Worldwide obesity has more than doubled since 1980 and at least 2.8 million people die each year as a result of being overweight or obese. An elevated body weight is the result of the interplay between susceptibility gene variants and an obesogenic environment, and recent evidence shows that epigenetic processes are likely involved. The growing availability of high-throughput technologies has made it possible to assess quickly the entire epigenome of large samples at a relatively low cost. As a result, vast amounts of data have been generated and researchers are now confronted to both bioinformatic and biostatistic challenges to make sense of such data in the context of obesity and its complications. In this doctoral thesis, we explored associations between the human blood methylome and obesity-associated gene variants as well as dietary fat quality and quantity. We used well described preprocessing techniques and statistical methods, along with publicly available data from consortiums and other research groups, as well as tools for pathway enrichment and chromatin state inference. We found associations between obesityassociated SNPs and methylation levels at proximal promoters and enhancers, and some of these associations were replicated in multiple tissues. We also found that contrary to dietary fat quantity, dietary fat quality associates with methylation levels in the promoter of genes involved in metabolic pathways. Then, using a gene-targeted approach, we looked at the impact of an acute environmental stress (sleep loss) on the methylation and transcription levels of circadian clock genes in skeletal muscle and adipose tissue of healthy men. We found that a single night of wakefulness can alter the epigenetic and transcriptional profile of core circadian clock genes in a tissue-specific manner. Finally, we looked at the effects of chronic maternal obesity and subsequent weight loss on the transcription of epigenetic machinery genes in the fetus and placenta of mice. We found that the transcription of epigenetic machinery genes is highly sensitive to maternal weight trajectories, and particularly those of the histone acetylation pathway. Overall, this thesis demonstrated that genetics, obesogenic environment stimuli and maternal programming impact epigenetic marks at genomic locations relevant in the pathogenesis of obesity.

Thesis advisor: Gabor T. Marth / Background: Due to the rapid development and application of next generation sequencing (NGS) techniques, large amounts of NGS data have become available for genome-related biological research, such as population genetics, evolutionary research, and genome wide association studies. A crucial step of these genome-related studies is the detection of genomic variation between different species and individuals. Current approaches for the detection of genomic variation can be classified into alignment-based variation detection and assembly-based variation detection. Due to the limitation of current NGS read length, alignment-based variation detection remains the mainstream approach. The Smith-Waterman algorithm, which produces the optimal pairwise alignment between two sequences, is frequently used as a key component of fast heuristic read mapping and variation detection tools for next-generation sequencing data. Though various fast Smith-Waterman implementations are developed, they are either designed as monolithic protein database searching tools, which do not return detailed alignment, or they are embedded into other tools. These issues make reusing these efficient Smith-Waterman implementations impractical. After the alignment step in the traditional variation detection pipeline, the afterward variation detection using pileup data and the Bayesian model is also facing great challenges especially from low-complexity genomic regions. Sequencing errors and misalignment problems still influence variation detection (especially INDEL detection) a lot. The accuracy of genomic variation detection still needs to be improved, especially when we work on low- complexity genomic regions and low-quality sequencing data. Results: To facilitate easy integration of the fast Single-Instruction-Multiple-Data Smith-Waterman algorithm into third-party software, we wrote a C/C++ library, which extends Farrar's Striped Smith-Waterman (SSW) to return alignment information in addition to the optimal Smith-Waterman score. In this library we developed a new method to generate the full optimal alignment results and a suboptimal score in linear space at little cost of efficiency. This improvement makes the fast Single-Instruction-Multiple-Data Smith-Waterman become really useful in genomic applications. SSW is available both as a C/C++ software library, as well as a stand-alone alignment tool at: https://github.com/mengyao/Complete- Striped-Smith-Waterman-Library. The SSW library has been used in the primary read mapping tool MOSAIK, the split-read mapping program SCISSORS, the MEI detector TAN- GRAM, and the read-overlap graph generation program RZMBLR. The speeds of the mentioned software are improved significantly by