Orientadores: Ronei Luciano Mamoni, Maria Heloisa Souza Lima Blotta / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T02:22:29Z (GMT). No. of bitstreams: 1 Ferreira_MariaCarolina_M.pdf: 3235698 bytes, checksum: 858844d8c2fb54db25e9acd5fcdbb327 (MD5) Previous issue date: 2009 / Resumo: Pacientes com paracoccidioidomicose (PCM) apresentam supressão da resposta imunológica celular, caracterizada por testes de hipersensibilidade do tipo tardio negativos a antígenos do P. brasiliensis, alta taxa de apoptose de linfócitos e alta expressão de CTLA-4 (CD152) e alta produção de citocinas supressoras (IL-10 e TGF-b). Esses dados indicam a possível participação de células T regulatórias (CD4+CD25+ - Tregs) na PCM. O objetivo desse trabalho foi verificar se, e por qual mecanismo as células T regulatórias estão envolvidas nessa imunossupressão. Para isso, analisamos o número, fenótipo, assim como a atividade funcional das células regulatórias do sangue periférico de pacientes com PCM antes e após o tratamento antifúngico efetivo. O número e o fenótipo das células T regulatórias foram avaliados por citometria de fluxo, e os resultados demonstraram que pacientes com PCM ativa (DA) apresentam um maior número dessas células que pacientes tratados (DT) ou controles normais (C). Além disso, observamos uma maior expressão de CD95L, CTLA-4, LAP-1 e GITR em células regulatórias do grupo DA em relação aos grupos DT e C. A expressão do RNAm (quantificado por PCR em tempo real) para FoxP3, IL-10 e TGF-ß em CMSP ex vivo também foram maiores no grupo DA que nos controles e pacientes do grupo DT. Com o intuito de comparar a atividade funcional das células T regulatórias, analisamos o efeito da cocultura com células T regulatórias na resposta proliferativa de CMSP estimuladas com ConA. As células regulatórias do grupo DA exibiram uma atividade supressora maior que as provenientes do grupo DT ou C, de uma maneira dose dependente. Para verificar se o mecanismo por meio do qual as células regulatórias exercem sua atividade é dependente de contato, utilizamos um sistema "transwell". Os resultados demonstraram que a atividade supressora das células T regulatórias do grupo controle é dependente de contato, mas que essa atividade para as células provenientes dos pacientes do grupo DA é apenas parcialmente revertida com esse sistema. A adição de citocinas supressoras recombinantes (IL-10 e TGF-ß), assim como de anticorpos neutralizantes para essas citocinas nas coculturas, mostrou que a produção de citocinas regulatórias pode ser outro mecanismo utilizado pelas células regulatórias do grupo DA. Em conclusão, o número aumentado de células T regulatórias no sangue periférico de pacientes com DA, expressando altos níveis de moléculas associadas à atividade regulatória, ao lado da alta expressão do RNAm para FoxpP3, IL-10 e TGF-b, sugere uma participação dessa população de linfócitos na imunossupressão observada em pacientes com PCM ativa. Além disso, nossos resultados indicam que as células T regulatórias dependem principalmente do contato intercelular para exercer seus efeitos supressores, e que a produção de citocinas supressoras também está envolvida nesse processo. / Abstract: Patients with paracoccidioidomycosis (PCM) present suppression of cellular immune response characterized by negative DTH to P. brasiliensis antigens, elevated apoptosis of lymphocytes, high expression of CTLA-4, and production of IL-10 and TGF-ß. Together these data point towards the involvement of regulatory T cells (CD4+CD25+ - Tregs) in PCM. The aims of this study were to verify whether and how Tregs are involved in this immunosuppression, through the analysis of the number and the phenotype, as well as, the functional activity of Tregs cells from peripheral blood of patients before and after antifungal treatment. The number and phenotype of Tregs cells were evaluated by flow cytometry, and the results showed that PCM patients with active disease (AD) present higher number of Treg cells than patients after treatment (TD) or healthy controls (C). Furthermore, we observed higher expression of CD95L, CTLA-4, LAP-1 and GITR on Tregs from AD group, than in cells from TD and C groups. mRNA expression (quantified by qRT-PCR) for FoxP3, IL-10 and TGF-ß in ex vivo PBMC, were also higher in AD group than in controls and in TD group. In order to compare the functional activity of Tregs, we analyzed the effect of Treg cells on the proliferative response of PBMC stimulated with ConA. Tregs from AD group exhibited stronger regulatory activity than cells from TD and C groups, in a dose dependent manner. To verify the possible mechanism through which Tregs suppress the cell proliferation we utilized a transwell system, which showed that the contact is mandatory for regulatory activity of Treg cells from C group, but had only a partial involvement in cells from AD patients. The addition of IL-10 and TGF-ß and anti cytokines in the co-cultures showed that the production of immunoregulatory cytokines may be other mechanism used by Tregs. In conclusion, the increased number of Treg cells in peripheral blood of patients with active disease, expressing high levels of regulatory markers and suppressive activity, besides the high expression of Foxp3, IL-10 and TGF-ß mRNAs suggest the potential participation of this population in the immunosuppression observed during the disease. Moreover, our results indicate that Tregs act mainly by contact to exert their inhibitory effects, but the production of immunoregulatory cytokines are also involved. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientadores: Vitor Baranauskas, Leonilda Maria Barbosa dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-13T15:52:46Z (GMT). No. of bitstreams: 1 Grecco_AnaCarolinaPimenta_M.pdf: 4028722 bytes, checksum: 6c6aa22381786380372d5a804c0a2fe1 (MD5) Previous issue date: 2009 / Resumo: Os nanotubos de carbono estão sob intenso estudo diante da possibilidade de serem utilizados em aplicações biomédicas. Estudos prévios descreveram que a inalação de nanopartículas de carbono induz resposta inflamatória no tecido pulmonar; entretanto o efeito dessas partículas na resposta imune adaptativa não está completamente entendido. Assim, três diferentes preparações de nanotubos de carbono (NT1, NT4, NT5) foram testadas in vivo na resposta de linfócitos T e B de camundongos C57Bl/6. Os nanotubos de paredes múltiplas NT1 e NT4 foram produzidos em nosso laboratório e seu efeito na resposta imune adaptativa foi comparado com uma preparação comercial de nanotubos (NT5). A preparação (NT4) demonstrou alguns efeitos citotóxicos, sendo inadequada para uso in vivo. As outras preparações não mostraram toxicidade quando administradas sistemicamente. Os nanotubos NT1 e NT5 induziram a uma significativa ativação de linfócitos T e B. A administração sistêmica dessas estruturas resultou no aumento da resposta proliferativa de linfócitos ao mitógeno inespecífico Con A, no aumento da expressão de mRNA de citocinas como TNFa, IL-6, IL-10 e significativa redução do TGFß. As nanoestruturas induziram significativo aumento da produção de anticorpos específicos para ovalbumina. Esses resultados enfatizam a importância de estudar a resposta imune induzida pelas nanopartículas antes funcionalizá-las com proteínas, DNA ou utilizá-las na oferta de fármacos. / Abstract: Carbon nanotubes are currently under scrutiny as new tools for biomedical applications. Previous studies have shown that inhalation of carbon nanoparticles elicited an inflammatory response in the lung tissue; however the effect of these particles in the adaptive arm of the immune response deserves more attention. Thus, three different preparations of carbon nanotubes (NT1, NT4, NT5) were tested on in vivo T and B lymphocytes response of C57Bl/6 mice. The multi-walled carbon nanotubes NT1 and NT4 were produced in our facilities and their effect on adaptive immune response was compared to commercial carbon nanotubes (NT5). The preparation (NT4) has demonstrated some cytotoxic effects and was inappropriate for use in vivo. No cytotoxicity was observed in the other preparations when administered systematically in vivo. Carbon nanotubes NT1 and NT5 lead to a significant activation of the T and B lymphocytes. The systemic administration of these structures resulted in increased proliferative response oflymphocytes to nonspecific mitogen ConA, augmented the mRNA cytokines expression such as TNFa, IL-6, IL-10 and significant decrease of TGFß. Nanostructures induced significant increase in the production of antibody to a specific antigen (OVA). These results emphasize the importance of studying the immune response induced by the nanoparticles before functionalizing them with proteins, DNA or for drug delivery purpose. / Mestrado / Eletrônica, Microeletrônica e Optoeletrônica / Mestre em Engenharia Elétrica

