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Orientadores: Maricilda Palandi de Mello, Celso Eduardo Benedetti, Fernanda Caroline Soardi Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A expressão do gene SRY (Sex Determining Region in chromosome Y) é responsável por desencadear a determinação testicular durante o desenvolvimento embrionário, a partir das gônadas ainda indiferenciadas. Mutações nesse gene são encontradas em muitos casos de anomalias do desenvolvimento gonadal. O projeto teve por objetivo principal a análise funcional do efeito de uma mutação na região promotora do gene SRY, sendo que essa mutação consiste em uma deleção de 3 pares de base em um dos sítios consenso de ligação do fator de transcrição Sp1 ao promotor do gene. O portador dessa mutação é um indivíduo com disgenesia gonadal pura 46,XY, sendo que outros membros da família apresentavam ambiguidade genital e o pai, também portador da mutação, possuía grave hipospadia ao nascimento. Para tentar esclarecer os efeitos desta mutação nos mecanismos moleculares de regulação da expressão do gene SRY, este trabalho primeiramente analisou a interação da proteína Sp1 com os sítios localizados na região promotora de SRY e o efeito da mutação nesta interação, através de ensaios de EMSA (Electrophoretic Mobility Shift Assay). Concluiu-se que os sítios Sp1A e Sp1B se ligam a duas moléculas de Sp1 e que a mutação no Sp1A praticamente abole esta ligação. Possivelmente a ausência dessa ligação impediu a formação de um complexo de transcrição, causando uma diminuição na expressão do gene SRY e levando à ausência de formação dos testículos e reversão sexual completa na paciente. Complementando, foi analisado o efeito dessa mutação na expressão através de ensaio de expressão com gene repórter, no qual o promotor normal se mostrou em média duas vezes mais eficiente na ativação da expressão da luciferase que o promotor mutante. Entretanto, mais experimentos de transfecção, inclusive com outras linhagens celulares, devem ser realizados para confirmação desse resultado. Além disso, foi analisado o efeito de uma nova mutação (localizada na região codificante do gene SRY) na ligação da proteína SRY com o DNA, através de ensaios de EMSA. Concluiu-se que a mutação E89K, associada com disgenesia gonadal pura 46,XY, reduziu em alto nível a atividade de ligação in vitro da proteína SRY mutante ao DNA, o que representa um forte indício de que atividade reduzida da proteína mutante não foi suficiente para desencadear o processo de determinação testicular. Paralelamente, foi feito o rastreamento de mutações no gene SRY e sua região promotora em novos casos de disgenesia gonadal, não tendo sido encontradas, porém, alterações nos pacientes analisados Abstract: The SRY (Sex Determining Region in chromosome Y) gene expression is responsible for testicular determination during embrionary development. Mutations in SRY are found in many cases of anomalies of gonadal development. This project analyzed the functional effect of a mutation in SRY promoter region; the mutation is a 3-bp deletion in a consensus binding site for Sp1 transcription factor. The patient presented 46,XY pure gonadal dysgenesis, and a family history of relatives with different levels of genital ambiguity. Her father shares the same mutation in the SRY promoter region. In order to investigate the effects of the mutation upon the molecular mechanisms that regulate SRY gene expression, the interaction of the Sp1 transcription factor with normal and mutant binding sites was analyzed by EMSA (Electrophoretic Mobility Shift Assay). Each of Sp1A and Sp1B normal sites binds to a single Sp1 molecule, whereas the 3-bp deletion in Sp1A abolishs the binding to this site. Probably, the lack of Sp1 binding to the Sp1A site prevented the formation of a stable transcription complex, reducing the level of SRY expression and leading to the absence of testicles and complete sex reversal in the patient. Parallely, the effect of the mutation was analyzed by a reporter gene assay, indicating that the normal promoter is almost two times more efficient than the mutant promoter in the activation of luciferase gene expression using HeLa cells. However, further transfection experiments with other cell lineages must be performed to confirm this result. In addition, the effect of a new mutation (E89K, located in the SRY gene coding region) was analyzed by testing the ability of the SRY mutant protein to bind its DNA consensus sequence. EMSA assays revealed that the E89K mutation, which is associated with 46,XY pure gonadal dysgenesis, strongtly reduced the SRY protein binding activity in vitro. This result is a strong evidence that the reduced activity of SRY mutant protein was not sufficient to trigger the testicular determination in the patient, leading to the pure gonadal dysgenesis phenotype. Screening of mutations in the SRY gene coding and promoter regions was also performed in six diferent patients with 46,XY gonadal dysgenesis. However, other mutations have not been identified Mestrado Genética Animal e Evolução Mestre em Genética e Biologia Molecular

Introduction: The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated. Methods: Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis. Results: A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release. Conclusions: The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension.