Submitted by Irvana Coutinho (irvana@ufpa.br) on 2012-12-12T14:17:30Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoPerfilMetilacao.pdf: 1830589 bytes, checksum: f88cbaf31829d2cb8cb979c72b380439 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-12-20T12:59:58Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoPerfilMetilacao.pdf: 1830589 bytes, checksum: f88cbaf31829d2cb8cb979c72b380439 (MD5) / Made available in DSpace on 2012-12-20T12:59:58Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoPerfilMetilacao.pdf: 1830589 bytes, checksum: f88cbaf31829d2cb8cb979c72b380439 (MD5) Previous issue date: 2012 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer gástrico continua sendo uma importante causa de morte dentre os tipos de câncer no Brasil e no mundo. A origem do câncer de estomago provém, assim como nos demais, de acúmulo de alterações genéticas. Portanto, se faz necessário saber quais alterações genéticas são importantes para desencadear a patogênese do câncer gástrico. O MNU, conhecido carcinógeno, quando ingerido de forma oral em doses determinadas desencadeia o desenvolvimento de adenocarcinoma gástrico do tipo intestinal, com aparecimento de estágios pré definidos característico deste tipo tumoral. Com base nestes conhecimentos, realizamos um experimento com 6 macacos da espécie Cebus apella induzidos à desenvolver câncer gástrico do tipo intestinal. Os animais ingeriam 16mg/kg de peso diariamente da droga, com desenvolvimento de lesões pré-neoplásicas em todos. Infelizmente, devido à toxicidade da droga, somente um animal sobreviveu ao tratamento e desenvolveu desenvolveu uma massa tumoral propriamente dita. Foram feitas avaliações periódicas dos animais, em dias pré-determinados (ao início, no 120º, 150º, 300º, 940º) com coletas de fragmentos da mucosa gástrica. Foram coletadas 20 amostras de tecido, distribuídas entre mucosa normal, gastrite, displasia, metaplasia e tumoral. Destas amostras extraímos DNA para as análises do gene CDH1. Não há na literatura sequencia deste gene para a espécie utilizada no estudo. Com este objetivo, utilizamos iniciadores construídos a partir de sequencias de CDH1 de Callithrix jacchus (espécie filogeneticamente) e seqüenciamos cerca de 342pb da região promotora de CDH1 de C. apella. As análises mostraram uma similaridade de 98% desta região com a dos humanos, com presença de vários sítios de ligação de fatores de transcrição (sp1, Ap2, NF-x, AREB6, Puf e CTF) além da presença do CAAT Box. Esta região ainda possui 30 sitios CpGs, o que indica que ela pode estar sofrendo regulação epigenética. Para se verificar como se encontrava o padrão de metilação desta região realizamos uma análise de MSP com iniciadores específicos e constatamos que houve predominância de alelos não metilados de CDH1 para todas as amostras pré neoplásicas e a amostra de adenocarcinoma encontra-se metilada. O que pode ser constatado com um ensaio de Imunohistoquímica, onde somente a amostra tumoral não expressava a proteína caderina. Desta maneira nossas análises sugerem que a metilação do gene CDH1 desempenha papel importante na tumorigênese gástrica em C. apella, e é um evento tardio, uma vez que não é observado nos outro tipos teciduais não tumorais E, partindo-se da similaridade entre as seqüências, podemos sugerir que o mesmo evento pode está ocorrendo em humanos. Esta metilação do promotor do CDH1 leva a um silenciamento deste gene o que desencadeia o estabelecimento de adenocarcinomas gástricos, fato apoiado pela literatura onde vários trabalhos apontaram que a hipermetilação do promotor da caderina está associada ao desenvolvimento do câncer gástrico. / The gastric cancer remains a major cause of death among cancers in the world. In Brazil, are expected around 500 000 new cases in 2012/2013. The origin of stomach cancer, as in others, arises from the accumulation of genetic alterations. Therefore, it is necessary to know which genetic changes are important in triggering the pathogenesis of gastric cancer. We know that the intestinal type develops through well defined stages. The MNU - a known carcinogen - when ingested in oral form at dosages determined triggers development of this histological type of gastric cancer. Based on this knowledge, we conducted an experiment with six specime of Cebus paella, induced to develop intestinal type gastric cancer. The animals consumed 16mg/kg of body weight daily drugs. All pre-neoplasic lesions developed. Due to drug toxicity, only one survived the entire treatment and developed a tumor. Periodic evaluations were made of animals in pre-determined days (the beginning, days 120, 150, 300, 