Orientador: Maria Heloisa Souza Lima Blotta / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T00:09:24Z (GMT). No. of bitstreams: 1 Soares_LannyCristinaBurlandy_D.pdf: 1993850 bytes, checksum: c3f425ae548a166847fe87f0b24ad2b0 (MD5) Previous issue date: 2008 / Resumo: Em doenças causadas por microorganismos intracelulares como a tuberculose, criptococose e listeriose foi demonstrado que as células T CD8+ contribuem de forma relevante para o controle da infecção. Em trabalho anterior verificamos um aumento do número de linfócitos T CD8+ no lavado broncoalveolar de pacientes com paracoccidioidomicose (PCM) pulmonar, sugerindo um papel para estas células, cuja ação efetora se dá por meio da produção de citocinas como o IFN-? e atividade citotóxica. O presente estudo teve por objetivo verificar o estado de ativação e a produção de moléculas citotóxicas por linfócitos do sangue periférico (CD4+ e CD8+) de pacientes com PCM, indivíduos com PCM-Infecção e controles, tanto ex-vivo como in vitro, após estimulação com leveduras de P. brasiliensis. A expressão dos marcadores de ativação e moléculas citotóxicas foi avaliada por citometria de fluxo. A análise ex-vivo mostrou que, de maneira geral, os pacientes apresentam menor freqüência de células positivas para moléculas citotóxicas (granzima A, B e perforina), em relação aos indivíduos com PCM-infecção. A estimulação com leveduras de P. brasiliensis levou a um aumento discreto de células ativadas (CD69+) e uma redução na expressão de grânulos citotóxicos. A adição de IL-15 às culturas mostrou elevação da freqüência de células CD69+ apenas no grupo com PCM-infecção e controles. Já as células T CD4+ e CD8+ dos pacientes foram ativadas apenas na ausência do fungo. O efeito da adição de IL-15 na expressão dos grânulos foi pouco expressivo em relação à granzima A e B, mas maior freqüência de células CD8+perforina+ foi observada em indivíduos com PCM-infecção, em relação aos pacientes. Menor expressão do receptor para IL-15 (IL-15Ra) foi detectada em células T CD4+ de pacientes com PCM comparada ao grupo PCM-infecção e aos controles. A dosagem da granulisina sérica pela metodologia de ELISA mostrou níveis inferiores nos pacientes com PCM, comparado aos outros grupos. Além disso, os resultados mostraram uma tendência a um aumento de granulisina nos pacientes após terapia antifúngica. Em conjunto os resultados mostraram que os linfócitos de pacientes com PCM encontram-se em um estado de menor ativação, expressam menores quantidades do receptor para IL-15 e produzem níveis basais de grânulos citotóxicos (granzima A, B, perforina e granulisina). Estes fatores, ao lado de outros mecanismos que comprometem a imunidade celular, poderiam resultar em atividade citotóxica deficitária e, portanto, menor capacidade de lisar o fungo / Abstract: CD8+ T cells play a pivotal role in host defense against diseases caused by intracellular pathogens such as tuberculosis, cryptococcosis and listeriosis. In a previous study we verified an increased number of T CD8+ cells in bronchoalveolar lavage of patients with pulmonary paracoccidioidomycosis (PCM), suggesting a role for them in the local immune response. CD8+ T cells effector functions include cytokines production, mainly IFN-? and cytotoxic activity. The aims of this study were to verify the activation state as well as the production of cytotoxic molecules by peripheral blood lymphocytes (CD8+ and CD4+) from patients with PCM, individuals with PCM-infection and controls, both ex-vivo and in vitro after stimulation with P. brasiliensis yeast cells. The expression of activation and cytotoxic molecules was evaluated by flow cytometry. The ex-vivo analysis showed that, in general, the patients presented a lower frequency