Orientador: Laura Sterian Ward / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T00:19:00Z (GMT). No. of bitstreams: 1 Silva_JoycedoRosarioda_D.pdf: 4211439 bytes, checksum: d230e48c7d6fd16a7f9c75667ad83f9a (MD5) Previous issue date: 2009 / Resumo: A maior expressão de MUC1 tem sido relacionada com o pior prognóstico de diversas malignidades como o câncer de mama e pâncreas. Aproximadamente 20% dos carcinomas diferenciados da tiróide (CDT) evoluem com recidivas locais e a distância. O nosso objetivo foi o de avaliar o gene da MUC1 nos pacientes com CDT e relacionar com aspectos clínicos e anatomo-patológicos. CASUÍSTICA E MÉTODOS: Selecionamos 150 pacientes portadores de carcinoma papilífero (CP), 57 oriundos do Hospital das Clínicas da UNICAMP, acompanhados por 67 (73,29±39,83) meses, de 1995 a 2008 e 93 pacientes do Hospital AC Camargo - Fundação Antônio Prudente em São Paulo acompanhados por 41,37 (32,5±34,30) meses, de 1998 a 2008. Realizamos análise da expressão do gene da MUC 1 por imunoistoquímica e por PCR em Tempo Real e comparamos com dados de evolução clínica e do anatomopatológico. RESULTADOS: Observamos a expressão da proteína MUC1 em 82,19% dos pacientes com CP, no entanto, sem diferenças estatísticas para os dados de evolução clínica e do anatomo-patológico. A análise do RNA-m de MUC1 se correlacionou com a menor expressão nos indivíduos que apresentaram metástases ao diagnóstico (p valor=0,0216). Observamos a pior evolução: no sexo masculino, quando havia metástases ao diagnóstico, na ausência de tiroidite e nos tumores maiores que 4 cm. A presença de invasão tumoral foi mais freqüente nos indivíduos com ausência de tiroidite em 47% dos casos (p=0,0132; OR 2,473 - 95%IC: 1,198-5,104). CONCLUSÃO: Não conseguimos correlacionar a análise do gene MUC1 com aspectos clínicos e anatomo-patológicos de pior prognóstico para o CDT. / Abstract: The over expression of MUC1 has been related with the worst prognosis in malignancies like breast and pancreas cancer. We know that around 20% of the patients with differentiated thyroid cancer (DTC) can develop local and/or distant recurrences and because of that we decide to analyze the MUC1 gene in patients with DTC and tried to relate it with clinical and pathological patterns of the thyroid cancer. PATIENTS AND METHODS: We selected 150 patients with Papillary Thyroid Cancer: 57 from the Clinical Hospital of Campinas State University, followed up for 67 (73,29±39,83) months, since 1995 to 2008 and 93 patients from the A. C. Camargo Hospital - Antonio Prudente Foundation - São Paulo for 41,37 (32,5±34,3) months since 1998 to 2008. We analyzed the MUC1 gene with the immunohistochemistry and the Real Time - PCR techniques and compared the results with clinical and pathological data. RESULTS: The MUC1 expression was positive in 82,19% of the patients with papillary thyroid cancer, however, when we compared with clinical and pathological data, there was not statistical significance. The MUC1 m-RNA analysis was correlated with the less expression of the gene in the individuals who had had metastases at the diagnosis. We could observe the worst outcome in the individuals of the male gender, in the presence of metastases at the diagnosis, in the absence of thyroiditis in the non-neoplasic tissue and in tumors larger than 4 cm. The presence of tumoral invasion was significant in the patients with metastases to the diagnosis and in the ones without thyroiditis in 47% (p=0,0132; OR 2,473 - 95% CI: 1,198-5,104). CONCLUSION: We conclude that MUC1gene analysis was not useful to determine aggressive tumors nor to predict prognosis in papillary thyroid carcinomas. / Doutorado / Clinica Medica / Doutor em Clínica Médica

A fase lútea tardia (FLT) é citada pela literatura como crítica para exacerbação de todos os transtornos psiquiátricos, o que pode significar maior consumo alcoólico por indivíduos dependentes e risco de recaídas para aqueles abstinentes. Paralelamente, é freqüente o relato clínico de aumento do consumo alimentar nesta fase. Conter a compulsividade e impulsividade alimentar é um dos inúmeros aspectos da atuação nutricional na recuperação de mulheres dependentes de álcool, uma vez que a eutrofia destas pacientes é importante para suas condições clínicas e psicológicas e, conseqüentemente, prevenir recaídas. Desta forma, conhecer a influência do ciclo menstrual feminino no consumo alimentar e alcoólico de mulheres em tratamento torna-se importante. OBJETIVOS: Comparar a composição alimentar e o consumo alcoólico entre a fase folicular (FF) e a FLT de mulheres dependentes de álcool. METODOLOGIA: estudadas 30 mulheres alcoolistas em tratamento no Programa de Atenção à Mulher Dependente Química (PROMUD) do Instituto de Psiquiatria do Hospital das Clínicas da Faculdade de Medicina da USP. Todas elas preencheram diários alimentares (que continham consumo alcoólico) durante dois meses. Foram aplicados também questionários relativos a dados sócio-demográficos, comportamento alimentar e comorbidades psiquiátricas. Os diários foram avaliados pelo Programa Virtual Nutri versão 1.0. RESULTADOS: A correlação do consumo energético (p=0,688), lipídico (0,500) e alcoólico (p= 0,673) entre a FF e FLT foi positivo. Foi possível dividir a amostra em dois clusters, onde constatou-se diferença significativa no consumo energético e de carboidratos entre os dois grupos, que possuíam tempo de tratamento e Índice de Massa Corpórea (IMC) diferentes, mas não estatisticamente significantes. CONCLUSÕES: Não parece existir diferença de consumo alimentar ou alcoólico em mulheres alcoolistas entre a FF e FLT. Se dividirmos a amostra em subgrupos, aquelas com mais tempo de tratamento IMC mais elevado tendem a ingerir mais álcool e não relatar corretamente seu consumo alimentar e alcoólico / The late luteal phase (LLF) of the menstrual cycle is often associated with exacerbation of psychiatric disorders. Increased food intake due to mechanism not yet established can be seen during