Nos últimos anos, notou-se aumento da incidência de carcinoma espinocelular de orofaringe (CECOF) associado ao HPV. Sabe-se que CECOF associado ao HPV apresenta melhor prognóstico do que CECOF não infectado por HPV. Inúmeros estudos em carcinoma cervical demonstram alterações de TLRs, isto provavelmente devido às associações das oncoproteínas E6 e E7 com estes receptores. Em humanos, existem 10 TLRs identificados, os quais colaboram na resposta imune contra bactérias, fungos e vírus, bem como colaboram na promoção ou regressão do tumor. Esta influência do TLR na carcinogênese tem sido alvo de inúmeros estudos devido à ligação entre inflamação e o câncer. O presente trabalho teve como objetivo verificar diferenças na expressão e função de receptores Toll-like em carcinoma espinocelular de orofaringe (CECOF). Para tal, foram utilizados trinta e sete espécimes diagnosticados como CECOF e a expressão imuno-histoquímica das proteínas p16 e TLR4 analisadas. Duas linhagens de CECOF HPV16 + e duas CECOF HPV-. foram utilizadas para análise da expressão de TLR1-10, IL-6 e IL-8, por qPCR. A detecção dos principais TLRs (TLR1, TLR2, TLR6 e TLR4) foi feita por citometria de fluxo. Para ativação da via de sinalização de TLR2, e posterior análise da expressão de IL6 e IL8, as células foram estimuladas com peptidoglicano. Para verificar a expressão e função de TLR4, as células foram estimuladas com LPS e LPS UP para posterior análise de IL-6 e IL-8, por ELISA. Os resultados demonstraram diferenças na expressão gênica de TLR1 e TLR6 entre as linhagens HPV- e o grupo HPV+ e diferenças na expressão proteica de TLR9. TLR2 apresentou aumento da expressão proteica em todas as linhagens e demonstra desencadeamento da resposta imune, com secreção de IL6 e IL8 nas linhagens HPV- (SCC72 e SCC89) e em uma das linhagens HPV+ (SCC2). Interessantemente, TLR4 não apresentou diferenças significativas na expressão gênica e proteica. Entretanto, as linhagens HPV+ não demonstraram resposta pró-inflamatória mesmo quando estimuladas com LPS e LPS ultra puro, agonista específico de TLR4. Assim, este trabalho contribui para estabelecer o perfil da expressão dos receptores Toll-like em linhagens celulares de CECOF HPV- e HPV+, e aponta para alterações ocorridas na via de sinalização mediada por TLR4. Além disso, nossos resultados abrem portas para futuros estudos na avaliação de alterações causadas no sistema imune inato pelo HPV, em carcinomas espinocelulares de orofaringe. / The incidence of HPV- associated oropharynx squamous cell carcinoma (OPSCC) has increased in recent years. HPV- associated OPSCC has a better prognosis than OPSCC not infected with HPV. In cervical carcinoma, HPV oncoproteins E6 and E7 influence the expression of Toll-like receptors (TLR). To date, 10 TLRs have been identified in humans and many are important for the detection of bacteria, fungi and viruses, as well as regression and tumor promotion. This influence of TLR in carcinogenesis has been the subject of numerous studies, due to the connection between inflammation and cancer. This study aimed to determine differences in the expression and function of Toll-like receptor in OPSCC. Thirty-seven tumours were selected and immunohistochemistry for p16 and TLR4 was performed. Two HPV16-associated OPSCC and two OPSCC not associated to HPVcell lines were used. qPCR was performed to analyze the expression of TLR1-10, IL-6 and IL-8. The detection of the main TLRs (TLR1, TLR2, TLR6 and TLR4) was performed by flow cytometry. For activation of the TLR2-signaling pathway and subsequent analysis of IL6 and IL8 expression, cells were stimulated with peptidoglycan. To verify the expression and function TLR4 cells were stimulated with LPS and LPS UP and subsequent analysis of IL-6 and IL-8 by ELISA. The results showed differences in the expression of TLR1 and TLR6 between the HPV- group and the HPV+ group and differences in protein expression of TLR9. TLR2 has increased protein expression in all cell lines and demonstrates the trigger of the immune response, with the secretion of IL6 and IL8 in HPV- cell lines (SCC72 and SCC89) and one HPV+ cell line (SCC2). Interestingly, TLR4 showed no significant differences in gene and protein expression. However, HPV+ cell lines showed no pro-inflammatory response even when stimulated with LPS and LPS UP, a specific agonist of TLR4. This work helps to establish the profile of the expression of Toll-like receptors in OPSCC (SCC72 and SCC89) and HPV- associated OPSCC (SCC2 and SCC90), in vitro, and points to changes in the signaling pathway mediated by TLR4. In addition, our results open new avenues for future studies to assess changes in the innate immune system caused by HPV in oropharynx carcinomas.

Adipocytokines released by the adipose tissue play an important role in the regulation of immune and inflammatory responses. In obesity their production is dysregulated, which is one of the major factors contributing to the onset of a chronic low-grade systemic inflammation representing a risk factor for the progression of other diseases, such as atherosclerosis or type-2 diabetes. The main goal of this thesis was to analyze the secretion of selected adipocytokines (adiponectin, IL6 and MCP1) by in-vitro differentiated adipocytes, isolated from the adipose tissue prior to and after a dietary intervention, and this under basal conditions and during stimulated lipolysis. In case of adiponectin, the secretion of its isoforms was analyzed too. The concentration of adiponectin, IL6 and MCP1 was determined by the ELISA method, the Western Blot method was used to determine the distribution of the adiponectin isoforms. The thesis also concentrates on the gene expression of ATF3, ATF4 and HSPA5, factors engaged in the ER stress in the course of the differentiation of adipocytes. The changes in the gene expression were measured by the quantitative Real Time PCR method. At the same time the development of the endoplasmic reticulum (ER) in the course of adipogenesis was monitored by indirect...

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.