Endometriose é uma doença de etiopatogenia complexa e multifatorial, caracterizada pela presença de tecido endometrial fora da cavidade uterina principalmente no peritônio pélvico e ovários, envolvendo predisposição genética, fatores ambientais, anatômicos, endócrinos e alterações imunológicas. Afeta 10 a 15 % das mulheres em idade reprodutiva e 35 a 50% das mulheres com infertilidade, dor pélvica ou ambos. Apesar de ser uma das doenças mais estudadas em ginecologia, sua etiologia ainda não está clara e várias são as teorias para explicá-la. O estudo de genes que regulam de alguma forma os processos envolvidos com a endometriose como o ID2 (proliferação celular), PRELP (matriz extracelular) e SMOC2 (angiogênese) pode ajudar a esclarecer o desenvolvimento da mesma. O objetivo desse trabalho foi analisar a expressão gênica destes genes em amostras teciduais pareadas de 20 mulheres, sendo 10 de endométrio eutópico e lesões endometrióticas peritoneais e 10 de endométrio eutópico e endometrioma ovariano com idade entre 18 e 40 anos e em 10 amostras de endométrio de mulheres sem endometriose (controle), padronizadas de acordo com a fase do ciclo menstrual em fase proliferativa. O estudo foi realizado através de técnicas de Biologia Molecular como a Transcrição Reversa (RT-PCR) e análise quantitativa da expressão gênica (PCR em tempo real). A análise estatística mostrou que não houve diferença na expressão gênica entre o endométrio de mulheres sem endometriose e o endométrio eutópico de mulheres com endometriose. O gene ID2 foi mais expresso na fase avançada da endometriose quando comparada a inicial e no endometrioma ovariano quando comparado ao endométrio eutópico de mulheres com endometriose e o PRELP na lesão peritoneal quando comparado ao endométrio eutópico de mulheres com endometriose. Nas análises realizadas com todas as lesões endometrióticas juntas, o SMOC2 foi mais expresso na lesão (peritônio e ovário) quando comparado ao endométrio eutópico de mulheres com endometriose. Os resultados citados demonstram que a expressão dos genes estudados pode sofrer influência do meio peritoneal, podendo ser alterada dependendo do local da implantação (ovário ou peritôneo). / Endometriosis is a complex disease and its etiology is multifactorial, characterized by the presence of endometrial tissue outside the uterine cavity especially in the pelvic peritoneum and ovaries, involving genetic predisposition, environmental factors, anatomical, endocrine and immunological changes. It affects 10 to 15% of women of reproductive age and 35 to 50% of women with infertility, pelvic pain or both. Despite being one of the most studied diseases in gynecology, its etiology remains unclear and there are several current theories to explain it. The study of genes that regulate the processes involved somehow with endometriosis as ID2 (cell proliferation), PRELP (extracellular matrix) and SMOC2 (angiogenesis) may help clarify its development. The aim of this study was to analyze the gene expression of these genes in tissue samples from 20 women paired with 10 endometrial tissue and 10 peritoneal endometriotic lesions 10 endometrial tissue and 10 ovarian endometriotic lesions in proliferative phase of the menstrual cycle. Sixteen samples of endometrium of women without endometriosis, in the same phase of the menstrual cycle were collected to control (C). The study was conducted through molecular biology techniques such as reverse transcription (RT-PCR) and quantitative gene expression analysis (real-time-time PCR). Statistical analysis showed no difference in gene expression between the endometrium of women without endometriosis and endometrium from women with endometriosis. Showed a greater expression of the ID2 gene in advanced stage of endometriosis when compared to the initial stage and ovarian endometrioma compared to eutopic endometrium of women with endometriosis and PRELP in peritoneal lesion compared to eutopic endometrium of women with endometriosis. In the analysis performed with all lesions the SMOC2 was expressed more in the lesions (ovarian and peritoneal) compared to the eutopic endometrium of women with endometriosis. The results cited show that the expression of the genes studied may be influenced through the peritoneal cavity, and may be modified depending on the implantation site (ovary or peritoneum).