LLF. A key component of nutritional intervention for the treatment of alcoholic women is to approach the so-called binge and impulsive eating behaviors. It is thus necessary to know the impact of the menstrual cycle on food and alcohol intake of women in alcohol dependence treatment. OBJECTIVES: To compare food composition and alcohol consumption between follicular phase (FP) and LLF in alcohol dependent women. METHODS: Thirty alcoholic women in treatment at the Women Drug Dependent Treatment Center (PROMUD) at Clínicas Hospital Psychiatry Institute of Universidade de São Paulo Medical School completed food diaries (including alcohol consumption) for two months. Data on sociodemographic characteristics, eating behaviors and psychiatric comorbidities were also collected using specific questionnaires. Food diaries were assessed using Virtual Nutri Program version 1.0. RESULTS: There was no statistically significant difference of energy and alcohol consumption, as well as macronutrient intake (carbohydrates, proteins and lipids), between FP and LLF. But when the study sample was divided into two groups according to nutritional variables, a significant difference was evidenced in energy and carbohydrate intake. Length of treatment and body mass index (BMI) differed in both subgroups but not statistically significant. CONCLUSIONS: There does not seem to be a difference in food or alcohol consumption in alcoholic women between FP and LLF. The two subgroups studied showed that longer treatment and higher BMI are associated with higher alcohol consumption and misreporting of food and alcohol intake

Os mecanismos envolvidos na etiopatogênese da infertilidade em pacientes com endometriose não foram totalmente elucidados. A infertilidade apresentada por pacientes com as formas moderada e grave (estadios III e IV, respectivamente) seria, parcialmente, decorrente de alterações anatômicas pélvicas associadas à endometriose. Entretanto, há evidências de que lesões sutis ou implantes endometrióticos em estágios iniciais (estágio mínimo e leve) também poderiam contribuir com a etiopatogênese da infertilidade. Uma pior qualidade oocitária pode estar envolvida nas menores taxas de implantação após fertilização in vitro encontradas nessas pacientes. Questionamos a possibilidade de haver alterações no microambiente folicular de pacientes inférteis com endometriose, as quais poderiam afetar a aquisição de competência oocitária e, consequentemente, comprometer a fertilidade natural e os resultados dos tratamentos de reprodução assistida em mulheres com esta doença. Sabe-se que, para ser competente e poder ser fertilizado, o oócito precisa estar maduro e ter um fuso morfologicamente funcional, que garanta a fidelidade da segregação cromossômica durante a meiose. Dessa forma, o objetivo deste estudo foi avaliar o potencial impacto de diferentes concentrações de fluido folicular (FF) de mulheres inférteis com e sem endometriose leve sobre a integridade do fuso, alinhamento cromossômico e organização dos microfilamentos de actina de oócitos bovinos maturados in vitro. Realizou-se um estudo experimental, onde amostras de fluido follicular foram consecutivamente obtidas de 22 pacientes inférteis (11 com endometriose leve e 11 com infertilidade por fator tubário e/ou masculino) submetidas à estimulação ovariana para injeção intracitoplasmática de espermatozóide. Oócitos bovinos imaturos foram submetidos à maturação in vitro (MIV) sem adição de fluido follicular (sem fluido) e com 4 concentrações (1%, 5%, 10%, e 15%) de duas amostras de fluido folicular (uma de paciente com endometriose e outra de paciente sem endometriose). Foram realizadas 11 MIVs e cada amostra de fluido follicular foi usada apenas uma vez. Os oócitos foram fixados, marcados por imunofluorescência para visualização morfológica de microtúbulos, cromatina e microfilamentos de actina e, então, analisados por microscopia confocal. A porcentagem de anormalidade de oócitos em MII (fuso normal e cromossomos desalinhados, fuso anormal e cromossomos desalinhados, fuso anormal e cromossomos alinhados) foi significativamente maior naqueles maturados com FF de pacientes com endometriose (1%: 55,56%, 5%: 63,26%, 10%: 54,54%, 15%: 48,84%) quando comparados com oócitos maturados com FF de pacientes controles (1%: 19,15%, 5%: 23,44%, 10%: 25%, 15%: 23,81%) e oócitos maturados sem fluido (23,53%), sem haver diferença entre as concentrações testadas em cada grupo. Pode-se concluir que oócitos bovinos maturados in vitro na presença de FF de mulheres inférteis com endometriose leve têm maior freqüência de anormalidade meiótica. Estes dados sugerem que o FF de mulheres com endometriose pode comprometer a qualidade oocitária por promover danos ao fuso e/ou cromossomos / The mechanisms involved in the etiopathogenesis of infertility in patients with endometriosis have not been fully elucidated. The infertility presented by patients with moderate and severe disease (stages III and IV, respectively) would be partly due to anatomical pelvic changes associated with endometriosis. However, there are evidences that subtle lesions or endometriosis implants in the early stages (stages I and II) might also contribute to the etiophatogenesis of infertility. Impaired oocyte quality may be involved in lower implantation rates after in vitro fertilization in these patients. We question if alterations in the follicular microenvironment of infertile patients with endometriosis might affect oocyte competence acquisition and compromise the natural fertility and assisted reproduction treatment outcomes in women with this disease. It is known that to be competent and capable of fertilizing, the oocyte must be mature and have a morphologically functional spindle, which ensure the fidelity of chromosome segregation during meiosis. Thus, the aim of this study was to evaluate the potential impact of different concentrations of follicular fluid (FF) of infertile women with and without mild endometriosis