T (TREC), la quantification des émigrants thymiques récents (ETR) et une évaluation par imagerie dans un sous-groupe de patients. Ce résultat était plutôt inattendu sachant que le thymus est largement décrit comme un organe tolérogène pour les réponses alloimmunes T.Afin de clarifier le rôle du thymus dans la réponse humorale alloimmune, nous avons mis en place un modèle murin d’allotransplantation aortique (AA) associé ou non à une thymectomie prophylactique. Nous avons démontré que la thymectomie réduisait la réponse médiée par les anticorps, avec une expansion concomitante des lymphocytes T régulateurs. En conclusion, ces données suggèrent que la suractivation du thymus pourrait promouvoir une réponse alloimmune humorale, un résultat inattendu car d’autres travaux indiquent que le thymus est plutôt un inducteur de tolérance. Le thymus pourrait avoir un double rôle, en promouvant la tolérance lymphocytaire T mais aussi, dans un contexte de suractivation, la survenue du RH par un mécanisme restant à élucider. D’autres études doivent être menées pour mieux comprendre l’interaction entre les différents contingents cellulaires thymiques et la modulation des fonctions du thymus au cours de la vie et dans les situations de réactivation du thymus. / The induction of tolerance remains the main goal of transplantation research because it would prevent the development of rejection and eliminate the morbidity associated with prolonged immunosuppression. In this setting, the thymus could be a crucial organ because it establishes the size and diversity of the naïve T-cell pool and is responsible for the generation of immunetolerance by shaping the T-cell repertoire through the process of negative selection. Inaddition to T lymphocytes, dendritic cells (DCs), epithelial cells and B lymphocytes play animportant role in the thymus as antigen-presenting cells (APCs). Therefore, it is a highlydynamic organ, functioning at the interface between the central and peripheral immunesystem. While it undergoes involution before adulthood, thymic function persists throughout life, though with a high degree of interindividual variability. In the field of heart transplantation, patients are often subjected to lymphodepleting therapy by antithymocyteglobulin (ATG), which is expected to reduce the risk of acute rejection but potentiallyr epresents a main contributor to iatrogenic thymic injury. Additionally, heart transplant (HTx)patients under go surgery during which the thymic remnants may be at least partially removedor surgically injured. Considering the variation in thymic function between individuals and possibility of thymic regeneration following lymphodepletion, we hypothesized that heart transplantation could be a clinical situation associated with a reactivation of thymic functionand that there could be a relationship between this function and the onset of rejection. First, I analyzed the influence of induction therapies on the repopulation kinetics of lymphocytes. I studied lymphocyte subpopulations in pre- and early post-transplant period ina cohort of patients receiving either basiliximab (IL-2 receptor antagonist) or ATG inductionand showed that ATG induced accelerated immunological senescence. This first part enabled the characterization of immune modifications induced by ATG before analysis of there partition of lymphocyte subpopulations in ATG-treated HTx patients classified according rejection on set. In this setting, I next demonstrated the occurrence of more efficient thymopoiesis in HTx patients who developed antibody-mediated rejection (AMR), by assessments of blood T cell receptor excision circle (TREC) levels, quantification of circulating recent thymic emigrants (RTEs) and imaging evaluation in a subset of patients. This finding was rather unexpected, as the thymus is widely described as a tolerogenic organfor alloimmune T cell responses. To clarify the role of the thymus in the humoral alloimmune response, we used a murine model of aortic allotransplantation (AA) associated or not withprophylactic thymectomy. We demonstrated that thymectomy decreased antibody-mediated responses, with a concomitant expansion of the regulatory T cell compartment. In conclusion, my data suggest that over-activation of the thymus could prompt humoral alloimmune responses, which was unexpected since previously published data indicate that the thymus rather induces T cell tolerance. Hence, the thymus might serve a dual role bypromoting T cell tolerance but, when over-activated, promoting AMR on set through a mechanism that remains to be elucidated. Further investigations are needed to better understand the complex interplay between the different thymic cell constituents and the modulation of thymic functions throughout life and in situations of thymic reactivation.