of granzime A, B and perforin-positive cells as compared to PCM-infection individuals. P. brasiliensis yeast cells stimulation led to a discrete increase in CD69+ cells and a reduction in cytotoxic granules expression in all groups. The addition of IL-15 to the cultures induced an increase in the frequency of CD69+ cells only in individuals with PCM-infection and controls. Differently, CD8+ and CD4+ T cells from PCM patients were activated only in the absence of fungal cells. The effect of IL-15 in granzyme A and B expression was low but a higher frequency of CD8+perforin+ was detected in individuals with PCM-infection than in patients with PCM. IL-15Ra expression was lower in CD4+ T cells from patients in relation to individuals with PCM-infection and controls. The detection of granulysin levels by ELISA showed lower levels in PCM patients than in individuals with PCM-infection and controls. Moreover, a tendency to a rise in granulysin levels was observed after antifungal therapy. Altogether the results showed that lymphocytes from PCM patients are poorly activated, express low levels of IL-15Ra and produce basal levels of cytotoxic granules (granzyme A, B, perforin and granulysin). These findings, in addition to other mechanisms that impair cellular immunity, may account to defective cytotoxic activity and consequently low capacity to kill the fungus / Doutorado / Ciencias Medicas / Doutor em Ciências Médicas

O vírus linfotrópico de células T humanas (HTLV-1) foi o primeiro retrovírus descrito e está etiologicamente ligado a duas principais doenças: a leucemia/linfoma de célula T do adulto (ATLL) e a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). Apenas 0,3 a 5% dos indivíduos infectados desenvolvem essas doenças associadas, enquanto a maioria permanece assintomática. A HAM/TSP é uma manifestação inflamatória do sistema nervoso central e o mecanismo pelo qual o HTLV-1 induz o surgimento de HAM/TSP ainda não está totalmente esclarecido. Atualmente, uma abordagem promissora no entendimento de mecanismos, bem como na fisiopatogênese das infecções virais tem sido a avaliação da função de microRNAs (miRNAs). Há poucos dados na literatura envolvendo estas moléculas na infecção pelo HTLV-1 em linfócitos T CD4+ bem como no estabelecimento da doença HAM/TSP. No presente estudo, foi avaliada a expressão de miRNAs dos linfócitos T CD4+ isolados de portadores sem HAM/TSP (HAC), pacientes HAM/TSP e indivíduos sadios (CT) por meio de PCR em tempo real. A análise do perfil de expressão dos miRNAs nessas células revelou que 56 e 10 miRNAs apresentavamse mais 1,5 vezes aumentados no grupo HAM/TSP e HAC, respectivamente. O miR- 125b-1-1 apresentou expressão significamente maior no grupo HAC e o miR-146a, no grupo HAM/TSP. A análise in silico de predição de alvo demonstrou que o gene IFNG era potencialmente alvo do miR-125b-1-1 e os genes IRAK1 e TRAF6 do miR- 146a. Foi demonstrado que a expressão do IFNG no grupo HAC era 1,3 vezes mais elevado que o grupo CT e 1,8 vezes mais elevado no grupo HAM que no grupo CT. Houve um aumento na expressão de TRAF6 de 15,7 e 1,5 vezes nos grupos HAM/TSP e HAC, respectivamente. Não foi observada diferença na expressão de IRAK1 entre os três grupos. O ensaios de superexpressão do miR-125b-1-1 alterou a expressão do IFNG e do miR-146a alterou a expressão do gene IRAK1 e sua proteína. Os resultados evidenciados neste trabalho ressaltam a importância dos miRNAs na modulação de genes e proteínas importantes durante a infeção pelo HTLV-1. A correlação entre o miR-125b-1-1 e gene IFNG sugere que este miRNA esteja envolvido nos mecanismos de desenvolvimento de HAM/TSP. Além disso, a interação entre o miR-146a e os genes IRAK1 e TRAF6 sugerem que este miRNA esteja relacionado a mecanismos de persistência viral da infecção pelo HTLV-1 em linfócitos T CD4+. / Human T-cell lymphotropic vírus type 1 (HTLV-1) was the first human retrovirus discovered and it is related with two major diseases: adult T cell lymphoma/leukaemia (ATLL) and HTLV-1 -associated myelopathy/tropical spastic paraparesis (HAM/TS). About 0.3 to 5% of infected individuals will develop HTLV-1 related diseases, while the majority will remain life-long