replacing their ordinary Smith-Waterman or banded Smith-Waterman module with the SSW Library. To improve the accuracy of genomic variation detection, especially in low-complexity genomic regions and on low-quality sequencing data, we developed PHV, a genomic variation detection tool based on the profile hidden Markov model. PHV also demonstrates a novel PHMM application in the genomic research field. The banded PHMM algorithms used in PHV make it a very fast whole-genome variation detection tool based on the HMM method. The comparison of PHV to GATK, Samtools and Freebayes for detecting variation from both simulated data and real data shows PHV has good potential for dealing with sequencing errors and misalignments. PHV also successfully detects a 49 bp long deletion that is totally misaligned by the mapping tool, and neglected by GATK and Samtools. Conclusion: The efforts made in this thesis are very meaningful for methodology development in studies of genomic variation detection. The two novel algorithms stated here will also inspire future work in NGS data analysis. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

No Brasil, a perspectiva de aumento do volume de exportações e a exigência dos diferentes mercados faz com que a adaptação da cadeia produtiva da carne seja necessária, assim como a mudança de conceitos e critérios de seleção dos animais, de forma a melhorar as características consideradas pelo consumidor como de primeira importância: aparência e a palatabilidade. Porém existem diversos fatores que podem influenciar na qualidade da carne: sexo, raça (genética), idade, nutrição e estresse durante a vida do animal. Esses fatores influenciam diretamente no produto final, pois têm relação com as mudanças que ocorrem durante o post mortem. Desta forma, este trabalho teve como objetivo geral avaliar a influencia do pH, da condição sexual e de polimorfismos genéticos em genes relacionados à qualidade da carne e a resposta ao estresse sobre a variação das características da carne em rebanho bovino comercial Simental Sul Africano x Nelore. Para isso o trabalho foi dividido em duas partes: (a) avaliar os parâmetros de qualidade da carne em dois diferentes tempos de maturação (48 horas e 15 dias), sendo estudadas as variações entre os sexos (fêmeas e machos castrados), e nos diferentes valores de pH (classe 1=pH ≤ 5,79 e classe 2=pH ≥ 5,8). (b) Associar SNPs de diferentes genes ou marcadores (CAPN, UOGCAST, NR3C1_1, NR3C2_1, TG5) ligados a características de qualidade da carne, visando compreender melhor os efeitos genéticos desses marcadores sobre as características da carne. Para esta pesquisa foram avaliados 485 bovinos (182 fêmeas e 303 machos) recriados e terminados em confinamento. Os resultados mostraram haver relação do sexo como parâmetros de cor e maciez. As classes de pH mostraram relação significativa com a cor da carne. Verificou-se incidência de carnes DFD (pH≥5,8) da ordem de 5,3%. Dos polimorfismos estudados foram encontradas associações significativas apenas para CAPN, NR3C1_1 e TG. O CAPN relacionado à cor e, NR3C1_1 e TG com as perdas de água por cocção (PAC). Portanto, é possível inferir que tanto o sexo quanto as classes de pH tem influencia direta com os parâmetros de qualidade da carne. Os polimorfismos dos genes CAPN, NR3C1_1 e TG propiciaram mudanças nas características físico-químicas da carne. / In Brazil, the prospect of increased exports and the requirement of different markets makes necessary adaptation of the meat production chain, as the change of concepts and criteria in animal selection to improve the characteristics that the consumer consider as most important: appearance and palatability. However, there are some factors that could influence meat quality: sex, breed (genetics), age, nutrition and stress during the life of the animal. These factors directly influence the final product, since they are related to changes that occur during post mortem period. The aim was to evaluate the pH influence, sexual condition and genetic polymorphisms on genes related to meat quality and stress response on meat quality variation of South African Simmental x Nellore beef cattle. Therefore, this research were divided into two parts: (a) to evaluate the meat quality characteristics in two different aging times (2 and 15 days), being studied variations within the sexual condition (females and castrated males), as well as within the pH range (pH ≤ 5.79 Class 1 and Class 2 ≥ pH 5.8). (b) Associate SNPs of different genes (CAPN, UOGCAST, NR3C1_1, NR3C2_1, TG5) with meat quality characteristics. For this research were evaluated 485 animals (182 heifers and 303 steers) recreated and finished in feedlot. The results shows relation of sex with color and tenderness and, from pH range with color. There was an incidence of DFD meat (pH≥5,8) of 5,3%. The CAPN polymorphism was associated with color meat and NR3C1_1 and TG with cooking loss. Therefore, it is possible to infer that both sex and pH value influence in the meat quality. The polymorphisms CAPN, NR3C1_1 and TG led to changes in the physicochemical characteristics of the meat.

Made available in DSpace on 2016-08-10T10:38:51Z (GMT). No. of bitstreams: 1 PAULA APARECIDA BORGES PERES.pdf: 1092863 bytes, checksum: 6b1ba024090a288ed778814bb2668c7f (MD5) Previous issue date: 2012-08-31 / Despite its obscure aetiopathology, fibromyalgia is defined by diffuse musculoskeletal pain and not joint, and heightened sensitivity to palpation of certain muscle points, mainly affecting women, in a ratio of 6 to 10 women for every man. Several studies have demonstrated the role of polymorphisms of genes serotonergic, dopaminergic and catecholaminergic in the etiology of fibromyalgia. Thus, genetic factors may be determinants in the pathogenesis of fibromyalgia and some environmental factors may trigger fibromyalgia in genetically predisposed individuals. In this context, it seems that the gene polymorphism T102C serotonin receptor (5-HT2A) is relevant in susceptibility to fibromyalgia. The objective of this study was to perform a metaanalysis in order to investigate the association between polymorphism of the serotonin receptor gene (5-HT2A), the T102C SNP in susceptibility to fibromyalgia. Five studies, between the years 1999 to 2011, about 5-HT2AT102C polymorphism in fibromyalgia were selected and used for making a meta-analysis. The epidemiological profile of fibromyalgia patients revealed a mean age of 46.7 (+ 5.9) years, with extremes ranging from 21 to 77 years. The proportion between genders showed a difference of approximately 7 women for every man diagnosed fibromyalgia. The average time in which patients suffering from fibromyalgia syndrome is 7.4 (+ 2.9) years. Allele frequencies for T and C, the gene for 5- HT2AT102C were, respectively, for patients: 337 (43.1%) and 445 (56.9%) and for the controls: 362 (51.6%) and 340 (48.4%), indicating a predominance of the C allele in patients with fibromyalgia (p=0.0013). When evaluating the genotypic frequencies, we found: 73 (18.7%) TT, 191 (48.8%) and TC 127 (32.5%) CC. The control group: 98 (27.9%) TT, 166 (47.3%) and TC 87 (24.8%) CC. The CC genotype appears more frequently in the patients fibromyalgia patients (p=0.0046). A meta-analysis generated a universe of simultaneous evaluation of 742 individuals (391 fibromyalgia patients and 351 controls). The applied tests indicated no significant differences between the case and control groups. Thus, the result of the meta-analysis suggests no correlation between polymorphism in question and susceptibility to fibromyalgia. We suggest the use of a broad panel of SNPs with the intention of increasing the chances of finding an association between genetic polymorphisms and susceptibility to fibromyalgia. / Apesar de sua etiopatologia obscura, a fibromialgia é definida por dores musculoesqueléticas difusas e não articulares e sensibilidade a palpação exacerbada em determinados pontos musculares, afetando principalmente mulheres, numa proporção de 6 a 10 mulheres para cada homem. Vários estudos têm demonstrado o papel relevante dos polimorfismos de genes serotoninérgicos, dopaminérgicos e catecolaminérgicos na etiologia da fibromialgia. Desta forma, fatores genéticos podem ser determinantes na patogênese da fibromialgia e alguns fatores ambientais podem desencadear a fibromialgia em indivíduos geneticamente predispostos. Neste contexto, parece que o polimorfismo T102C do gene do receptor da serotonina (5-HT2A) pode ser relevante na suscetibilidade à fibromialgia. Assim, o objetivo do presente trabalho foi o de realizar uma meta-análise com o intuito de investigar a associação entre o polimorfismo do gene do receptor da serotonina (5- HT2A), no SNP T102C, na suscetibilidade à fibromialgia. Cinco estudos, entre os anos de 1999 a 2011, sobre o polimorfismo 5-HT2AT102C em fibromialgia foram selecionados e utilizados para a confecção de uma meta-análise. O perfil epidemiológico dos pacientes fibromiálgicos revelou uma idade média de 46,7 (+ 5,9) anos, com os extremos variando de 21 a 77 anos. Adiconalmente, a proporção entre gêneros apontou uma diferença de aproximadamente 7 mulheres com fibromialgia para cada homem diagnosticado. O tempo médio em que os pacientes sofrem da síndrome fibromiálgica é de 7,4 (+ 2,9) anos. As frequências alélicas para T e C, para o gene 5-HT2AT102C, foram, respectivamente, para os pacientes: 337 (43,1%) e 445 (56,9%) e para os controles: 362 (51,6%) e 340 (48,4%), indicando um predomínio do alelo C em pacientes com fibromialgia (p=0,0013). Ao avaliar as frequências genotípicas, encontrou-se: 73 (18,7%) TT, 191 (48,8%) TC e 127 (32,5%) CC. No grupo controle foram: 98 (27,9%) TT, 166 (47,3%) TC e 87 (24,8%) CC. O genótipo CC aparece em maior frequência no grupo dos pacientes fibromiálgico (p=0,0046). A meta-análise gerou um universo de avaliação simultânea de 742 indivíduos (391 pacientes com fibromialgia e 351 controles). Os testes aplicados não indicaram diferenças significativas entre os grupos caso e controle. Assim, o resultado da meta-análise sugere ausência de correlação entre o polimorfismo em questão e a suscetibilidade à fibromialgia. Sugerimos a utilização de um amplo painel de SNPs com a intenção de aumentar as chances de se encontrar associação entre polimorfismos gênicos e suscetibilidade à fibromialgia.

A fissura labiopalatina é a anomalia craniofacial mais comum nos seres humanos e em relação à cavidade bucal a agenesia dentária se apresenta mais prevalente em indivíduos com fissuras labiopalatinas do que na população em geral. Esses fenótipos têm sido considerados decorrentes de alterações do desenvolvimento embrionário e ocorrem como resultado da interação de fatores genéticos e ambientais, caracterizando uma etiologia multifatorial. Tem sido apontado que a função anormal de alguns genes que possuem papel na formação craniofacial e dentária poderia estar relacionada à etiologia desses fenótipos. Dentre os genes candidatos para esses fenótipos têm se destacado o MSX1 entre outros. Dessa forma, o objetivo do trabalho foi avaliar a associação entre o polimorfismo no gene MSX1 (rs12532) com os fenótipos fissura labiopalatina e agenesia dentária não sindrômicos isolados ou em associação. A amostra foi composta por 222 indivíduos divididos em 4 grupos: grupo 1, indivíduos com fissura e agenesia dentária; grupo 2, indivíduos com fissura sem agenesia dentária; grupo 3, indivíduos com agenesia dentária sem fissura e grupo 4, controle (sem agenesia e sem fissura). Foi realizada a extração do DNA genômico a partir da saliva coletada dos indivíduos. O polimorfismo no gene MSX1 (rs12532) foi estudado por meio de Reação em cadeia da Polimerase em tempo real (PCR Real Time) utilizando o ensaio Taqman (Applied Biosystems). O teste do qui-quadrado (p<0,05) e o cálculo da razão de chances (IC=95%) foram utilizados na análise estatística. O polimorfismo no gene MSX1 esteve associado aos indivíduos dos grupos com agenesia associada à fissura e agenesias isoladas, porém não houve associação para os indivíduos do grupo com fissuras isoladas. Os resultados sugerem que o polimorfismo no gene MSX1 (rs12532) exerce um papel na suscetibilidade das agenesias dentárias na população brasileira em indivíduos com e sem fissura. / Cleft lip and palate is the most common craniofacial anomaly in humans and, in relation to oral cavity, tooth agenesis is significantly more prevalent in individuals with cleft lip and palate than in the general population. Cleft lip and palate and tooth agenesis phenotypes are considered changes in embryonic development and occur as a result of interaction between environmental and genetic factors, featuring a multifactorial etiology. It has been suggested that abnormal function of some genes that have role in craniofacial and tooth formation, could be related in the etiology of these phenotypes. Among the candidate genes for these phenotypes, MSX1 has been highlighted. The aim of this study was to investigate the association between the MSX1 gene polymorphism (rs12532) with nonsyndromic cleft lip and palate and tooth agenesis phenotypes isolated or in association. The sample was comprised of 222 individuals divided into 4 groups: group 1, cleft lip and palate with tooth agenesis; group 2, cleft lip and palate without tooth agenesis; group 3, tooth agenesis without cleft and group 4, a control group without tooth agenesis or cleft. Genomic DNA extraction was performed from the saliva collected from the individuals. The MSX1 gene polymorphism (rs 12532) was studied using real time PCR, Taqman method. The chi-square (p< 0.05) and odds ratio tests (CI= 95%) were performed for statistical analyses. The MSX1 polymorphism was associated with cleft lip and palate with tooth agenesis and isolated tooth agenesis groups, but no association was found between the polymorphism and isolated cleft lip and palate group. This suggests that MSX1 gene polymorphism (rs12532) plays a role in the susceptibility for tooth agenesis in the Brazilian population with and without cleft lip and palate.

Depuis que Parsons et col. ont découvert en 2008 que le gène de l’isocitrate déhydrogénase 1 (IDH1) est fréquemment muté dans les glioblastomes (12%), de nombreuses équipes ont étudié la prévalence et les caractéristiques des mutations des gènes IDH1 et 2 dans les gliomes.Les mutations du gène IDH1 sont observées dans environ 40% des gliomes. La mutation d’IDH1 la plus fréquentes dans les gliomes (>90% des cas) est la mutation R132H. La fréquence des mutations IDH1 et 2 est inversement corrélée au grade des gliomes (grade II ~80%, III ~50%, and IV ~10%). Les mutations IDH1/2 ont une valeur diagnostique ainsi que pronostique (associées à une meilleure survie). Pendant ce travail de thèse nous avons dans une première partie analysé la distribution de ces mutations IDH1/2 dans les différents gliomes, leur association avec d’autres altérations génétiques, ainsi que leur valeur diagnostique et pronostique dans une cohorte de 1332 patients atteints de gliomes. Nous confirmons sur cette très grande cohorte les données de la littérature et affinons la valeur pronostique des mutations IDH1/2. Dans une seconde partie, nous avons mis en évidence dans les gliomes un polymorphisme (SNP) du gène IDH1 (SNP rs 11554137; C (cytosine) substituted by T (thymin)) précédemment observé dans les leucémies myéloïdes aigues. Ce SNP, codon 105, est localisé dans le même exon que le codon 132 fréquemment muté, et nous avons montré qu’il est associé à une moins bonne survie des patients atteints de gliomes. Les mutations du codon 132 causent une baisse de l’activité enzymatique normale d’IDH1/2 qui est remplacé par le gain d’une nouvelle. Les protéines IDH1/2 mutés, au lieu de produire de l’alpha-cétoglutarate de façon NADP dépendante, réduisent de façon NADPH dépendante l’alpha-cétoglutarate en 2-hydroxyglutarate (2HG). Une forte concentration de 2HG et une baisse de la quantité de NADPH peuvent sensibiliser les tumeurs au stress oxidatif et donc potentialiser l’effet de la radiothérapie, ce qui pourrait expliquer la meilleure survie de ces patients. Nous avons donc dans une troisième partie étudié in vitro l’impact de la mutation IDH1R132H sur la survie après radiothérapie de cellules tumorales exprimant de façon stable ce gène muté. Les résultats obtenus montrent que dans certaines conditions ces cellules pourraient être plus radiosensibles que les mêmes cellules exprimant le gène IDH1 non-muté.Dans ce travail de thèse, nous avons donc étudié le gène IDH1 dans les gliomes de patients et tenté par une approche fonctionnelle in vitro d’évaluer l’impact de la mutation IDH1R132H sur la radiosensibilité des cellules tumorales. / Since Parsons et al. (2008) found the frequent mutations of IDH1 (12%) in GBMs, various reports have studied the prevalence and characteristic of IDH1 and IDH2 mutations.The mutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in nearly 40% of gliomas. The frequency of IDH1 mutations are inversely connected with grade II (~80%), III (~50%), and IV (~ 10%) gliomas. Importantly, the status of IDH1 mutations is associated with a better outcome and demonstrated a diagnostic value. We analyzed also these mutations in distribution, association with tumor-derived other genetic alterations and the diagnostic and prognostic value in a cohort of 1332 glioma patients.A synonymous single nucleotide polymorphism [SNP rs 11554137; C (cytosine) substituted by T (thymin)] has been studied in gliomas patients. The SNP rs 11554137 (in codon 105) are located in the same exon with the IDH1 R132 mutations (in codon 132). And gliomas patients with SNP rs 11554137: C>T had a poorer outcome than patients without SNP rs 11554137. This was observed a similarly adverse effect in survival in patients with AML. Mutations in codon 132 can cause a decrease of IDH1/2 activity and also gain a new enzyme function for the NADPH dependent reduction of alpha-ketoglutarate to 2-hydroxyglutarate. High 2HG and low NADPH levels might sensitize tumors to oxidative stress, potentiating response to radiotherapy, and may account for the prolonged survival of patients harboring the mutations. So we studied further the alterations of function in IDH1R132H mutant cells in vitro. Based on the decrease of defence and the increase of impairing factors in tumor cells, we found that the tumors harbouring IDH1 mutations may have an elevated radiosensitivity. In the present study, we described the impact of IDH1 mutations in gliomas and search for new perspectives for the treatment strategy.

INTRODUÇÃO: O Câncer de Próstata (CaP) é a sexta neoplasia mais comum no mundo correspondendo a aproximadamente 10% do total de cânceres. No Brasil, o CaP é a neoplasia maligna não cutânea mais comum entre os homens. A hereditariedade é um dos principais fatores de risco do CaP, que se caracteriza pela herança de mutações em genes de susceptibilidade de alta penetrância que quando transmitidos aos descendentes aumentam o risco de desenvolvimento de tumores. Os andrógenos e estrógenos influenciam o desenvolvimento, maturação e manutenção da próstata, afetando a proliferação e a diferenciação. Isso tem despertado grande interesse no papel desses hormônios esteroides no desenvolvimento e manutenção tanto da próstata normal quanto maligna. Os Polimorfismos de Nucleotídeo Único (SNPs) são variantes de risco genéticos associados com uma série de doenças, incluindo o câncer. Considerando que a história familiar e que os componentes hormonais constituem fatores de risco para o desenvolvimento do CaP, acredita-se que a identificação de polimorfismos envolvidos nesses processos possa ter um papel relevante para auxiliar no desenvolvimento de ferramentas alternativas para a detecção precoce e para a definição do prognóstico desta neoplasia. OBJETIVOS: Analisar polimorfismos (SNPs) relacionados com histórico familiar e com fatores hormonais em amostras de sangue de pacientes com CaP e em homens saudáveis. Além disso, correlacionar os resultados da genotipagem com parâmetros clínico-patológicos. MÉTODOS: O estudo foi composto por 185 pacientes diagnosticados com CaP, sendo 97 casos esporádicos e 72 com histórico familiar (dois parentes de primeiro grau). O grupo controle foi composto por 70 amostras de sangue de indivíduos saudáveis, que comprovadamente não possuíam CaP e fazem acompanhamento com intuito preventivo. Foram selecionados 13 polimorfismos para análise: rs10486567, rs10993994, rs9364554, rs5945572, rs2735839, rs4430796, rs7501939, rs138213197, rs1271572, rs2987983, rs8072254, rs4919743 e rs3808330. A genotipagem foi realizada através da técnica de PCR em tempo real (qPCR) e correlacionada com o histórico familiar de CaP, PSA pré-operatório, graduação histológica de Gleason e estadiamento patológico. RESULTADOS: Analisamos a frequência dos polimorfismos selecionados e encontramos as seguintes correlações em nossos casos: os SNPs rs10486567 e rs9364554 aumentam a chance de desenvolvimento do CaP enquanto que o SNP rs8072254 diminui o risco. Com relação à hereditariedade, o SNP rs1271571 apresentou associação com o CaP esporádico. Na comparação com os fatores prognósticos encontramos que o SNP rs3808330 foi mais frequente em indivíduos que possuíam PSA < 10; o SNP rs7501939 foi mais frequente em indivíduos com menor escore de Gleason e ausência de recidiva e o SNP rs5945572 foi mais frequente em indivíduos com menor escore de Gleason na peça. CONCLUSÕES: De uma forma geral encontramos polimorfismos que parecem