Endometriose é uma doença de etiopatogenia complexa e multifatorial, caracterizada pela presença de tecido endometrial fora da cavidade uterina principalmente no peritônio pélvico e ovários, envolvendo predisposição genética, fatores ambientais, anatômicos, endócrinos e alterações imunológicas. Afeta 10 a 15 % das mulheres em idade reprodutiva e 35 a 50% das mulheres com infertilidade, dor pélvica ou ambos. Apesar de ser uma das doenças mais estudadas em ginecologia, sua etiologia ainda não está clara e várias são as teorias para explicá-la. O estudo de genes que regulam de alguma forma os processos envolvidos com a endometriose como o ID2 (proliferação celular), PRELP (matriz extracelular) e SMOC2 (angiogênese) pode ajudar a esclarecer o desenvolvimento da mesma. O objetivo desse trabalho foi analisar a expressão gênica destes genes em amostras teciduais pareadas de 20 mulheres, sendo 10 de endométrio eutópico e lesões endometrióticas peritoneais e 10 de endométrio eutópico e endometrioma ovariano com idade entre 18 e 40 anos e em 10 amostras de endométrio de mulheres sem endometriose (controle), padronizadas de acordo com a fase do ciclo menstrual em fase proliferativa. O estudo foi realizado através de técnicas de Biologia Molecular como a Transcrição Reversa (RT-PCR) e análise quantitativa da expressão gênica (PCR em tempo real). A análise estatística mostrou que não houve diferença na expressão gênica entre o endométrio de mulheres sem endometriose e o endométrio eutópico de mulheres com endometriose. O gene ID2 foi mais expresso na fase avançada da endometriose quando comparada a inicial e no endometrioma ovariano quando comparado ao endométrio eutópico de mulheres com endometriose e o PRELP na lesão peritoneal quando comparado ao endométrio eutópico de mulheres com endometriose. Nas análises realizadas com todas as lesões endometrióticas juntas, o SMOC2 foi mais expresso na lesão (peritônio e ovário) quando comparado ao endométrio eutópico de mulheres com endometriose. Os resultados citados demonstram que a expressão dos genes estudados pode sofrer influência do meio peritoneal, podendo ser alterada dependendo do local da implantação (ovário ou peritôneo). / Endometriosis is a complex disease and its etiology is multifactorial, characterized by the presence of endometrial tissue outside the uterine cavity especially in the pelvic peritoneum and ovaries, involving genetic predisposition, environmental factors, anatomical, endocrine and immunological changes. It affects 10 to 15% of women of reproductive age and 35 to 50% of women with infertility, pelvic pain or both. Despite being one of the most studied diseases in gynecology, its etiology remains unclear and there are several current theories to explain it. The study of genes that regulate the processes involved somehow with endometriosis as ID2 (cell proliferation), PRELP (extracellular matrix) and SMOC2 (angiogenesis) may help clarify its development. The aim of this study was to analyze the gene expression of these genes in tissue samples from 20 women paired with 10 endometrial tissue and 10 peritoneal endometriotic lesions 10 endometrial tissue and 10 ovarian endometriotic lesions in proliferative phase of the menstrual cycle. Sixteen samples of endometrium of women without endometriosis, in the same phase of the menstrual cycle were collected to control (C). The study was conducted through molecular biology techniques such as reverse transcription (RT-PCR) and quantitative gene expression analysis (real-time-time PCR). Statistical analysis showed no difference in gene expression between the endometrium of women without endometriosis and endometrium from women with endometriosis. Showed a greater expression of the ID2 gene in advanced stage of endometriosis when compared to the initial stage and ovarian endometrioma compared to eutopic endometrium of women with endometriosis and PRELP in peritoneal lesion compared to eutopic endometrium of women with endometriosis. In the analysis performed with all lesions the SMOC2 was expressed more in the lesions (ovarian and peritoneal) compared to the eutopic endometrium of women with endometriosis. The results cited show that the expression of the genes studied may be influenced through the peritoneal cavity, and may be modified depending on the implantation site (ovary or peritoneum).

<p>E and Id proteins are members of the basic helix-loop-helix (bHLH) transcription regulator family. These proteins control a broad range of lymphocyte biology, from the development of multiple lineages to execution of their effector functions. With the development of new experiment models, novel functions of E and Id proteins continued to be discovered. In this thesis, I focused my study on the role of Id2 in gamma delta T cells and CD4<super>+</super> alpha beta T cells, as well as the role of Id3 in B cells.</p><p> Id proteins have been shown to control gamma delta T cell development. Id3 knockout mice demonstrate a dramatic expansion of innate-like Vgamma1.1<super>+</super> Vdelta6.3<super>+</super> T cells in the neonatal stage, suggesting that Id3 is an inhibitor of their development. Interestingly, Id3 knockout mice with a B6/129 mix background have much less expansion of the Vgamma1.1<super>+</super> Vdelta6.3<super>+</super> T cells compared to mice with pure B6 background. Genetic studies showed that this difference is strongly influences by a chromosome region very close to the Id2 locus. Using the Id2<super>f/f</super> CD4Cre<super>+</super> mice, I found that Id2 is also an inhibitor of gamma delta T cell development. Deletion of Id2 alone is sufficient to enhance the maturation of these cells in the thymus and induce a moderate expansion of gamma delta T cells in the periphery. This study demonstrated the delicate balance of transcription control in cells of the immune system.</p><p> The Id2<super>f/f</super> CD4Cre<super>+</super> mice also enabled me to study the role of Id2 in peripheral CD4<super>+</super> alpha beta T cell functions, which was difficult in the past because Id2 knockout mice lack lymph node development. I found that CD4 T cells in these mice have a profound defect in mounting immune responses, demonstrated by a complete resistance to induction of experimental autoimmune encephalomyelitis (EAE). I found that Id2-deficient CD4 T cells fail to infiltrate the central nervous system, and the effector CD4 T cell population is smaller compared to that in control mice. Id2 is important for the survival and proliferation of effector CD4 T cells, and this phenotype was correlated with an increased expression of <italic>Bim</italic> and <italic>SOCS3</italic>. This study revealed a novel role of Id2 in the functioning of CD4<super>+ </super>alpha beta T cells.</p><p> Switching my focus to B cells, recent next generation sequencing of human Burkitt lymphoma samples revealed that a significant proportion of them have mutations of Id3. This finding suggests that Id3 may be a tumor suppressor gene in the lymphoid system. Utilizing various Id3 knockout and conditional knockout mouse models, I showed that Id3 deficiency can accelerate lymphoid tumor genesis driven by the over-expression of oncogene c-Myc. This work may lead to development of a more realistic mouse model of human Burkitt lymphoma, allowing more mechanistic studies and perhaps preclinical tests of new therapies.</p> / Dissertation

Les cellules T CD8+ jouent un rôle primordial dans le contrôle des infections virales en limitant la dissémination des cellules infectées. Lors de l’infection chronique par le virus HIV, les cellules T CD8+ HIV-spécifiques ne se différencient pas en cellules effectrices fonctionnelles capables de tuer les cellules infectées par le virus ; ces cellules ne sont plus capables de proliférer ou de produire l’ IL-2. Ces cellules expriment PD-1 et l’engagement de PD-1, par son ligand, aboutit a plusieurs de ces déficits fonctionnels des cellules T . Le rôle de PD-1 dans la régulation d'évènements transcriptionnels contrôlant la différentiation et l'obtention des fonction effectrices des cellules T CD8+ reste à démontrer. Id2 joue un rôle central dans la différenciation des cellules T CD8+ effectrices. Nous avons émis l’hypothèse que le défaut de maturation observé chez les cellules T CD8+ PD-1 high HIV-spécifiques (CD8+PD-1hi) au cours de l’infection chronique par le virus HIV pouvait être lié à la diminution d’expression du régulateur Id2. Nous avons ainsi démontré que l'engagement de PD-1 contribuait à une diminution d'expression de Id2 et de ses cibles transcriptionnelles. La surexpression de Id2 de ces cellules a permis de restaurer l'expression de marqueurs tels que Granzyme B et Bcl-2 et diminuir l’expression du marqueur de maturation de CD27. La famille des cytokines à chaine gamma joue un rôle clef dans la survie et l’homéostasie des cellules T. Dans ce travail, nous avons démontré que l’IL-15 était unique grâce à ses capacités de stimulation de l’expression d’Id2 et ses propriétés favorisant la survie ainsi que la différenciation des cellules T CD8+ effectrices. l’IL-15 induit la prolifération de toutes les populations de cellules T mémoires provenant de donneurs sains. L’addition de cette cytokine aux sous-populations cellulaires Ttm et Tem a permis leur différenciation en cellules effectrices capables de produire Granzyme B alors que la stimulation par l’IL-15 des cellules Tcm ne favorise pas leur différenciation. Un test de cytotoxicitié par cytométrie en flux nous a permis de confirmer que la stimulation de cellules T CD8+ HIV spécifiques par l’IL-15 favorisait l’expression de Id2 et restaurait les fonctions cytotoxiques des cellules T CD8+ HIV spécifiques. En conclusion, nous avons pour la première fois dans cette thèse défini les mécanismes moléculaires impliqués dans la modulation de l’expression du régulateur transcriptionnel Id2 par l’IL-15. Nous avons également révélé comment l’engagement de PD-1 conduisait a une altération de l’expression et de la fonction d’Id2 et favorisait la diminution des fonctions effectrices des cellules T CD8-HIV spécifiques. Une perspective de traitement avec des agents tels que l’IL-15 ou le bloquage de PD-1, en combinaison avec les traitements conventionnels, pourrait contribuer à une meilleure stimulation des réponses immunes favorisant ainsi la réactivation des cellules T CD8+ et permettant la destruction de cellules T CD4+ infectées de manière latente. / CD8+ T cells play a fundamental role in controlling viral replication and dissemination by killing virus-infected cells. However during chronic HIV infection HIV-specific CD8+ T cells fail to differentiate to functional cytotoxic effector cells and develop functional defects such as loss of IL-2 secretion, decreased proliferation and express high levels of PD-1. Persistent expression of PD-1 and triggering by its ligand results in immune dysfunction; it is not known how PD-1 signaling influences transcriptional events involved in T cell differentiation and effector function. We found that the transcriptional regulator Id2 was downregulated in PD-1hi HIV-specific CD8+ T cells when compared to PD-1low CMV-specific CD8+ T cells from the same HIV-infected donors. Since Id2 has been shown to play a central role during differentiation of effector CD8+T cells, we hypothesized that skewed maturation of the PD-1hi HIV-specific CD8+ T during chronic HIV infection could result from decreased levels of Id2. We found that signals transduction pathways downstream of PD-1 ligation inhibited the expression of Id2; transfection of PD-1hi effector cells from HIV infected individuals with a Tat-Id2 construct could reverse an apoptotic fate associated with the exhausted phenotype. Finally, overexpression of Id2 restored expression of Granzyme-B and Bcl-2 and led to a decreased expression of the T cell maturation marker CD27. Although the extrinsic signals and costimulation needed to activate cell proliferation and effector function are well known, signal-transduction pathways that regulate differentiation of memory cells to effector cells are beginning to be understood. Thechain family of cytokines is essential for the survival and homeostasis of T cells; they have pleiotropic effects on the differentiation of effector and memory virus-specific CD8+ T cells. IL-15 was unique among gamma-chain cytokines in upregulating the expression of Id2 and promoting the survival and differentiation of effector memory CD8+ T cells. IL-15 induced proliferation of all memory subsets from healthy subjects but only induced differentiation, Granzyme-B production, and cytotoxic effector function in CD8+ Ttm and Tem cells. Stimulation of Tcm with IL-15 failed to induce their differentiation; this was associated with their decreased ex vivo levels of IL-15R when compared to Tem and Ttm subsets. Finally, we developed a single cell flow-cytometry cytotoxicity assay, and found that stimulation of CD8+T cells from HIV chronically infected subjects with peptide plus IL-15 induced the differentiation of tetramer+ CD8+ Ttm cells and restored Id2 expression and their cytotoxic activity . Overall, we illustrate in this thesis, for the first time, the molecular mechanisms of effector T cell differentiation mediated by IL-15 and its downstream transcriptional regulator Id2; we reveal how PD-1 engagement leads to alteration of the Id2 pathway leading to decreased effector function of the HIV-specific CD8+ T cells. Immunotherapy with agents such as IL-15 or PD-1 blocking antibody that increase levels of Id2 expression , in combination with HAART, should trigger the functional re-activation of HIV-specific CD8+ T cells and the killing of latently HIV-infected CD4+ T cells.