asymptomatic carriers of the virus. HAM/TSP is an inflammatory manifestation of central nervous system and the mechanism involved in HAM/TSP development is noy well elucidated. Currently, a promising approach on understanding the mechanisms as well as physiopathogenesis of viral infections has been the evaluation of the role of microRNAs (miRNAs) roles. There are few data involving CD4+ T cells miRNA expression in HTLV-1 infection as well as HAM/TSP establishment. To identify miRNAs differentially expressed in CD4+ T cells among non-infected individuals (CT), asymptomatic (HAC) and HAM/TSP patients we applied quantitative real time PCR. The analysis of miRNA expression profile in these cells showed 56 and 10 miRNAs upregulated 1.5 times in HAM/TSP and HAC groups, respectively. miR- 125b-1-1 was upregulated in HAC group and miR-146a in HAM/TSP. In silico analysis of target prediction showed that IFNG was a potentially miR-125b-1-1 target and IRAK1 and TRAF6 were miR-146a targets. IFNG expression was 1.3 higher in HAC than CT group and 1.8 higher in HAM/TSP than CT group. It was observed that TRAF6 expression was 15.7 and 1.5 times higher in HAM/TSP and HAC groups, respectively. There was no difference of IRAK1 expression among the three groups. Overexpression assays of miR-125b-1-1 altered IFNG expression and overexpression of miR-146a altered IRAK1 gene and protein expression. The results revealed that miRNAs modulate genes and proteins during HTLV-1 infection. miR- 125b-1-1 and IFNG gene correlation suggests that miR-125b-1-1 seems to contribute to HAM/TSP development. Besides, miR-146a and IRAK1 and TRAF6 interaction suggests that miT-146a seems to contribute to HTLV-1 establishment in CD4+ T cells.

Breast cancer (BC) is the most common cancer in women. It is a highly heterogeneous disease in terms of histology, therapeutic response and patient outcomes. Early and accurate detection of breast cancer is crucial as the patient prognosis varies greatly depending on the diagnosis of the disease. Patient outcomes have been linked to the presence of tumor infiltrating lymphocytes (TIL) in solid tumors. In human BC, higher TIL infiltration is associated with a better prognosis and also predicts relevant responses to pre-operative chemotherapy. TIL are primarily composed of T cells, albeit around 20% of BC patients (pts) show significant B cell infiltration, and can organize in tertiary lymphoid structures (TLS) located in the peritumoral stroma, which are associated with survival in HER2+ and triple negative BC patients. Further, these studies revealed that CD4+ follicular helper T (Tfh) cells producing CXCL13 were specifically associated with peritumoral TLS. CXCL13 is an important B cell chemoattractant whose function is to recruit B cells to the germinal center (GC) in secondary lymphoid organs and TLS, where they can mature and differentiate into memory or antibody-producing B cells. The principal objective of this thesis project was to investigate the role of CXCL13 and Tfh cells play in the development and/or maintenance of GC-like structures in BC-associated TLS.Further understanding of the factors that promote TLS formation in vivo could provide important insight for treatment decisions in BC. CXCL13 expression was originally identified as an important signal associated with TLS that was predictive for patient outcomes. We investigated factors capable of inducing CXCL13 expression in CD4+ T cells isolated from peripheral blood, using flow cytometry analysis. Treatment with TGFβ1 alone, or together with several cytokines (IL12, IL21, and in particular IL2 blockade), increased CXCL13 expression in activated CD4+ T cells. Similar to our characterization of Tfh TIL in fresh tumor tissues, these CXCL13-producing CD4+ T cells were CXCR5 negative and expressed the Tfh markers PD-1 and ICOS. The positive correlation, in treated cells and fresh TIL, between CXCL13-producing CD4+ T cells and FoxP3-expressing regulatory CD4+ T cells, and the diminished chemokine production upon depletion of the latter population, suggest a possible positive relationship between regulatory CD4+ T cells and CXCL13-producing CD4+ T cells.We then derived a GC-associated B cell gene signature for integration in our previously published Tfh cell gene signature, including CXCL13 gene. The combined GC gene signature was tested for its ability to sensitively detect BC-associated TLS using a qRT-PCR-based assay on two different cohorts, a primary BC set (n=83) and a retrospective series (n=52) of formalin-fixed paraffin-embedded (FFPE) BC tissues. These data revealed a correlation between gene signature expression and the extent of TLS scored by trained pathologists on dual-immunohistochemistry stained (CD3+CD20 for T and B cells, respectively) FFPE tissue sections. In addition, the high GC signature expression predicted better overall and disease-free survival of BC pts in our retrospective BC cohort, as well as in public microarray data.This thesis research has demonstrated that CXCL13-producing CD4+ T cells lacking CXCR5 differentiate and exert their function in IL-2-limited but TGF-β1-rich conditions. Furthermore, we developed a GC-associated gene signature able to detect TLS in BC and predict BC pts better survival. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Neuropilin-2 (NRP2) is a single transmembrane receptor and was first found in the nervous system to play a role in axon guidance. Interestingly, NRP2 was also found on many tumor cells and various studies showed that NRP2 is associated with a poor prognosis in different cancers and is involved in migration and therapy resistance. We investigated the prognostic potential of NRP2 in the pancreatic ductal adenocarcinoma (PDAC) and found out that in contrast to other kinds of cancer a high expression of NRP2 is associated with a longer cancer specific survival. We hypothesized that this effect could be either triggered through an expression of different interaction partners of NRP2. Both semaphorine 3F and VEGFs can bind to NRP2 but have different effects on cancer cells. Semaphorine 3F was found to have a great potential as a cancer inhibitor in pancreatic cancer whereas VEGFs are often associated with a worse prognosis. Both compete for the binding to NRP2. Furthermore, we found high expression of NRP2 in tumor-associated macrophages (TAMs) in PDACs. Until now, NRP2 expression and function is poorly analyzed in the immune system. Therefore, we next focused on the investigation of NRP2 in the immune system during cancer progression. We used LysM:cre-NRP2LoxP/LoxP (conditional knock-out of NRP2 in macrophages) and Vav:cre-NRP2LoxP/LoxP (conditional knock-out in all immune cells) for our experiments. We showed that NRP2 is upregulated during the differentiation/maturation of macrophages. Next, we injected LLC cells subcutaneously to analyze the effect of NRP2 knock-out in macrophages (LysM:cre) or in the all immune cells (Vav:cre). No difference was detected in tumor size, but the vascularization was impaired in both mouse models. Different tumor models with extended tumor growth times and metastasis should be performed next to proof the importance of NRP2 in immune cells during tumor progression. Due to the broad expression of NRP2 in the immune system we used the Vav:cre-NRP2LoxP/LoxP mouse to investigate the role of NRP2 during an immune response. We used a mild allergic inflammation model of the lung and analyzed the different immune cell populations. Interestingly, T cells and eosinophils were reduced during the inflammation indicating, that the conditional knock-out of NRP2 is inhibiting the immune response. We further analyzed the role of NRP2 in T cells and found out, that the expression of NRP2 is very different in the various T cell populations. CD8+ T cells express ca. 10 times as much mRNA for NRP2 compared to CD4+ T cells. Also, the CD4 subpopulation showed a diverse expression of NRP2. Th2 and Th17 express a lot of NRP2 and Treg and Th1 very low levels. These results suggest an important role of NRP2 in certain cells. The knock-out of NRP2 in Th2 cells leads to an upregulation of IL-13, IL-5 and IL10. We first showed the importance of NRP2 during an immune response and found interesting regulations in immune cell populations and important cytokines. More work needs to be done to understand the functions of NRP2 during an immune response.

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.

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Immunological correlates of anger conditions were investigated. Participants were 33 females and 36 males, ranging from 25 to 55 years old. Percentages of total T-lymphocytes, suppressor-T, helper-T, and ratio of helper-T to suppressor-T cells were measured. Differences were found between males and females for Anger Control and Anger Expression. For females, total T-cell percentages correlated with State Anger, Angry Temperament, Anger Out, and the combination of State Anger/Angry Reaction. Suppressor-T cell percentages correlated with State Anger, Trait Anger, Angry Temperament, Anger Out, Anger Expression, and the combination of Angry Temperament/Anger In. Helper-T cells correlated with State Anger, Angry Temperament, Angry Reaction, Anger Out, and Anger Control. Mindbody appears to function in a unified fashion.

Le cytomégalovirus (CMV) est l’agent responsable de l’infection opportuniste la plus fréquemment rencontrée en transplantation d’organe. Chez les receveurs séronégatifs qui reçoivent un rein provenant d’un donneur séropositif, 50 % de ces patients peuvent développer une virémie, et 30 % une maladie. A court terme, malgré les traitements anti-viraux, elle est responsable d’une morbidité non négligeable. A long terme, le CMV est associé à une augmentation de la fréquence des sténoses artérielles, plus d’infections associées, plus de rejet aigu, plus de lésions de fibrose interstitielle et d’atrophie tubulaire, une moins bonne survie des greffons et des patients. La cohabitation et la coévolution du CMV avec l’homme depuis des milliers d’années ont aboutie à un état d’équilibre entre le virus et son hôte. Le virus s’est profondément adapté à son hôte afin d’échapper à la réponse immune. En réponse à cela, la réponse immunitaire anti-CMV occupe une part unique et majeure au sein de la réponse immune de l’hôte. Les lymphocytes T CD8+ spécifiques du CMV représentent par exemple 10.2% des lymphocytes T CD8+ mémoires. Avec l’âge, ils s’accumulent et peuvent représenter jusqu’à 30% du pool total de lymphocyte T CD8+. Le système immunitaire sous la contrainte du virus s’est donc refaçonné de façon à garder le contrôle du virus. Depuis 1999, un nouvel acteur de cette réponse immunitaire a été identifié : les lymphocytes T gamma delta Vdelta2-negative. Ces cellules sont impliquées habituellement dans la lutte contre les différents stress d’origine microbien et non microbien (tumeur). Elles interviennent plutôt localement (dans les épithéliums) par différents mécanismes et sont désormais considérées comme des effecteurs intermédiaires entre l’immunité innée et l’immunité adaptative. Leur expansion dans le sang est associée à la guérison de la maladie et à la résolution de l’infection à CMV. Elles ont par ailleurs in vitro une réactivité croisée contre des cellules infectées par le CMV et des cellules tumorales. Les lymphocytes T gamma delta Vdelta2-negative sont donc une représentation supplémentaire de l’énorme impact du CMV sur le système immunitaire de l’hôte. Dans ce travail, nous avons pu étendre et approfondir leur rôle en transplantation d’organe. Nous avons tout d’abord décrit que les lymphocytes T gamma delta Vdelta2-negative avaient un phénotype et une cinétique d’expansion exactement superposable aux lymphocytes T CD8+ spécifiques du CMV in vivo. Nous avons ensuite observé que l’expansion des lymphocytes T gamma delta Vdelta2-negative induits pas l’infection à CMV s’associait à une survenue moindre de cancer à long terme chez les patients transplantés rénaux. Nous avons pu montrer que leur activation était sous la dépendance d’une interaction entre leur TCR et un ligand. Enfin, une autre voie d’activation dépendante du CD16, faisant intervenir les complexes immuns CMV-IgG anti-CMV a aussi été identifiée. Nos