on spindle integrity, chromosomes alignment and actin microfilaments organization of bovine oocytes in vitro matured. We performed an experimental study, where FF samples were consecutively obtained from 22 infertile patients (11 with mild endometriosis and 11 with tubal or male factors of infertility) submitted to ovarian stimulation for intracytoplasmic sperm injection. Immature bovine oocytes were submitted to in vitro maturation (IVM) without FF and with 4 concentrations (1%, 5%, 10%, and 15%) of 2 samples of FF (1 from a woman with endometriosis and one from a woman without endometriosis). We performed 11 IVM and each FF sample was used only once. The oocytes were then fixed, stained by immunofluorescence for morphological visualization of microtubules, chromatin and actin microfilaments, and then, analyzed by confocal microscopy. The percentage of abnormal MII oocytes was significantly higher for those matured with FF from patients with endometriosis (1%: 55.56%, 5%: 63.26%, 10%: 54.54%, 15%: 48.84%) when compared with oocytes matured with FF from patients without endometriosis (1%: 19.15%, 5%: 23.44%, 10%: 25%, 15%: 23.81%) and those matured without FF (23.53%), with no differences among the tested concentrations in each group. We can conclude that bovine oocytes matured in vitro in the presence of FF from infertile women with mild endometriosis have higher frequency of meiotic abnormalities. These data suggest that FF from women with endometriosis may compromise oocyte quality by promoting spindle and/or chromosomal damage

As técnicas de cultivo de folículos e oócitos in vitro, com o objetivo de se obter oócitos maduros para procedimentos de Reprodução Assistida (RA), têm sido aplicadas em diferentes contextos. O sucesso destes procedimentos está diretamente relacionado ao sistema de cultivo utilizado. A utilização de células da granulosa (CG) humanas cultivadas in vitro como um suporte para o co-cultivo destes oócitos imaturos e folículos tem sido descrita por alguns autores. A criopreservação destas células, considerando-se o contexto de sua obtenção em procedimentos de RA, permitiria a viabilização da aplicação destas células na prática clínica diária. Sendo assim, o objetivo deste estudo foi padronizar o congelamento de células da granulosa (CG) humanas para aplicação em sistemas de co-cultivos de folículos e oócitos imaturos. Foram obtidas CG de 20 voluntárias em tratamento de reprodução assistida, células de 10 voluntárias foram cultivadas em meio ?-MEM suplementado para interrupção da luteinização e congeladas após 48 horas em container \"Cryostep\" (grupo 2C- 2 cultivos) (etapa 2) e células de 10 voluntárias foram congeladas em container \"Cryostep\" sem cultivo prévio (grupo CD- congelamento direto) (etapa 3). Após o descongelamento estas células foram (re)cultivadas por 144 horas, com troca de meio em 48, 96 e 144 horas para avaliações da produção de estradiol (E2) e progesterona (P4) (ng/mL). Verificamos redução na contagem celular e na viabilidade celular tanto no método de congelamento direto (CD) quanto no método com dois cultivos (2C) após o descongelamento (p<0,05), e isso se refletiu na produção de estradiol e progesterona que foi maior nas culturas de células frescas em relação às células criopreservadas (p<0,05). Porém, a relação de E2/célula foi mantida após o descongelamento, sugerindo que esta redução na produção se deve à redução no número de células, as que sobrevivem se mantém normofuncionantes (p=0,23).O CD foi mais eficiente pois permitiu uma maior recuperação celular e uma melhor viabilidade quando comparado ao grupo 2C. A relação estradiol/progesterona foi mantida em todos os tempos de cultivo, fresco, CD e 2C (p>0,05), indicando que a característica funcional destas células foi preservada após o descongelamento. Concluímos que a criopreservação de CG humanas obtidas durante a captação de oócitos compromete a contagem celular e a viabilidade geral da cultura, entretanto, a capacidade funcional e a característica destas células se mantêm preservadas (manutenção das relações E2/célula e E2/P4) / Follicle and oocyte in vitro culture techniques, aiming to obtain mature oocytes for Assisted Reproductive Treatments (ART), have been applied to different contexts. The success of these procedures depends on the culture system used. The use of human granulosa cells (GC) in co-culture systems for follicle and oocyte maturation have been described by some authors. The cryopreservation of these cells, considering the context in which they are obtained during ART, would enable the usage of these cells in such procedures in daily clinical practice. Thus, the objective of this study was to standardize the freezing protocol for human granulosa cells (GC) for future applications in co-culture systems for follicle and oocyte maturation. Twenty volunteers submitted to ART donated their granulosa cells after oocyte retrieval, 10 were cultivated previously in order to interrupt the luteinization process and then frozen \"Cryostep\" container (group 2C- two cultures) (step 2) and 10 were directly frozen with no previous culture in the \"Cryostep\" container (group DF- direct freeze) (step 3). After thawing these cells were (re)cultured for 144 hours, with medium exchange at 48, 96 and 144 hours to evaluate the estradiol (E2) and progesterone (P4) production (ng/mL). After thawing, there was a reduction in the cell number (p<0,05) and cell viability in both methods, the direct freezing (DF) and the two cultures (2C) (p<0,05); this had an impact in the production of estradiol and progesterone, which were higher in fresh cultures than in the frozen ones (p<0,05). However, the E2/cell ratio was maintained after thawing (p=0.23), suggesting that this impairment in steroid production was probably due to the reduction in the cell count. The cells that survive remain functionally normal. The DF was more efficient since it allowed greater cell recovery and better viability when compared to 2C. The estradiol/progesterone ratio was maintained in all culture times, in the fresh, DF or 2C groups (p>0.05), indicating that the functional characteristic of these cells was preserved post-thawing. We conclude that cryopreservation of human GC obtained during oocyte retrieval compromises the cell count and the overall viability of the culture; however, the functional capacity and the characteristic of these cells are preserved (maintenance of E2/cell and E2/P4 relations)

