Orientador: Vera Fernanda Martins Hossepian de Lima / Coorientador: Danísio Prado Munari / Coorientador: Rodrigo Pelicioni Savegnago / Coorientador: Marcos vinícius Barbosa da Silva / Banca: Lenira El Faro Zadra / Banca: Joslane Noely dos Santos Gonçalves Cyrillo / Banca: Ricardo da Fonseca / Banca: João Ademir de Oliveira / Resumo: Considerando painéis de SNP (do inglês Single Nucleotide Polymorphism, polimorfismo de nucleotídeo único) objetivou-se descrever o perfil genômico populacional de bovinos leiteiros da raça Gir por meio da caracterização de linhagens desta população e inferir sobre a variabilidade genética utilizando informações de animais provenientes de diversas fazendas do Brasil. Após o controle de qualidade de genótipos e amostras, foram obtidos 1987 SNP bialélicos em cromossomos autossomos presentes nos painéis HD e 50K SNP, em um painel resultante de SNP em comum dos dois painéis, 21K SNP. Distâncias genéticas entre os marcadores foram obtidas e, por meio da decomposição espectral, foram gerados os componentes principais. A análise multivariada de componentes principais, em um contexto de estrutura populacional, permite inferir sobre a variabilidade genética utilizando informações de parentesco entre os animais. A determinação do número de agrupamentos foi realizada com o procedimento k-means, utilizando a distância genética entre os animais, que permite identificar subgrupos genéticos na população, com moderada-alta qualidade de agrupamento, com coeficiente correlação cofenética equivalente a 0,66. O parentesco, heterozigosidade esperada (He) e observada (Ho), coeficientes de endogamia, desequilíbrio de ligação (LD) e tamanho efetivo populacional, baseados exclusivamente em informações genômicas também foram obtidos. Os resultados evidenciaram quatro diferentes agrupamentos genéticos, ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective was to describe the genomic structure of Gir dairy cattle population through the characterization of lineages of this population and infer about the genetic variability considering information from animals from several farms in Brazil using panels of SNP (Single Nucleotide Polymorphism). After quality control of genotypes and samples, we obtained 1987 biallelic SNPs on autosomal chromosomes present in the HD panels and 50K SNP, in a resultant panel of SNPs in common from to two panels, 21K SNP. Genetic distances between the markers were obtained and, through spectral decomposition, the principal components were generated. Multivariate analysis of principal components, in a context of population structure, allows inferring about genetic variability using relatedness information between animals. The determination of the number of clusters was performed using the k-means procedure, using the genetic distance between the animals, which allows the identification of genetic subgroups in the population, with moderate-high quality of grouping, with a coefficient correlation coefficient equivalent to 0.66. Expected heterozygosity (He) and observed (Ho), coefficients of inbreeding, linkage disequilibrium (LD) and effective population size (Ne) based exclusively on genomic information were also obtained. The results evidenced four different genetic groups, defined as breeding lineages of the Gir dairy breed of Brazil, indicating that the use of a few breeders or their descendants with greater intensity and also due to the different origins of the ancestors, formed the different genetic groups, which are composed of animals from various farms. There was little relationship between the animals and this was reflected in the average inbreeding coefficient, and the average inbreeding obtained for all the individuals was 0.017, evi... (Complete abstract click electronic access below) / Doutor

As leucemias constituem o câncer mais comum da infância, representando 30% de todas as neoplasias infantis. Dentre elas, a leucemia linfoblástica aguda (LLA) é a mais freqüente, atingindo 75% dos casos pediátricos de leucemias. A ocorrência da LLA tem sido relacionada com a exposição a alguns fatores ambientais (químico, físico e biológicos) e maternos (uso de drogas e dieta) tanto no desenvolvimento intra-útero como após o nascimento. No entanto, o processo de leucemogênese, particularmente com relação à importância da susceptibilidade genética herdade e fatores ambientais, ainda não foi elucidado. As enzimas do citocromo P450 (CYP), assim como outras enzimas das fases I e II do metabolismo estão envolvidas na biotransformação de uma variedade de xenobióticos presentes na alimentação, no cigarro, nas drogas, nas bebidas alcoólicas e nos poluentes ambientais. Polimorfismos em genes responsáveis