travaux depuis 10 ans ont donc démontré que les lymphocytes T gamma delta Vdelta2-negative occupaient une place majeure dans la réponse immune anti-CMV au même titre que les lymphocytes T CD8+. L’intégration de ces cellules à l’immunologie anti-CMV devrait permettre de mieux comprendre certains effets indirects induits par le virus, et pourrait être utile dans le suivi de la réponse immune anti-CMV en transplantation d’organe. L’identification de leur ligand pourrait permettre enfin de tester assez rapidement de nouveaux protocoles d’immunothérapie anti-virale ou anti-tumorale. / Cytomegalovirus (CMV) infection is the most frequent opportunistic infection encountered in solid-organ transplantation. Fifty percent of seronegative kidney transplant recipients (KTR) who receive a kidney from a seropositive donor may develop a CMV infection which causes a disease in 30% of cases. In the long term, CMV is associated with an increased incidence of arterial stenosis, more opportunistic infections, more acute rejection episodes, more interstitial fibrosis and tubular atrophy, and a poorer graft and patient survivals. For thousands years, the co-evolution between the CMV and the immune system allowed to a state of equilibrium between the virus and the host. The virus has deeply adapted to its host in order to escape the immune response. In response, the anti-CMV immune reaction takes up an important and unique place. For example, the CMV-specific CD8+ T cells represent an average 10.2% of the memory CD8+ T cell compartment in CMV-seropositive healthy individuals. With age, these cells accumulate and can represent around 30% of CD8+ T lymphocytes. Therefore under the long-lasting pressure of the virus, the immune system has redesigned in order to keep the control of the virus. Since 1999, a new actor was identified within the immune system: the Vdelta2-negative gamma delta T cells. These cells are involved against various microbial and non microbial stresses. They act locally in epithelia by different mechanisms and are now considered as intermediate effectors between innate and adaptive immunity. In vivo in KTR, their blood expansion is associated with the resolution of CMV infection. In vitro, they share a cross-reactivity against CMV-infected cells and tumor cells. Therefore, the Vdelta2-negative gamma delta T cells are new representatives of the huge impact of CMV on the host immune system. In this work, we were able to extend and get further insight into their role in organ transplantation. In vivo, we first described that Vdelta2-negative gamma delta T cells displayed a phenotype and an expansion kinetic similar to that of CMV-specific CD8+ T cells. Next, we observed that the CMV-induced Vdelta2-negative gamma delta T cells expansion was associated with a lower occurrence of cancer in long-term KTR. In vitro, experiments of transfer of gamma delta TCR allowed us to show that their activation against tumor ligands was TCR-dependent and that different tumor ligands could be recognized by each Vdelta2-negative gamma delta TCR studied. In addition, we observed that the recognition of CMV-infected cells was not only TCR-dependent, but under the dependence of a multi molecular complex involving co stimulatory signals. Finally, we also identified a new CD16-dependent pathway of activation in gamma-delta T cells, involving IgG-opsonised CMV. In summary, Vdelta2-negative gamma delta T cells take up a major place within the anti-CMV immune response in addition to CD8+ T lymphocytes. The integration of these cells to the anti-CMV immunology should provide a better understanding of some indirect effects of the virus and could be useful to monitor the immune response against CMV in solid-organ transplant recipients. Moreover, identification of their ligands could provide interesting tools for new protocols of anti-CMV and anti-tumor immunotherapy.