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of this study was to investigate the factors controling the expression of angiotensin II (AngII) receptors and to determine the physiological role of AngII in granulosa cells. The AGTR2 receptor was localized in granulosa (and theca) cells from follicles of different sizes. Bovine ovaries were collected at a local abattoir and small follicles (2-5mm) were isolated for harvesting granulosa cells. The cells were cultured in free medium serum in non-luteinizing conditions without FSH (control group) or with graded doses of FSH or IGF1. In other cultures, cells were cultured with or without IGF1 and bone morphogenetic protein-7 (BMP-7), and fibroblast growth factor-2 (FGF-2). Treatment with FSH, IGF1 and BMP-7 increased (P<0.05) estradiol secretion and AGTR2 mRNA expression relative to control cultures. In contrast, none of these treatments affected AGTR1 receptor expression. Addition of FGF-2 significantly decreased estradiol secretion but did not affect AGTR1 or AGTR2 expression. Cells were cultured with FSH plus graded doses of FGF-7 or FGF-10, and the effects of these factors on AGTR2 protein levels were measured by Western blot. AGTR2 protein levels decreased in the groups treated with FGF-7 (10 and 100ng/ml) and FGF-10 (all concentrations; P<0.05), and estradiol secretion was significantly inhibited by the highest dose of each FGF (P<0.05). Bovine follicles greater than 5 mm diameter were dissected and granulosa and theca cells were separated for RNA extraction, and follicle fluid assayed for estradiol (E2) and progesterone (P) content. Non-atretic follicles (P less than 100ng/ml) were classed as estrogenic (E2 greater than 100ng/ml) or non-estrogenic (E2 less than 40ng/ml). There were no differences in AGTR1 receptor expression in theca and granulosa cells between estrogenic and nonestrogenic follicles. Likewise, there were no changes in AGTR2 receptor expression in theca cells with follicle state. However, AGTR2 receptor mRNA levels were significantly higher in granulosa cells of estrogenic compared to non-estrogenic follicles (P<0.01), and AGTR2 receptor mRNA was correlated with E2 concentrations in follicular fluid. To determine the physiological consequences of AT activation in granulosa cells, cells from small (2-5mm) bovine follicles were cultured in serum-free medium with FSH ± AngII. The addition of AngII had no effect on estradiol or progesterone secretion, but significantly inhibited protease nexin-1 (PN-1) mRNA levels and protein secretion (P<0.05). PN-1 is an inhibitor of proteases involved in extracellular matrix remodeling and follicle rupture. Bovine granulosa cells from large (>10 mm) follicles were cultured for 6h, 12h and 24h with LH (100ng/ml) or AngII, with or without angiotensin receptor blocker (losartan for AGTR1 and PD123,319 for AGTR2). These cells expressed Ptgs2 under basal culture conditions, which was not upregulated by either LH or AngII alone. However, LH and AngII in combination significantly enhanced Ptgs2 (P<0.05) mRNA and protein accumulation. Similarly, expression of the proteolytic enzymes uPA and tPA, and their inhibitor, PN-1, were upregulated by the combination of LH and AngII but not by either factor alone. The addition of AGTR blockers inhibited the effect of AngII. In conclusion, AGTR2 receptor is present in granulosa bovine cells, and mRNA and protein are regulated by FSH, IGF-1, BMP-7, FGF-7 and FGF-10 in bovine granulosa cells in vitro, AGTR2 but not AGTR1 receptor mRNA levels are regulated during follicular growth in cattle, and that AngII regulates granulosa PN-1 secretion. These data suggest that AngII is a physiological co-factor necessary for the expression of genes in granulosa cells that are critical for ovulation. / O objetivo deste trabalho foi estabelecer o controle de expressão dos receptores de angiotencina II (AngII) e determinar a ação fisiológica da AngII em células da granulosa (CG) cultivadas in vitro. Os receptores de AngII tipo 1 (AGTR1) e tipo 2 (AGTR2) foram localizados em folículos de bovinos de diferentes tamanhos. Verificouse que as CG de bovinos provenientes de folículos entre 2- 5 mm e cultivadas com FSH, IGF-1, BMP-7 apresentaram aumento na expressão do receptor AGTR2 (P<0,05) em relação ao grupo controle (GC), bem como aumento da secreção de estradiol (E2; P<0,05). Em contraste, as CG tratadas com 10 ng/ml de FGF-2 ou 10 e 100 ng/ml de FGF-7 e FGF-10 apresentaram uma redução na secreção de E2 (P<0,05), porém somente os grupos FGF-7 e 10 nas doses 10 e 100 ng/ml reduziram (P<0,05) a expressão do receptor AGTR2. Para os dois experimentos, não houve diferença na expressão do receptor AGTR1 entre o GC e os grupos tratados. As CG e células da teca (CT) foram coletadas de ovários provenientes de abatedouro para extração de RNA e o fluido folicular para dosagem de E2 e progesterona P4. Esses folículos foram classificados como dominantes (FD; P4<100ng/ml e E2>100ng/ml) e atrésicos (FA; P4>40ng/ml). A expressão dos receptores AGTR1 e AGTR2 foi mensurada por RTPCR. Não houve diferença na expressão do receptor AGTR1 entre FD e FA. No entanto, o receptor AGTR2 apresentou um aumento (P<0,05) na expressão em CG de FD em relação a FA. A expressão do receptor AGTR1 se manteve constante em CG e CT de FD e FA. Para determinar os afeitos da AngII através da ativação de seus receptores CG foram cultivadas em meio livre de soro com FSH e/ou AngII. A AngII não apresentou efeito na secreção de E2 ou P4, mas inibiu (P<0.05) mRNA