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    Study the functional impact of the BRCA2 portuguese founder mutation in mammary epithelial progenitors

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    Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2022As muta√ß√Ķes BRCA1 e BRCA2 s√£o as principais precursoras de cancro heredit√°rio da mama e ov√°rio. BRCA1 e BRCA2 s√£o prote√≠nas supressoras de tumor, cruciais para manter a estabilidade gen√≥mica, nomeadamente atrav√©s da repara√ß√£o de double-strand DNA breaks (DSBs) por recombina√ß√£o hom√≥loga. As prote√≠nas BRCA est√£o expressas na maioria dos tecidos. Dado que o seu papel fisiol√≥gico parece relevante de forma ub√≠qua, por que √© que as muta√ß√Ķes germinativas do gene BRCA est√£o associadas maioritariamente a cancro da mama e ov√°rio? Uma hip√≥tese √© que a muta√ß√£o BRCA2 esteja associada a neoplasias em tecidos dependentes de estrog√©nio por um efeito sinerg√≠stico entre a defici√™ncia na repara√ß√£o de DSBs e o dano no DNA induzido pelo estrog√©nio. Para testar esta hip√≥tese, propusemo-nos a estudar o impacto funcional da muta√ß√£o BRCA2 portuguesa (BRCA2 156_157insAlu) em progenitores mam√°rios epiteliais. O objetivo deste projeto √© tripartido: desenvolver um modelo de progenitores epiteliais do tecido mam√°rio epiteliais a partir de c√©lulas estaminais pluripotentes induzidas (iPSCs), caracterizar estes progenitores epiteliais mam√°rios, e finalmente estudar as diferen√ßas funcionais entre os progenitores mam√°rios com a muta√ß√£o BRCA2 portuguesa e os progenitores saud√°veis como controlo. iPSCs obtidas a partir de uma portadora da muta√ß√£o BRCA2 portuguesa em heterozigotia foram diferenciadas em c√©lulas progenitoras de epit√©lio mam√°rio. Estas c√©lulas foram tratadas com estrog√©nio e metabolitos de estrog√©nio (2-OHE2 e 4-OHE2), de modo a comparar a presen√ßa de DSBs (atrav√©s da quantifica√ß√£o de gamaH2AX) nas c√©lulas com muta√ß√£o BRCA2 e BRCAwild type, utilizando a t√©cnica de imunofluoresc√™ncia. Observ√°mos que a percentagem de c√©lulas com DSBs foi superior em progenitores epiteliais mam√°rios derivados de iPSCs com a muta√ß√£o BRCA2 portuguesa, comparativamente √†s c√©lulas BRCA wild-type. Contudo, n√£o houve diferen√ßa entre os progenitores epiteliais mam√°rios tratados com estrog√©nio, metabolitos de estrog√©nio e mock solvent, tanto nas c√©lulas com BRCA mutado como controlo.BRCA1 and BRCA2 mutations are the main drivers of hereditary breast and ovarian cancer. BRCA1 and BRCA2 are tumor suppressor proteins, with crucial roles in maintaining genomic stability. It is well-established that BRCA1 and BRCA2 proteins play an important part in the repair of double-strand DNA breaks (DSBs) by homologous recombination. BRCA1 and BRCA2 are expressed in most tissues. Given that BRCA physiological roles as tumor suppressor proteins seem relevant ubiquitously, why are germline BRCA gene mutations mainly associated to breast and ovarian cancers? One hypothesis is that BRCA2 mutation causes cancer in estrogen-related tissues by a synergistic effect between DSBs repair deficiency and estrogen-induced DNA damage and genomic instability. To test this hypothesis, we studied the functional impact of the BRCA2 Portuguese founder mutation (BRCA2 156_157insAlu) in mammary epithelial progenitors. The aim of this project is threefold: to develop a model of mammary epithelial progenitors derived from induced pluripotent stem cells (iPSCs), then to characterize these mammary epithelial progenitors, and finally to study the functional differences between the mammary epithelial progenitors with BRCA2 Portuguese founder mutation, and BRCA wild-type control. iPSCs generated from a heterozygous carrier of the BRCA2 Portuguese founder mutation were differentiated into mammary epithelial progenitors and characterized. Finally, the mammary epithelial progenitors were treated with estrogen and estrogen metabolites (2-OHE2 and 4-OHE2), to compare the presence of DSBs (through the quantification of gammaH2AX) in both BRCA2 mutated and healthy control cells, using immunofluorescence. We observed that the percentage of cells with DSBs was higher in mammary epithelial cells derived from iPSCs with BRCA2 Portuguese founder mutation, compared with BRCA wild-type control. However, conversely to what we expected, there was no significant difference between mammary epithelial cells treated with estrogen, estrogen metabolites and mock solvent, in both BRCA mutated and BRCA wild-type control
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