ter um papel relevante no desenvolvimento do CaP, na transmissão familiar e a fatores prognósticos. Estes importantes polimorfismos, ainda não haviam sido estudados na população brasileira e nosso trabalho identificou correlações ainda não demonstradas na literatura / BACKGROUND: Prostate Cancer (PCa) is the sixth most common cancer worldwide accounting for around 10% of all cancers. In Brazil, the PCa is the most common non-skin malignancy among men. Heredity is one of the main risk factors for PCa, which is characterized by mutations of heritage in highpenetrance susceptibility genes that when transmitted to offspring increase the risk of tumor development. Androgens and estrogens influence the development, maturation and maintenance of the prostate, affect proliferation and differentiation. This has aroused great interest in the role of these steroid hormones in the development and maintenance of both normal and malignant prostate. Single Nucleotide Polymorphisms (SNPs) are variants of genetic risk associated with a number of diseases including cancer. Considering that the family history and hormonal components are risk factors for the development of PCa, we believe that the identification of polymorphisms involved in these processes may have an important role to assist in the development of alternative tools for early detection and to define the prognosis of this cancer. OBJECTIVES: To analyze polymorphisms (SNPs) associated with family history and hormonal factors in patient blood samples with PCa and in healthy men. In addition, to correlate the results of genotyping with clinical-pathological parameters. METHODS: The study consisted of 185 patients diagnosed with PCa, divided into 97 sporadic cases and 72 with a family history. The control group consisted of 70 blood samples from healthy individuals who had no proven PCa and do it for preventive purposes. We selected 13 polymorphisms for analysis: rs10486567, rs10993994, rs9364554, rs5945572, rs2735839, rs4430796, rs7501939, rs138213197, rs1271572, rs2987983, rs8072254, rs4919743 and rs3808330. Genotyping was performed by PCR in real time (qRT -PCR) and correlated with family history of PCa, preoperative PSA, Gleason histologic grading and pathological staging. RESULTS: We analyzed the frequency of the selected polymorphisms and found the following correlations in our cases: SNPs rs10486567 and rs9364554 increase the chance of developing PCa while the SNP rs8072254 decreases the risk. Regarding heredity, the SNP rs1271571 presented association with sporadic PCa. In comparison with the prognostic factors we found that the SNP rs3808330 was more frequent in patients who had PSA < 10; SNP rs7501939 was more frequent in patients with lower Gleason score and no recurrence and the SNP rs5945572 was more frequent in subjects with lower Gleason score on the surgical specimen. CONCLUSIONS: In general we found that polymorphisms that appear to have a relevant role in the development of PCa in family transmission and in prognostic factors. These important polymorphisms had not yet been studied in the Brazilian population and our work has identified correlations yet not demonstrated in the literature

<p>Analysis of genetic relatedness is of great importance in forensic casework such as immigration and identification cases. The conventional methods for relationship testing are not sufficient in the most complicated cases, because more genetic markers are required to obtain results with satisfactory statistical security. This study demonstrates that microarrays, which can be used to genotype thousands of single nucleotide polymorphisms (SNPs), could be a promising solution to this problem. The microarray technique used in this study performed very well on blood samples and also worked well in combination with whole genome amplification, but did not generate any results when used on severely degraded materials.</p><p>Markers suitable for relatedness analysis were selected from the microarray and were successfully tested on families with known genetic relations. Although a maximum of 64 autosomal markers were used, there is a great potential of selecting the hundreds or thousands of markers that may be required in some cases of relatedness investigation.</p>