e proteína para a protease nexin-1 (PN-1). Considerando a redução de expressão da PN-1 envolvida no controle do remodelamento da matriz extracelular (RME), é possível especular um efeito de AngII sobre RME durante o desenvolvimento folicular. Em um terceiro experimento, as CG de folículos grandes (>10mm) foram cultivadas durante 6h, 12h e 24h na presença de Ang (10-5) com ou sem LH (100ng/ml). A combinação de AngII e LH aumentou significativamente (P<0,05) a expressão de mRNA e proteína para COX-2, ativador do plasminogênio tipo U e T, bem como para PN-1. Entretanto, AngII ou LH não aumentaram a expressão de COX-2. O aumento da expressão destes gene indica uma função de AngII no processo de ovulação através das CG. Em um segundo momento, verificou-se através de qual receptor a AngII atua para controlar a expressão desses genes. As CG foram cultivadas por 6h com LH e/ou AngII com ou sem inibidores específicos para o receptor AGTR1 (losartan) e AGTR2 (PD123,319). Os resultados demonstraram que a presença do inibidor de AGTR2 bloqueou o efeito da associação de LH e AngII em relação ao grupo controle (P<0.05), demonstrado que a ação da AngII é mediada pelo receptor AGTR2 em CG. Em conclusão, o receptor AGTR2 está presente nas células da granulosa de bovinos e o mRNA para o receptor AGTR2 é regulado durante o crescimento folicular. Além disso, a expressão do mRNA e a tradução da proteína para o AGTR2 são reguladas por FSH, IGF-1, BMP-7, FGF-7 e FGF-10 em CG de bovinos cultivadas in vitro. Os dados também sugerem que AngII regula a proteína PN-1 em CG e age como um co-fator fisiológico necessário para a ovulação.

<p>Over the past decade, studies using murine models have led to the demonstration that CD4+ T helper (Th) cells play a critical role in the control of cancer progression. Additional support for their importance comes from the growing body of recent clinical/translational research data demonstrating the importance of tumor-infiltrating T and B lymphocytes in long-term patient survival for various types of cancer, including breast cancer (BC). As the key population coordinating adaptive immune responses, the role(s) played by individual Th subsets in cancer immunity remains largely controversial. The Th1 subset has uniquely been shown to have a clear anti-tumor effect, guiding CD8+ cytotoxic T cells-mediated direct tumor cell lysis through IFN-γ secretion. Although the negative regulatory role played by Treg cells has been extensively studied in cancer, its prognostic value along with that of Th2 and Th17 cells have not been clearly demonstrated in patients. T follicular helper (Tfh) cells, a recently characterized Th subset that plays a primary role in the generation of B cell memory in secondary lymphoid organs, have not been previously described infiltrating solid tumors. The principal objective of this thesis was to perform an in-depth characterization of tumor-infiltrating CD4+ T cells (TIL) and Th subsets in human BC, where very little is currently known.<p>Using whole genome microarrays, we analyzed the gene expression profiles of TIL relative to their counterparts from the axillary lymph nodes and peripheral blood. Applying a novel approach, we compared TIL profiles with public microarray data for Th subsets, demonstrating: 1) the presence of all major Th subsets (Th1, Th2, Th17, Treg as well as Tfh) in the TIL, 2) the TIL are effector memory rather than central memory cells, 3) the TIL are concomitantly activated and suppressed and 4) TIL from tumors with extensive lymphoid infiltrates are more activated/less suppressed in the TCR/CD3 signaling pathway, producing higher levels and a wider panel of Th cytokines than TIL from minimally-infiltrated tumors.<p>We also performed in vitro experiments to study tumor microenvironment effects on TIL by treating normal CD4+ T cells from healthy donor blood with primary tumor supernatants (SN). Tumor SN largely reproduces the TIL profile in normal Th cells, totally suppressing their activation and inhibiting their cytokine production. Intriguingly, the highly restricted number of cytokines induced by tumor SN included several tumor-promoting factors, such as IL-8 and TNF. SN from an extensively-infiltrated tumor was found to be less immune-suppressive than SN from minimally-infiltrated tumors. In line with this, TIL from minimally-infiltrated tumors are closer to SN-treated (suppressed) activated donor cells whereas TIL from extensively-infiltrated tumors are more similar to activated cells without SN treatment.<p>These results led us to further investigate the observed differences between TIL from extensive and minimally-infiltrated tumors. Genes characterizing Th1 and Tfh cells were enriched in the extensively-infiltrated tumors. PD-1hiCD200hi Tfh cells were specifically detected in extensively-infiltrated tumors by flow cytometry and these cells were determined to be the major source of the chemokine CXCL13. Immunohistochemical analysis demonstrated highly-organized tertiary lymphoid structures (TLS) within the tumor, containing a CD4+/CD8+ T cell zone and a B cell zone with reactive germinal centers where Tfh cells and follicular dendritic cells (FDC) are resident. Their presence suggests the origin of an effective memory anti-tumor immune response.<p>Finally, we generated Tfh- and Th1-specific gene signatures reflecting differences between extensive and minimal TIL and tested their prognostic value in large-patient-scale public data sets. Our Tfh signature predicts better 10-year disease-free survival for all BC subtypes, outperforming the Th1 signature, suggesting that Tfh cells play a more central role than Th1 cells in anti-tumor immunity. CXCL13 is the determinant gene of our Tfh signature, showing particularly strong prognostic power for the HER2+ subtype. Additionally, these signatures also predict a better response to neoadjuvant chemotherapy.