940) during which samples were made of the gastric mucosa. We collected 20 samples of tissue, distributed among normal mucosa, gastritis, dysplasia, metaplasia, and tumor. DNA extracted from these samples for further analyzes of the CDH1 gene. There is no sequence of this gene in the literature for the species under study. So the first step was to get this sequence. Using the initiators constructed from sequences of CDH1 Callithrix jacchus (species phylogenetically closer to C. apella) we get a sequence of 342pb. There is a similarity of 98% with humans, the presence of binding sites for transcription factors like (sp1, Ap2, NF-x, AREB6, Puf and CTF) and the presence of CAAT Box. The sequence has 30 CpG sites could suffer epigenetic regulation. We also performed MSP analysis with specific primers and found that the region analyzed, there was predominance of the unmethylated CDH1 alleles for all samples pre-neoplastic and adenocarcinoma sample is methylated. When we compared these data with immunohistochemistry revealed that only tumor sample did not express the protein cadherin. Our analyzes suggest that methylation of the CDH1 gene plays an important role in gastric tumorigenesis in C. apella. And, from the similarity between sequences, we suggest that the same occurs in humans. This cadherin promoter methylation leads to inactivation of the gene and establishment of gastric adenocarcinomas. The cadherin promoter hypermethylation has been reported in several articles associated with the development of gastric cancer.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-04-17T23:12:48Z No. of bitstreams: 1 AnaRafaelaDeSouzaTimoteo_TESE.pdf: 3659954 bytes, checksum: d2c8b061166b6be5547b4452cf6fed7b (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-04-20T21:54:51Z (GMT) No. of bitstreams: 1 AnaRafaelaDeSouzaTimoteo_TESE.pdf: 3659954 bytes, checksum: d2c8b061166b6be5547b4452cf6fed7b (MD5) / Made available in DSpace on 2017-04-20T21:54:51Z (GMT). No. of bitstreams: 1 AnaRafaelaDeSouzaTimoteo_TESE.pdf: 3659954 bytes, checksum: d2c8b061166b6be5547b4452cf6fed7b (MD5) Previous issue date: 2016-12-12 / A S?ndrome de c?ncer de mama e ov?rio heredit?rio corresponde a 10-15% de todos os casos diagnosticados de c?ncer de mama no mundo. A maioria das muta??es germinativas s?o identificadas nos genes BRCA1 e BRCA2, contudo, a aplica??o de pain?is multig?nicos tem aumentado o n?mero de variantes patog?nicas detectadas em outros genes supressores de tumor. De acordo com a vers?o atual do protocolo americano NCCN (National Comprehensive Cancer Network), as muta??es em BRCA1 e BRCA2, TP53 e PTEN conferem alto risco de desenvolver c?ncer de mama, e muta??es em CDH1, CHEK2, PALB2, ATM e BRIP podem aumentar em 20% o risco para o desenvolvimento desta doen?a. Neste estudo foram analisados 157 indiv?duos com hist?rico pessoal e/ou familiar de c?ncer de mama. O DNA gen?mico foi isolado a partir de sangue perif?rico por meio de extra??o ? base de solu??o salina e as amostras foram analisadas usando o sequenciamento de nova gera??o (NGS). Foram identificadas 15 variantes patog?nicas e 4 VUS (Variants of Uncertain Significance) em 27 indiv?duos (27/157; 17%), dos quais tr?s s?o assintom?ticos. Foram identificadas sete novas variantes em 4 genes: BRCA1_c.3409A>G; BRCA2_g.26826_30318del, BRCA2_c.5800C>T; BRCA2_c.5228G>A; BRCA2_c.5305delG; ATM_c.634delT e ATR_c.3043C>T. Sessenta e oito por cento (13/19; 68%) de variantes foi detectada nos genes BRCA1 e BRCA2, enquanto 32% (6/19) foram identificados nos genes de risco moderado ATM (2/19); ATR (1/19); CDH1 (1/19); MLH1 (1/19) e MSH6 (1/19). Os indiv?duos foram separados em dois grupos para a an?lise comparativa: portadores de muta??o nos genes de alto risco e nos genes de risco moderado. Entre os tr?s indiv?duos assintom?ticos, duas variantes est?o presentes nos genes de risco moderado ATM e MLH1. Entre os indiv?duos com c?ncer de mama, dezoito pacientes (18/24; 75%) apresentaram muta??es em genes de alto risco, enquanto seis (6/24; 25%) s?o portadores de muta??es em genes de risco moderado. Ambos os grupos apresentaram alta incid?ncia de c?ncer de mama precocemente (83% dos indiv?duos). O grupo de portadores de muta??o nos genes de alto risco apresentaram maior ocorr?ncia de tumores de alto grau (83% vs 67%, P = 0,0090). No grupo de indiv?duos com muta??es em genes de risco moderado, os tumores apresentaram um fen?tipo mais agressivo com c?ncer bilateral (33% versus 11%, P = 0,0002), ocorr?ncia de met?stases (33% vs 5,6%, P <0,0001) e ?bito (33% vs 5,6%, P <0,0001). Ao todo, 1/3 de variantes foram identificadas em genes de risco moderado em pacientes com c?ncer mais agressivo. Estes resultados refor?am a import?ncia da aplica??o de an?lise multig?nica em indiv?duos em situa??o de risco para c?ncer de mama, especialmente em uma popula??o heterog?nea como brasileira. / Hereditary breast and ovarian cancer (HBOC) corresponds to 10-15% of all diagnosed cases of breast cancer in the world. The majority germline mutations are identified in BRCA1 and BRCA2 genes, however the application of multigene panels has increased the number of pathogenic variations detected in DNA repair genes. According to the current version of NCCN (National Comprehensive Cancer Network) Guideline, mutations in BRCA1, BRCA2, TP53 and PTEN confers high risk to develop breast cancer, and mutations in CDH1, CHEK2, PALB2, ATM and BRIP can increases over than 20% this risk. We analyzed 157 individuals with personal and/or familial breast cancer history. Genomic DNA was isolated from peripheral blood through saline-based extraction and samples were analyzed using next-generation sequencing (NGS). We identified 15 pathogenic variants and 4 VUS (Variants of Uncertain Significance) in 27 individuals (27/157; 17%), in which three are asymptomatic. Seven novel variants in 4 genes were identified: BRCA1_c.3409A>G; BRCA2_g.26826_30318del, BRCA2_c.5800C>T; BRCA2_c.5228G>A; BRCA2_c.5305delG; ATM_c.634delT and ATR_c.3043C>T. Sixty-eight percent (13/19; 68%) of variants was detected in BRCA1 and BRCA2 genes, while 32% (6/19) were identified in moderate risk genes ATM (2/19); ATR (1/19); CDH1 (1/19); MLH1 (1/19) and MSH6 (1/19). The individuals were separated in two groups for comparative analysis: high-risk genes and moderate risk genes. Among three asymptomatic individuals, two present variants in moderate risk genes ATM and MLH1. Among breast cancer individuals, eighteen patients (18/24; 75%) presented mutations in high-risk genes, while six (6/24; 25%) harbored mutations in moderate risk genes. Both groups had a high incidence of early-onset breast cancer, 83%. The group of individuals harboring variants in high-risk genes presented a greater occurrence of high-grade tumors (83% vs. 67%, P= 0.0090). In the group of individuals harboring mutation in moderate risk genes, tumors presented a more aggressive phenotype with bilateral cancer (33% vs. 11%, P= 0.0002), occurrence of metastasis (33% vs. 5.6%, P<0.0001) and incidence of deaths (33% vs. 5.6%, P<0.0001). Altogether, 1/3 of variants were identified in moderate risk genes in patients presenting a more aggressive phenotype. These results reinforce the importance of applying multigene analysis in individuals at-risk for breast cancer, especially in a heterogeneous population as Brazilian.

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Bypass or resection of the stomach and oesophagus, has long been recognised to result in profound changes in the handling of ingested nutrients. This results in significant morbidity after radical surgery for oesophago-gastric cancer, in particular post-prandial hypoglycaemia, altered appetite, early satiety and noxious post-prandial symptoms. By profiling and challenging the gut hormone axis in healthy volunteers and patients who had undergone total or subtotal gastrectomy, or oesophagectomy, this thesis explores the possible causative mechanisms for the challenges faced by this patient population. In the surgical groups, an oral glucose tolerance test (OGTT) resulted in enhanced secretion of satiety and incretin gut hormones (GLP-1, GIP, PYY) and insulin, followed by hypoglycaemia in a cohort of patients. Continuous glucose monitoring of gastrectomy participants over two weeks of normal lifestyle identified an increased incidence of day and night time hypoglycaemia. RNAseq and mass spectrometry based peptidomics of human and murine enteroendocrine cells in the pre- and post-operative populations revealed no significant change in the underlying cellular pathways for nutrient sensing and gut hormone secretion, indicating that the altered hormone secretion is primarily driven by accelerated nutrient transit, rather than adaptive changes in the gut. Finally, specific blockade of the GLP-1 receptor in post-gastrectomy patients using Exendin 9-39 normalised insulin secretion and prevented reactive hypoglycaemia after an OGTT. In conclusion, profound changes in gut hormone secretion as a result of enhanced nutrient transit after foregut surgery likely underlie the early and late post-prandial symptoms seen in this group, and therapies specifically targeting the gut hormone axis, and GLP-1 in particular, could be the first targeted treatments for post-gastrectomy syndromes.