Neonatal immune responses are typically deficient against a wide variety of antigens, including alloantigens, vaccine antigens, and infectious agents. These responses are characterized by Th2-skewed cytokine production, and deficient Th1 and cytotoxic responses. However, these deficient responses can be boosted to adult levels by the use of strong, Th1 promoting agents. This demonstrates that neonates are capable of developing mature immune responses under specific conditions. Using two different murine models, we have found that neonates develop robust Th and cytotoxic responses, which under some antigenic conditions significantly exceed those of adults. First, using a model of early life exposure to noninherited maternal antigens (NIMA), we found that murine neonates develop robust in vivo cytotoxic responses to low doses of alloantigens. Importantly, primary in vivo cytotoxic responses to alloantigen developed during the neonatal period, and persisted into adulthood. Neonates developed similar memory cytotoxic responses to donor spleen cells, bone marrow, and stem cell-enriched (Lin-) bone marrow cells, suggesting that the exposure dose is more important than the type of transplanted donor cell for the development of cytotoxicity. NIMA-exposed neonates also developed vigorous primary and memory allospecific Th1/Th2 responses which exceeded the responses of adults. These findings suggest that early exposure to low levels of NIMA may lead to long term immunological priming of all arms of T cell adaptive immunity. Second, we characterized the phenotype and function of neonatal recent thymic emigrants (RTE). RTE are the predominant cell type in murine neonates, and are present at higher frequencies within the neonatal CD4+ compartment than in adults. Our data demonstrate that RTE from murine neonates and adults are phenotypically and functionally distinct. In particular, although the magnitude of RTE cytokine responses from both age groups is dependent on the conditions of activation, neonatal RTE consistently exhibited higher levels of effector cytokine production than adult RTE. In particular, activation of neonatal RTE in the presence of IL-7 lead to greatly increased IFNgamma production, while adult responses were not altered. Overall, neonatal RTE responses were more plastic than those of adult RTE, as both Th1 and Th2 responses were altered in neonates using various activation conditions, while only Th2 responses were consistently changed in adults. Finally, in contrast to adult RTE, neonatal RTE proliferated in response to IL-7 stimulation at very early timepoints. This was associated with faster kinetics of IL-7Ralpha downregulation and higher levels of pSTAT5 in neonatal RTE. These quantitative and qualitative differences in neonatal RTE populations may largely explain the diverse responses that are elicited in neonates in response to different antigens, especially under those conditions in which Th1 responses are enhanced (i.e., exposure to NIMA alloantigens). Taken together, these data demonstrate that neonatal T cell responses are actually highly plastic, instead of intrinsically deficient. Furthermore, if given optimal stimulation conditions, neonatal responses can actually exceed those produced by adults.

<p>Thymus atrophy is highly inducible by stress and prolonged thymus atrophy can contribute to T cell deficiency or inhibit immune recovery after acute peripheral T cell depletion. Little is known regarding the mechanisms driving thymic involution or thymic reconstitution after acute stress. Leptin deficiency in mice results in chronic thymic atrophy, suppressed cell-mediated immunity, and decreased numbers of total lymphocytes, suggesting a role for leptin in regulating thymopoiesis and overall immune homeostasis. Exogenous leptin administration during stress has been shown to protect against thymic damage, yet the mechanisms governing these thymostimulatory effects are currently undefined. Studies herein define the impact of endotoxin-induced thymic damage in the stromal and lymphoid compartment of the thymus and systemic glucocorticoid and cytokine responses in the animal. We report here the novel finding that leptin receptor expression is restricted to medullary thymic epithelial cells in the normal thymus. Using a model of endotoxin-induced acute thymic involution and recovery, we have demonstrated a role for the metabolic hormone leptin in protection of medullary thymic epithelial cells from acute endotoxin-induced damage. We also demonstrated that systemic leptin treatment decreased endotoxin-induced apoptosis of double positive thymocytes and promoted proliferation of double negative thymocytes in vivo through a leptin receptor isoform b-specific mechanism. Leptin treatment increased thymic expression of IL-7, an important soluble thymocyte growth factor produced by medullary thymic epithelial cells. We also found leptin to inhibit systemic glucocorticoid and pro-inflammatory cytokine responses. Using leptin-deficient and leptin receptor-deficient mice in our stress model, we found that endotoxin-induced thymic atrophy was exacerbated in the absence of leptin, despite an inability to mount a proper pro-inflammatory cytokine response. Together, these data support a model in which leptin can function to protect the thymus gland from stress-induced acute damage in part by reduction of systemic corticosteroid and pro-inflammatory cytokine responses, and intrathymically through a mechanism orchestrated by medullary thymic epithelial cells and their soluble mediators (e.g. IL-7). Taken together, these studies suggest a physiological role for leptin signaling in the thymus for maintaining healthy thymic epithelium and promoting thymopoiesis, which is revealed when thymus homeostasis is perturbed by stress.</p> / Dissertation

The traffic of T cells, that occur when bone marrow-derived T cell precursors penetrate the thymus, with a phenotype of immature cells, and its subsequent output, as mature cells, depends of the thymic blood vessels that consists mainly of thymic endothelial cells. The expression of ligands and receptors of ECM in thymic endothelial cells, as well as its role in the processes of migration and emigration of the T cells from the thymus remains unclear. Our goal was to evaluated the expression of some of ECM ligands and their respective receptors in the strain of thymic endothelial cells tEnd.1. In addition, we aimed to study the role of the components of the ECM in the tEnd.1 cells and thymocytes interaction. First, it was detected by confocal immunofluorescence the presence of ECM proteins fibronectin and laminin, which were also observed through flow cytometry. The presence of their respective receptors, VLA5 and VLA6, were highlighted by immunoperioxidase and flow cytometry. Assays of adhesion showed that the use of antibodies anti-VLA5 and anti-VLA6 seems to block at least partially, the adhesion of thymocytes to endothelial cells. In addition, the transendothelial migration assay showed less migration in absolute cell number of the thymocytes pre-treated with antibodies anti-receptors of ECM, although this migration was not statistically significant. Finally, when the phenotype of the transmigrated thymocytes was observed, there was not difference in the migration pattern among them. This investigation shows that the thymic endothelial cells are able to express fibronectin and laminin proteins, as well as, their respective receptors VLA5 and VLA6, and suggests that these molecules could participate in the processes of intrathymic lymphocytes migration. / Os precursores das células T sofrem diferenciação e seleção intratímica através das interações com os componentes do microambiente tímico. Este é constituído em sua grande maioria por células epiteliais tímicas (TEC), macrófagos, células dendríticas, fibroblastos e os produtos de secreção dessas células, tais como hormônios, interleucinas e moléculas de matriz extracelular (ECM). O tráfego de células T, que a princípio penetram no timo provenientes da medula óssea, com fenótipo de células imaturas e, a sua posterior saída, como células maduras, depende terminantemente dos vasos sanguíneos que irrigam o órgão. Tais vasos são constituídos principalmente de células endoteliais tímicas. Tendo em vista que a expressão de ligantes e receptores de ECM em células endoteliais tímicas, assim como sua função nos processos de migração e emigração de células T do timo permanece pouco estudada, neste trabalho foi avaliada a expressão de alguns ligantes de ECM e seus respectivos receptores em uma linhagem de células endoteliais tímicas, tEnd.1. Além disso, estudou-se também o papel destes componentes de ECM na interação entre as células tEnd.1 e os timócitos. A princípio, foi detectado por imunofluorescência confocal a presença das proteínas de ECM fibronectina e laminina, que foram também observadas através de citofluorimetria, enquanto a presença de seus respectivos receptores, VLA5 e VLA6, foram evidenciados por imunoperoxidase e citofluorimetria. Ensaios de adesão sugeriram que o uso de anticorpos anti-receptores de ECM, anti-VLA5 e anti-VLA6, inibem pelo menos parcialmente, a adesão de timócitos às células endoteliais tEnd.1. Por fim, ensaios de migração transendotelial demonstraram claramente uma menor migração em números absolutos dos timócitos pré-tratados com anticorpos anti-receptores de ECM, embora essa diminuição não tenha sido estatisticamente significativa. Tomando em conjunto, os dados neste trabalho demonstraram que as células endoteliais tímicas da linhagem tEnd.1 são capazes de expressar tanto ligantes de ECM, como seus respectivos receptores, sugerindo ainda, a participação destas moléculas nos processos de migração de linfócitos intratímicos.

Orientadores: Liana Verinaud, Wilson Savino / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T05:50:49Z (GMT). No. of bitstreams: 1 Gameiro_Jacy_D.pdf: 7674475 bytes, checksum: 85104c8f2befe30630bf49436c0fb352 (MD5) Previous issue date: 2009 / Resumo: O timo é o órgão linfóide primário responsável pelo amadurecimento dos linfócitos T. O processo de maturação é dependente da integridade do microambiente e da migração coordenada por elementos de matriz e quimiocinas dos precursores hematopoiéticos nos distintos nichos tímicos. Entretanto, a literatura mostra que profundas alterações no microambiente tímico, e conseqüentemente no desenvolvimento e amadurecimento dos linfócitos T, podem ocorrer como resultado de algumas patologias. No presente trabalho, analisamos a indução de atrofia e as alterações no microambiente tímico frente a três diferentes agentes indutores de atrofia, sendo dois agentes patogênicos, P.brasiliensis e P.berghei e uma doença metabólica, a diabetes induzida por aloxana. Nos três modelos estudados foi observada a atrofia do órgão com diferente intensidade nas alterações de componentes do microambiente tímico. Importantes moléculas associadas com a migração intratímica mostraram-se alteradas com modificações significativas nos elementos de matriz e nas quimiocinas bem como na expressão dos seus respectivos receptores. Ainda, a migração ex vivo também se mostrou alterada na atrofia induzida pelos agentes patogênicos, mas não no modelo de atrofia induzido por aloxana, sugerindo que as alterações desencadeadas são específicas de cada condição patológica analisada. As modificações nos elementos estudados sugerem alterações no padrão de migração intratímica e na exportação de timócitos, com comprometimento da função tímica, levando a maturação desequilibrada dos timócitos, com conseqüências para a resposta imune periférica. / Abstract: Thymus is the primary lymphoid organ responsible for differentiation of Tlymphocytes. This process is dependent of thymic microenvironment integrity and coordinated migration of hematopoietic precursors by chemokines and extracellular matrix elements. However, the literature shows that deep alterations in thymus microenvironment with modifications in thymocyte development may occur such as result of some pathological disorders. In this study we analyzed the atrophy induction and alterations in thymic microenvironment in three different models of thymus atrophy. We studied two models of infectious diseases, P.brasiliensis and P.berghei, and a metabolic disorder, alloxan induced diabetes. We have observed thymus atrophy in all models with different alterations levels in thymus microenvironment elements. Important molecules associated with intrathymic migration were been altered with significant modifications in extracellular matrix elements, chemokines and their specific receptors. Besides, ex vivo migration was altered in thymus atrophy induced by pathogenic agents, but no alterations were observed in diabetic mice suggesting that different pathological conditions studied, leads to singular alterations in thymus compartment. The modifications in thymic molecules observed in our models, suggest impaired thymus functionality and alterations in thymocyte migration patterns These alterations can lead T cell maturation imbalance with consequences in Tlymphocyte immune response. / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

Orientador: Liana Verinaud, Dea Maria Serra Villa-Verde / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T21:07:32Z (GMT). No. of bitstreams: 1 Loyola_PatriciaResendeAloNagib_D.pdf: 6019241 bytes, checksum: 171494f158eff0d6b54b029e9583a35c (MD5) Previous issue date: 2009 / Resumo: O timo é o órgão linfóide primário onde os precursores de células T, chamados de timócitos, sofrem processos de diferenciação, seleção e proliferação. Todos estes processos são seqüencialmente dependentes de distintos microambientes no interior do órgão delimitados morfológica e fenotipicamente. Por isso a manutenção do microambiente é essencial para a funcionalidade do órgão. Recentemente, o timo tem sido visto como um órgão alvo de diversas infecções e patologias. No presente trabalho, foi avaliado o comprometimento do timo em três diferentes modelos experimentais capazes de induzir sua atrofia, a saber, infecção experimental pelo fungo P. brasiliensis, infecção experimental por Plasmodium berghei e diabetes induzida quimicamente. Foram avaliadas as subpopulações celulares tímicas, a expressão de citocinas essenciais à maturação dos timócitos e o possível papel do hormônio leptina no estabelecimento e manutenção da atrofia tímica. Sucintamente, os resultados demonstraram que nos três modelos, houve diminuição de celularidade com alterações significativas na freqüência dos subtipos de timócitos, principalmente queda de DP, e menor número de TNCs/animal . No modelo de diabetes além destas alterações, foi detectado a queda dos níveis de leptina, aparentemente, devido á queda da insulina. No modelo de PCM, não houve alteração nos níveis de leptina, entretanto, houve