<p>This thesis research has demonstrated that a previously undetected Th subset, Tfh cells, infiltrates solid tumors and shown that their presence signals enhanced anti-tumor immunity.<p><p> <p>Durant cette dernière décennie, des travaux menés dans des modèles murins ont permis de mettre en évidence le rôle crucial joué par les lymphocytes T auxiliaires CD4+ (Th) dans le contrôle de la progression des cancers. De plus, de nombreuses études cliniques et/ou translationnelles récentes corroborent ces observations en montrant une corrélation entre l’importance de l’infiltration intra-tumorale par les lymphocytes T et B et la survie à long terme des patients atteints de différents types de cancer, dont le cancer du sein. En tant que chefs d’orchestre de la réponse immune adaptative, les rôles spécifiques des sous-populations des cellules Th restent controversés. Les Th1 sont la seule population exerçant une claire réponse anti-tumorale, qui est liée à la sécrétion d’IFN-γ, une cytokine primordiale à l’action des lymphocytes T cytotoxiques CD8+. Bien que le rôle néfaste des T régulateurs (Treg) a été largement étudié dans le cancer, leur implication pronostique ainsi que celle des Th2 et Th17 n’ont pas encore été clairement démontrées. La présence d’une sous-population de CD4, les T auxiliaires folliculaires (Tfh), cellules clés dans la différenciation des lymphocytes B mémoires au sein des organes lymphoïdes secondaires, n’a jamais été décrite dans les cancers solides. Le but principal de ce travail est de caractériser les sous-populations des lymphocytes T CD4+ infiltrant la tumeur (TIL) en prenant comme modèle le cancer du sein humain. A l’heure actuelle, il existe très peu de données sur les TIL CD4 dans ce type de cancer.<p>Nous avons d’abord établi le profil génique des TIL en les comparant avec ceux provenant des ganglions axillaires ou du sang périphérique. En appliquant une nouvelle approche, nous avons comparé les profils des TIL avec les données publiques de sous-populations de Th et démontré que :1) toutes les sous-populations de cellules Th (Th1, Th2, Th17, Treg et Tfh) infiltrent la tumeur, 2) les TIL ont un phénotype plus proche de celui des cellules mémoires effectrices que des cellules mémoires centrales, 3) les TIL sont simultanément activés et supprimés et 4) les TIL provenant des tumeurs massivement infiltrées («extensives») par des lymphocytes sont mieux activés et moins supprimés que les TIL des tumeurs peu infiltrées («minimales») dans la voie de signalisation TCR et produisent des cytokines d’une quantité plus élevée et d’une répertoire plus large.<p>Nous avons également effectué des expériences in vitro pour étudier l’effet de l’environnement tumoral sur les TIL en traitant des CD4 normaux (provenant des donneuses saines) par le surnageant (SN) extrait des tumeurs fraiches. Le SN tumoral induit un profil génique proche de celui des TIL en inhibant l’activation et la production de cytokines de ces cellules stimulées. Curieusement, parmi le peu de cytokines induites par le SN tumoral, des facteurs pro-tumoraux comme IL-8 et TNF sont détectés. Le surnageant provenant d’une tumeur «extensive» est moins immunosuppresseur que ceux des tumeurs «minimales». Conformément, les TIL provenant des tumeurs «minimales» ont un profil génique proche des cellules normales activées et traitées (supprimées) par le SN tumoral tandis que les TIL des tumeurs «extensives» ressemblent aux cellules activées non traitées.<p>Ces résultats nous avaient guidés à investiguer plus profondément les différences observées entre les TIL des tumeurs «extensives» et «minimales». Les gènes caractéristiques des Th1 et Tfh sont enrichis dans les tumeurs «extensives». Les cellules Tfh PD1hiCD200hi sont spécifiquement détectées par cytométrie de flux dans les tumeurs «extensives» et sont identifiées comme les producteurs principaux de la chimiokine CXCL13. L’examen par immunohistochimie a permis de détecter des structures lymphoïdes tertiaires (TLS) dans la tumeur, composées d’une zone T (CD4 et CD8) et d’une zone B au sein de laquelle se trouve parfois un centre germinatif actif contenant des Tfh et des cellules dendritiques folliculaires (FDC). La présence de ces structures suggère l’origine d’une réponse immune mémoire anti-tumorale.<p>Finalement, nous avons établi des signatures géniques spécifiques aux Tfh et Th1 et recherché leur impact pronostique dans deux bases de données publiques à grande échelle. Notre signature Tfh est positivement corrélée avec la survie à 10 ans des patientes de tous les sous-types de cancer du sein, et est plus performante que la signature Th1. Ceci suggère que les Tfh pourraient jouer un rôle plus crucial que les Th1 dans la réponse immune anti-tumorale. CXCL13 est le gène déterminant de notre signature Tfh et son expression est fortement associée à une meilleure survie chez les patientes du sous-type HER2+. De plus, ces signatures prévoient également une meilleure réponse à la chimiothérapie néoadjuvante (préopératoire).<p>Cette étude a démontré qu’une nouvelle sous-population de CD4, les Tfh, infiltre la tumeur solide et leur présence indique l’existence d’une immunité anti-tumorale renforcée.