por codificar essas enzimas de metabolismo têm sido associados com um aumento na susceptibilidade a diferentes tipos de câncer e a doenças hematológicas em adultos e crianças. Similarmente, a capacidade diferencial de crianças portadoras de leucemia aguda para metabolizar carcinógenos e drogas quimioterápicas, a qual é influenciada pelos polimorfismos dos genes que codificam enzimas de metabolização, podem modificar a resposta à terapia. Sendo assim, é importante a realização de investigações epidemiológicas moleculares em crianças portadoras de leucemia, pois estes estudos poderão fornecer informações sobre a etiologia e os mecanismos que levam a esta doença, e sobre as respostas adversas ou inadequadas a certos agentes terapêuticos, com o intuito de buscar tratamentos mais específicos e eficazes. O presente trabalho teve por objetivo estimar a freqüência dos polimorfismos dos genes CYP2D6, EPHX1, MPO (responsáveis pelo metabolismo de xenobióticos) e TS (associado à síntese de DNA) e investigar a associação destes polimorfismos com o efeito do tratamento quimioterápico. Foram genotipados através da técnica de PCR-RFLP 132 pacientes portadores de LLA e 300 indivíduos controles. Analisando o gene CYP2D6 foi observada uma prevalência significante do alelo CYP2D6*3 no grupo dos pacientes. Em relação ao gene EPHX1, o alelo polimórfico *2 foi mais freqüente no grupo de indivíduos controles, assim como a repetição tripla (3R) do gene TS. O genótipo heterozigoto para o gene TS e o homozigoto selvagem para o gene MPO foram mais freqüentes em indivíduos do sexo feminino, sugerindo uma associação destes genótipos com o sexo. Não foi encontrada nenhuma associação dos polimorfismos estudados com a resposta ao tratamento quimioterápico. Com base nestes dados, é possível sugerir uma associação destes polimorfismos com a susceptibilidade ao desenvolvimento da LLA, destacando a importância da sua determinação para o prognóstico bem como para o caráter preventivo relacionado ao risco aumentado de doenças associadas à exposição ambiental. / The leukemias are the most common type of cancer in children, representing above 30% of all the pediatric malignancies. Among them, the acute lymphoblastic leukemia (ALL) is the most frequent, with a percentage of 75% of the total pediatric cases of leukemia. Its occurrence is closely related to the exposure to chemical, physical and biological environmental factors and so maternal factors either, such as drugs, alcohol even in the uterine phase or after birth. Despite all the investigations made until now, little is known about the leucemogenous process, in particular about the importance of herdable genetic susceptibility and environmental factors. The citochrome P450 enzymes, as well as other phase I and II enzymes are involved on the biotransformation process of a huge variety of xenobiotics on food, smoke, alcohol, drugs and chemical poluents. Polymorphisms on these genes have been associated to the increased susceptibility to different kind of adult cancers and hematological diseases in adults and child. Similarly, the differential capacity of children with ALL to metabolize carcinogen compounds and chemotherapy drugs, witch is influenced by polymorphisms on genes that encode metabolizing enzymes, can modify the individual risk of relapse and therapy response. Thus, molecular epidemiological investigations in children with acute leukemia became so important to help clinicians answer questions about the etiology and the right mechanisms that induce this malignance and about the adverse responses to therapy found in huge number of patients, trying to reach more specific treatments. So, the present study objective is to evaluate the polymorphism frequency on the following genes CYP2D6, EPHX1 and MPO (xenobiotic metabolizing genes) and TS gene (DNA synthesis) in patients with ALL and in controls individuals. We analyzed 132 patients and 300 health controls by PCR-RFLP technique. The CYP2D6*3 variant was more frequent on the case group. The EPHX1*2 and the triple repeat (3R) of TS gene were more frequent on the control group. The heterozygous genotype for the TS gene and the homozygous for the MPO gene were more prevalent on the female group, suggesting an association of these polymorphisms with the gender. We did not find any association with the studied polymorphisms and the response to therapy. Beyond these data, is it possible to suggest an association of these polymorphisms and the leukemia development susceptibility, emphasizing the importance of the polymorphism determination for prognosis and for the prevention, related to the increased risk of the diseases related to environmental exposure.