queda na expressão gênica de IL-7 e TGF-ß, fatores timo-estimulantes e inibição da capacidade migratória. Em animais infectados pelo P. berghei, houve queda de leptina e queda na expressão dos genes específicos para IL-7 e TGF-ß, além de aumento na atividade de migração Mediante os dados obtidos em cada modelo, sugere-se que no caso do modelo de PCM a atrofia de grau leve que foi observada deve-se à perda de células por diminuição da expressão das citocinas timo-estimulantes. Em malária acredita-se que a intensa atrofia ocorra por perda da região cortical devido, à queda na expressão das citocinas IL-7, TGF-ß e nos níveis séricos de leptina. Na diabetes induzida, o grau intermediário de atrofia pode ser correlacionado apenas à queda dos níveis séricos de leptina. / Abstract: The thymus is the primary lymphoid organ in which T cell precursors, called thymocytes, undergo processes of differentiation, selection, and proliferation. All these processes are sequentially dependent on distinct thymic microenvironments, which in turn, are delimited morphologically and phenotypically. For these reasons, the maintenance of this microenvironment is crucial for thymus functionality. Recently, the thymus has been seen an organ targeted by several infections and pathologies. In this study, we evaluated thymus compromise under three experimental models capable of inducing atrophy, as follows: (1) experimental infection by the fungus Paracoccidioides brasiliensis (PCM), (2) experimental infection by Plasmodium berghei, and (3) chemically-induced diabetes. We evaluated subpopulations of thymic cells, the expression of cytokines that are essential to thymocyte maturation, and the possible role of the leptin hormone in the establishment of thymic atrophy. Briefly, the results showed that in all three models there was a decrease of cellularity along with significant alterations in the frequency of each thymocyte subtype, especially a decrease in DP, and lower numbers of TNCs per animal. In the diabetes model, besides these alterations, we detected a decrease in leptin levels, apparently related to the insulin drop. In the PCM model, there were no alterations in leptin levels. However, we observed a drop in the expression of IL-7 and TGF- ß, both thymus-stimulant factors, as well as an inhibition of migratory ability. In P. berghei-infected animals there was a drop in leptin as well as in the expression of IL-7 and TGF- ß genes; besides an increase in migration activity. According to the data obtained within each model, we suggest that for the PCM model, the low degree of observed atrophy is a consequence of the loss of cells due to a drop in thymus-stimulant cytokine expression. In malaria, the intense atrophy happens due to the loss of the cortex region, generated by the drop in the expression of IL-7 cytokines, TGF- ß, and seric levels of leptin. As for the induced diabetes, the intermediate degree of atrophy can be correlated only to the drop in seric levels of leptin. / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

O sistema linfoide é composto de órgãos linfoides primários e secundários. O timo é um órgão linfoide primário responsável pela maturação, diferenciação e seleção da linhagem linfocitária do tipo T que é responsavel pela imunidade celular do individuo. Para cumprir estas funções, o timo possui uma disposição peculiar das suas células epiteliais morfologicamente distintas e de suas estruturas vasculares. Seus vasos sanguíneos possuem um papel na oxigenação tecidual e no processo de migração das células precursoras de linfócitos T para o interior do parênquima tímico e por isso apresentam uma arquitetura típica caracterizada por vasos de grande calibre, localizados na junção cortico-medular e uma fina rede de ramos e anastomoses que se estendem para o córtex. Este processo de estruturação e arquitetura vascular ainda possui sua base molecular desconhecida, assim como os mecanismos que provocam a involução do órgão. O VEGF é um fator angiogênico que atua na formação vascular e na modulação de funções relacionadas à vascularização, sendo um importante marcador da angiogênese. A fim de se melhor compreender o comportamento vascular na formação e involução tímica, propôs-se avaliar a expressão gênica e proteica deste fator durante fases de desenvolvimento e involução do órgão, além da quantificação da vascularização do timo pela técnica estereológica, análise do parênquima tímico pela técnica de microscopia eletrônica de varredura e análise dos tipos celulares presentes em cada estágio etário. Para tal utilizou-se amostras de timo de gato em quatro estágios de desenvolvimento fetal (35, 45, 55, 65), e dois estágios pós-natal (6 meses e 1 ano) para a realização da imuno-histoquímica, PCR em tempo real e MEV,e para a técnica estereológica 2 estágios pós-natal (6 meses e 1 ano). Na microscopia eletrônica de varredura foram observados os timócitos de diferentes tamanhos, em estágios de maturação distintos. As proteinas do VEGF-A e dos receptores Fit-1 e KDR foram identificadas no timo de gatos em todas as