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Haematological cancers like leukemia and lymphoma are characterized by genetic abnormalities, specifically chromosomal translocations. Analyses of the translocation breakpoint regions in patients have shown that some loci in the genome are more susceptible to breakage than others. However, very little is known about the mechanism of generation of many such chromosomal translocations. In the present study, we have attempted to understand the mechanism of fragility of three regions, which are prone to breaks during translocations in follicular lymphoma (FL) and T-cell leukemia. The t(14;18) translocation in FL is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3’ UTR of the BCL2 gene and are broadly classified into three clusters, namely major breakpoint region (mbr), minor breakpoint cluster region (mcr) and the intermediate cluster region (icr). The RAG complex has been shown to cleave BCL2 mbr by recognizing an altered DNA structure. In the present study, by using a gel based assay, nature of the non-B DNA structure at BCL2 mbr was identified as parallel intramolecular G-quadruplex. Various studies including circular dichroism (CD), mutagenesis, DMS modification assay and 1H NMR showed the presence of three guanine tetrads in the structure. Further, evidence was also found for the formation of such a G-quadruplex structure within mammalian cells. In an effort to characterize the mechanism of fragility of mcr, a unique pattern of RAG cleavage was observed in a sequence dependent manner. Three independent nicks of equal efficiency were generated by RAGs at the cryptic sequence, “CCACCTCT”, at mcr and at a cytosine upstream of it, unlike a single specific nick at the 5’ of heptamer during V(D)J rearrangement. Interestingly, RAG nicking at mcr occured in the presence of both Mg2+ and Mn2+. Using recombination assay, followed by sequencing of the junctions, we find that mcr can recombine with standard RSS in vivo, albeit at a very low frequency. Mutations to this novel motif abolish recombination at the mcr within the cells. In order to determine the prevalence of t(14;18) translocation in the healthy Indian population, nested PCR approach followed by Southern hybridization was used. Results showed 34% prevalence of t(14;18) translocation in the Indian population. Although, no gender based difference was observed, an age dependent increase was found in adults. Further, presence of the t(14;18) transcripts was also detected. The mechanism underlying the fragility of the t(10;14) translocation involving HOX11 gene in T-cell leukemia is not known. Using primer extension assays on a plasmid DNA containing HOX11 breakpoint region, presence of consistent pause sites corresponding to two G-quadruplex forming regions, flanking the patient breakpoints, were detected. These replication blocks were dependent on K+ ions. Native gel shift assays, mutation analysis, S1 nuclease and CD, further revealed formation of intermolecular G-quadruplexes, unlike the BCL2 mbr. Further, sodium bisulfite modification assay indicated the presence of such structures in the genomic DNA within cells. Hence, we propose that two independent G-quadruplex structures formed in the HOX11 gene could interact with each other, thereby resulting in fragility of the intervening sequences, where majority of the patient breakpoints are mapped. Overall, this study has attempted to understand the role of both sequence and structure of DNA, in generating chromosomal fragility during t(14;18) translocation in FL and t(10;14) translocation in T-cell leukemia. These results may facilitate future studies in unraveling the mechanism leading to genomic instability in other lymphoid cancers.

De nos jours, la cryoconservation est une technique très largement utilisée dans les protocoles d’assistance à la reproduction ou comme outil pour la sauvegarde des ressources génétiques. Toutefois, la chienne est un modèle animal complexe pour l’application des biotechnologies de la reproduction du fait de ses nombreuses singularités anatomiques et physiologiques. L’objectif de notre travail était d’étudier et de développer une méthode de cryoconservation des ressources génétiques chez la chienne par le biais de deux types de ressources : les embryons et le tissu ovarien. Après avoir mis au point une méthode de collecte d’embryons, nous nous sommes appliqués à la constitution d’un stock d’embryons cryoconservés en prévision d’un transfert embryonnaire. L’étude et le développement d’un protocole de cryoconservation du tissu ovarien ont été abordés après avoir adapté et validé nos méthodes d’analyses in vitro. L’utilisation de plans d’expériences factoriels fractionnaires a permis de mettre en évidence les facteurs les plus influents sur la qualité de la réserve folliculaire (nature du cryoprotecteur pénétrant, cinétique de congélation, étapes d’équilibration) et de proposer un protocole de cryoconservation. La combinaison du DMSO incorporé en un seul bain d’équilibration avec une vitesse de congélation de 0,3°C/min est apparue comme la combinaison la plus appropriée à la cryoconservation de tissu ovarien chez la chienne et a permis d’observer, après xénogreffe de tissu ovarien cryoconservé, une reprise de la croissance folliculaire et de l’activité hormonale du tissu greffé / Nowadays, cryopreservation is widely used in animal assisted reproduction or safeguarding of genetic resources. Nevertheless, the bitch is a complex animal model concerning the use of this biotechnology, due to numerous anatomical and physiological peculiarities. The aim of our research work was to investigate and develop a method of cryopreservation of genetic resources in the bitch by exploring two kinds of resources: embryos and ovarian tissue. After the setting up of a method for embryo collection, we have built up a stock of cryopreserved embryo for subsequent embryo transfer. After a preliminary validation of our in vitro assessment methods, the investigation and development of a cryopreservation protocol has been conducted. The use of fractional experimental design allowed us to highlight the main factors affecting the follicular pool quality (CPA nature, freezing rate and equilibration steps). The combination of DMSO incorporated in a unique equilibration bath with a freezing rate of 0.3°C/min appeared to be suitable for the cryopreservation of bitch ovarian tissue. Finally, Follicular growth and hormonal activity resumption have been observed after xenotransplantation of cryopreserved bitch ovarian tissue