<p>In this thesis, different genetic tools are used to investigate pre- and postzygotic barriers to gene exchange and their role in speciation in the pied flycatcher (<i>Ficedula hypoleuca</i>) and the collared flycatcher (<i>F. albicollis</i>). This species complex consists of four genetically distinct clades that apparently diverged in allopatry (I). Sequencing of introns from autosomal and Z-linked genes from the two species reveals signs of selection on the Z-chromosome. Sexual selection acting on Z-linked genes might explain this pattern (II). By using large-scale genotyping of single nucleotide polymorphisms (SNPs), introgression is observed at autosomal- but not Z-linked loci, mostly from the pied- to the collared flycatcher. Male plumage characters and genes involved in hybrid fitness are largely mapped to the Z-chromosome (III). By studying mate choice of female hybrids I show that there is a link between female preferences and the Z chromosome (IV). The rate of introgression in island versus clinal hybrid zones is consistent with regional differences in hybrid fertility. Asymmetric gene flow from allopatry on the islands may oppose reinforcement, leading to introgression and a partial breakdown of postzygotic isolation. Adaptive introgression may explain the high rate of introgression observed at one of the genetic markers (V). For late breeding female collared flycatchers it appears to be adaptive to use pied flycatchers as social fathers but conspecific males as genetic fathers. Additionally, females in mixed species pairs may reduce hybridization costs by producing an excess of male hybrid offspring that are more fertile than females (VI).</p><p>In conclusion, the Z-chromosome appears to play a major role in flycatcher speciation. Sexual selection and reinforcement are important mechanisms in the divergence of these birds. However, gene flow from allopatry, introgression of adaptive genes and adaptive hetrospecific pairing by late breeding collared flycatcher females may work in the opposite direction.</p>

<p>The studies in this thesis describe the development of a microarray based minisequencing system and its application to highly parallel genotyping of single nucleotide polymorphisms. The technical developments included identification of a three-dimensional microarray surface coating with high binding capacity for oligonucleotides modified with amino groups as the most optimal one for the system. The system was also established for multiplexed, reproducible quantitative analysis of SNP alleles both on the level of DNA and RNA. The sensitivity of the system to distinguish SNP alleles present as a minority in a mixed sample was found to be 1-6%. </p><p>The microarray based minisequencing system was applied in a pharmacogenetic study on antihypertensive drug response. A panel of 74 SNPs located in candidate genes related to blood pressure regulation were genotyped in DNA samples from hypertensive patients that had been treated with the antihypertensive drugs irbesartan or atenolol. Multiple regression analysis of the genotype data against the reduction in blood pressure identified genotype combinations of four to five SNPs that explain 44-56% of the reduction in blood pressure in the two treatment groups. The genotypes of two individual SNPs in the angiotensinogen (AGT) gene and a SNP in the low density lipoprotein receptor (LDLR) gene appeared to be associated to reduced blood pressure after treatment with atenolol, while a SNP in the apolipoprotein B (APOB) gene was associated to blood pressure reduction after irbesartan treatment. The genotype of one SNP in the adrenergic alpha-2A-receptor gene (ADRA2A) was related to the reduction in left ventricular mass following atenolol treatment while the genotypes of two SNPs, one in the APOB gene and one in the AGT gene were related to the reduction in left ventricular mass in the patients treated with irbesartan.</p>