fases do desenvolvimento foram localizadas no citoplasma de células epiteliais e no interior dos corpúsculos tímicos. A expressão do mRNA no período de 1 ano de idade a expressão do mRNA do VEGF e seus receptores tem um aumento significativo, coincidindo com a diminuição do Nvasc e do Nv(vasc) podendo causar um estado de hipóxia no órgão levando a um aumento compensatório de sistema VEGF. A curva de crescimento vascular obedece a um padrão de desenvolvimento e involuçãio do órgão. / The lymphoid system is composed by primary and secondary lymphoid organs. The thymus is a primary lymphoid organ responsible for maturation, differentiation and selection of the lymphoid T cell lineage that is responsible for cellular immunity. To accomplish these functions has a peculiar arrangement with morphologically distinct epithelial cells and vascular structures. The blood vessels have a role in tissue oxygentation and the migration of T cells into the thymic parenchyma, therefore they presents large vessels in cortico-medullary junction and a fine network branches to the cortex. This process has its molecular basis unknown as well as the involution process of the thymus. VEGF is an angiogenic factor that plays a role in the formation and modulation of vascular functions, being an important marker of angiogenesis. We proposed to evaluate the gene and protein of VEGF during the thymus development and involution, stereological quantification and scanning electronic microscopy. Samples of cat´s thymus from 35, 45, 55, 65 days of development and 6 months and 1 year of age. In scanning electronic microscopy different stages maturation thymocytes were observed. Protein expression of VEGF and its receptors were identified in all development stages in epithelial cells, endothelial cells and thymic corpuscles. The VEGF mRNA expression and its receptors in 1 year old animals was significantly increased, coinciding with the decreasing Nvasc and the Nv(vasc) causing a hypoxic condition in the thymus resulting in a compensatory increase of VEGF system. The vascular growth curve follows a pattern of development and involution of the organ.

Themis est une protéine de signalisation des récepteurs des lymphocytes T (TCR) essentielle pour la sélection positive des cellules T. La fonction moléculaire de Themis a été controversée mais de récentes études suggèrent qu'il est un régulateur positif des voies de signalisation des TCR. Nous avons montré dans une étude préliminaire que Themis interagit avec des déubiquitinases et qu'il est ubiquitiné dans les thymocytes. L'objectif de ma thèse était de caractériser les mécanismes moléculaires qui régulent l'ubiquitination de Themis et de déterminer si ces processus affectent la fonction de Themis durant le développement des lymphocytes T. Nous avons montré que si l'expression des ARNm codant pour Themis diminue dans les stades précoces de la sélection positive, son expression protéique est parallèlement augmentée, suggérant une stabilisation de Themis par des modifications post-traductionnelles durant cette étape. Nous avons montré que la déubiquitinase USP9X déubiquitine Themis pour stabiliser son expression durant la stimulation des TCR. L'ensemble de nos résultats proposent qu'USP9X soit activé durant la stimulation des TCR grâce à son recrutement dans les complexes proximaux des TCR par l'intermédiaire de l'adaptateur Grb2 et Themis, entrainant la stabilisation de l'expression de Themis. Nous pensons que ce mécanisme est important pour maintenir l'expression de Themis durant la sélection positive afin de favoriser l'induction d'un signal des TCR soutenu, requis pour l'efficacité de ce processus. / The protein Themis is a new actor of the T cell receptor (TCR) signaling essential for the positive selection of T cells. The molecular function of Themis has been controversial but recent findings suggest that it acts as positive regulator of TCR signaling. We demonstrated in an initial research that Themis interacts with deubiquitylases and is covalently associated to ubiquitin chains in thymocytes. The aim of my PhD project was to characterize the molecular process that regulates the ubiquitination of Themis and to investigate how these post-translational modifications affect Themis function during T cell development. We demonstrated that Themis mRNA expression is progressively decreased after positive selection whereas Themis protein expression is enhanced at the early stages of positive selection, suggesting that Themis is stabilized by post-translational modifications during positive selection. We demonstrated that USP9X allows the deubiquitination of Themis and its stabilization following TCR engagement. Ours results suggest that USP9X is activated during TCR engagement following its recruitment to proximal signaling complexes through Grb2 and Themis, leading to the deubiquitination and stabilization of Themis expression. We believe that this mechanism is important to sustain Themis expression during positive selection and to promote durable TCR signals required for the efficiency of this process.