Les lymphomes B sont des cancers du système lymphatique se développant à partir des cellules B. Il devient évident que le développement des cellules B malignes dépend d’interactions avec les cellules immunes dans leur microenvironnement. Nous avons étudié la caractérisation des lymphocytes T intra tumoraux afin de comprendre leur contribution dans la lymphomagenèse et leur potentiel thérapeutique dans les lymphomes B comme le lymphome diffus à grandes cellules B (DLBCL), le lymphome folliculaire (FL) et le lymphome Hodgkinien classique (cHL)Nous avons mis en évidence une différence importante, quantitative et qualitative, entre la composition immunitaire de différents lymphomes B, notamment au niveau des lymphocytes T intra tumoraux. Le FL se caractérise par une accumulation des lymphocytes T régulateurs (Tregs) exprimant ICOS, pouvant supprimer les cellules B lymphomateuses. La génération des Tregs ICOS+ est favorisée par le contact avec les cellules B lymphomateuses exprimant ICOSL. Quant à lui, le DLBCL a beaucoup de lymphocytes TCD8 coexprimant PD1 et TIM3 possédant un état de dysfonctionnement dit « épuisement », lymphocytes dont la proportion est corrélée à leur niveau de dysfonctionnement et à leur capacité de réponse au blocage des récepteurs inhibiteurs. Enfin, dans certains lymphomes B, en particulier le cHL, nous avons découvert une sous population de TCD8, dite « TFH-like » pour leur similarité phénotypique et fonctionnelle avec les lymphocytes T auxiliaires folliculaires (TFH). Ces données indiquent l’hétérogénéité des composants immunitaires entre différents lymphomes B et sont une piste pour une future thérapie ciblée dans le traitement du lymphome. / B-cell lymphomas represent a heterogeneous group of cancers that affect B cells in the lymphatic system. It has become evidence that malignant B cells depend on various interactions with microenvironmental immune cells for their development. Our study focuses on characterization of intra-tumoral T cells in order to understand their contribution in pathogenesis and their therapeutic potentials in the most frequent B cell-lymphoma such as Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma (FL) and classical Hodgkin lymphoma (cHL).During this work, we have demonstrated a significant quantitative and qualitative difference between different B-cell lymphoma immune composition, especially between their intra-tumoral T cells. FL is characterized by the accumulation of regulatory T cells (Tregs) expressing ICOS, with ability to suppress lymphoma B cells. Generation of Tregs ICOS+ is prompted by cell contact with the lymphoma B cells expressing ICOSL. On the other hand, DLBCL have high level of TCD8 coexpressing PD1 and TIM3 displaying an exhaustion state, which proportion is correlated with their dysfunction level and with their responsiveness to inhibitor receptors blockade. Finally, in some B-cell lymphoma cases, especially cHL, we found the existence of a TCD8 subset, called TFH-like due to their phenotypic and functional similarity with follicular helper T cells (TFH).These data show heterogeneity of immune components between the different B lymphomas, and give opportunity for targeted therapy in lymphoma treatment

Made available in DSpace on 2016-01-26T18:55:27Z (GMT). No. of bitstreams: 1 dissertacao Bianca.pdf: 325135 bytes, checksum: 8bbf94808bc25e72a05aba3d84bca76a (MD5) Previous issue date: 2008-11-14 / The aim of the study was to evaluate the effect of bovine somatotropin in follicular deviation of sixteen Tabapuã cows. The animals received an ear implant of progesterone and 1 mg of estradiol benzoate, IM (day 0). On day 5, the females were divided into 2 groups: GI (control, n=8) and GII (treated with 500 mg bST, n=8). On day 10, the implants were removed and injected 500 µg of PGF2&#945; in all cows. Only cows with follicles bigger 9 mm received 300 µg of GnRH. Ultrasound examinations were performed each 12 hours. There was no statistical difference between the follicular deviation in GI (2.4 days) and GII (2,1 days). At the divergence moment, FD and FS of GI were 6.28±0.42 and 6.26±0.41 mm, respectively, and FD and FS of GII were 6.08±0.72 and 6.12±0.39 mm. The mean maximum diameter of FD after ovulation was at 110.0±8.43 hours in GI and 115.2±8.98 hours in GII. FS reached the maximum diameter at 55.0±20.0 hours in GI and 76.8±10.46 hours in GII. The mean maximum diameter reached by FD and FS in GI was 8.85±0.41 and 6.5±0.42 mm, respectively, and GII 9.83±0.63 and 6.87±0.35 mm. There was no significant difference (p> 0.05) in diameter of FD and FS, neither in growth rates (mm/12 hours) of the FD before and after the deviation, neither in the moment of follicle deviation / O objetivo do presente estudo foi avaliar o efeito da somatotrofina bovina na divergência folicular em vacas da raça Tabapuã. Foram utilizadas 16 vacas da raça Tabapuã, inicialmente receberam implante de progestágeno auricular concomitante à aplicação IM de 1mg de benzoato de estradiol (dia 0). No dia 5, dividiram-se as fêmeas em 2 grupos: G-I (controle, n=8) e as vacas do G-II foram tratadas com 500 mg bST (n=8). No dia 10 foi feita a retirada do crestar concomitante a aplicação de 500 &#61549;g de PGF2&#61537; e apenas nas vacas com folículos> 9 mm aplicação de 300 &#61549;g de GnRH. Foram realizados exames ultrassonográficos a cada 12 horas por 5 dias. Não houve diferença estatística entre os momentos de divergência folicular, no G-I foi de 2,4 dias e no G-II 2,1 dias. Nesse momento o FD e FS mediram 6,28 &#61617; 0,42 e 6,26 &#61617; 0,41 mm no G-I e 6,08 &#61617; 0,72 e 6,12 &#61617; 0,39 mm no G-II. O FD atingiu diâmetro máximo após a ovulação em média 110,00 ± 8,43 horas no GI e 115,20 ± 8,98 horas no GII. Já o FS atingiu o diâmetro máximo às 55,00 ± 20,00 e 76,80 ± 10,46 horas, respectivamente. A média do diâmetro máximo atingido pelo FD e FS no GI foi respectivamente 8,85 ± 0,41 e 6,50 ± 0,42 mm e no GII foi 9,83 ± 0,63 e 6,87 ± 0,35 mm. Não houve diferença significativa (p>0,05) no diâmetro do FD e FS e nem nas taxas de crescimento (mm/12h) do FD antes e após a divergência folicular. Concluímos que a aplicação de bST não afetou o diâmetro folicular, a taxa de crescimento do FD e FS antes e após a divergência, nem tampouco, o momento da divergência folicular