Uma das principais causas da disfunção erétil (DE) pode ser relacionada com o déficit de óxido nítrico (NO) no corpo humano. O principal componente para a produção do NO é o aminoácido L-arginina que é utilizado pelas enzimas óxido nítrico sintase neuronal (nNOS), endotelial (eNOS) e induzida (iNOS) para sua produção. A dimetilarginina assimétrica (ADMA) atua como inibidor endógeno dos três subtipos de NOS citadas acima e é metabolizada pelas enzimas dimetilarginina dimetilaminohidrolase 1 e 2 (DDAH1 e DDAH2). Diversos estudos têm relacionado a alteração na expressão ou atividade das enzimas DDAH bem como alterações em seus genes, com distúrbios onde a sinalização de NO é prejudicada. Os objetivos deste estudo foram investigar a associação de variantes genéticas dos genes DDAH1 (rs1554597 e rs18582) e DDAH2 (rs805304 e 805305) com a predisposição à disfunção erétil (DE), scores de função erétil e concentrações plasmáticas de nitrito e ADMA. Também verificar se estes marcadores bioquímicos estão relacionados aos scores de função erétil. Foram selecionados 130 pacientes com DE clínica e 98 participantes controles saudáveis sem DE. A função erétil dos voluntários foi avaliada através do questionário Índice Internacional de Função Erétil (IIEF). Os genótipos dos rs1554597, rs805304 e rs805305 foram obtidos através da técnica de reação em cadeia da polimerase (PCR) seguida de digestão enzimática, e do rs18582 apenas por técnica de PCR alelo específica. No grupo Pacientes, foram encontradas associações do gene DDAH1 com as concentrações plasmáticas de ADMA: o rs1554597 teve os genótipos TT e TC associados positivamente (TT: ? 0,13 e P = 0,008; TC: ? 0,09 e P = 0,016;) e o genótipo CC associado negativamente (? -0,22 e P <0,001); já o rs18582 teve o genótipo GG associado positivamente (? 0,22 e P <0,001) e o genótipo AA associado negativamente (? -0,16 e P = 0,001); o haplótipo TG foi associado positivamente (? 0,12 e P = 0,016) e o haplótipo CA negativamente (? -0,18 e P = 0,002). Com relação ao nitrito, associações dos haplótipos do gene DDAH2 foram encontradas, o haplótipo CC foi associado negativamente (? -0,03 e P = 0,045) e o haplótipo AG foi associado positivamente (? 0,03 e P = 0,045).O rs18582 teve o genótipo GG associado positivamente com as concentrações plasmáticas de nitrito, no modelo aditivo (? 0,15 e P = 0,009) e no modelo dominante (? 0,08 e P = 0,009), e os genótipos GA ou AA associados negativamente com as concentrações plasmáticas de nitrito, apenas no modelo dominante (? -0,08 e P = 0,009). Não foi encontrada nenhuma outra associação significativa no estudo / One of the main causes for erectile dysfunction (ED) is related to nitric oxide (NO) deficiency in human body. The main substrate for NO synthesis is the amino acid L-arginine, which is processed by NO synthases (NOS) from three subtypes for its production: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). Asymmetric Dimethylarginine (ADMA) acts as an endogenous inhibitor of the three subtypes of NOS and is metabolized by enzymes dimethylarginine dimethylaminohydrolase types 1 and 2 (DDAH1 and DDAH2). Several studies associate the altered expression or activity of DDAH enzymes, as well as their genes, with diseases with hampered NO signaling. The objectives of this study were to investigate the association of genetic variants of DDAH1 (rs1554597 and rs18582) and DDAH2 (rs805304 and 805305) with vulnerability to develop ED, with altered scores of erectile function and with altered plasma concentrations of nitrite and ADMA. We also investigated whether these biochemical markers associated with erectile function scores and ED risk. We selected 130 patients with clinical ED and 98 healthy controls without ED. Erectile function was assessed through the International Index for Erectile Function (IIEF) questionnaire. Genotypes for rs1554597, rs805304 and rs805305 were obtained with polymerase chain reaction (PCR) followed by enzyme restriction (RFLP), while rs18582 was determined using Allele-Specific oligonucleotide PCR (ASO-PCR). At patients group, we found association of variants in DDAH1 gene with plasma ADMA levels: TT and TC genotypes of rs1554597 were associated with increases in ADMA (TT: ? 0.13 e P = 0.008; TC: ? 0.09 e P = 0.016;), while CC genotype was associated with decreases in ADMA (? 0.22 e P <0.001); regarding rs18582, GG genotype associated with increases in ADMA (? 0.22 e P <0.001), while AA genotype associated negatively (? -0.16 e P = 0.001); besides, haplotype TG was also associated with ADMA increases (? 0.12 e P = 0.016), while CA haplotype associated negatively with ADMA levels (? -0.18 e P = 0.002). Regarding nitrite, associations of the haplotypes of the DDAH2 gene were found, the haplotype CC was negatively associated (? -0,03 and P = 0,045) and the haplotype AG was positively associated (? 0,03 and P = 0,045) .O rs18582 had the GG genotype positively associated with plasma nitrite concentrations in the additive model (? 0.15 and P = 0.009) and in the dominant model (? 0.08 and P = 0.009), and negatively associated genotypes GA or AA with plasma nitrite concentrations, only in the dominant model (? -0.08 and P = 0.009). We found no further significant associations in our study

Ce travail de thèse s’articule en deux parties. Dans une première partie (aspects descriptifs, cohortes SU.VI.MAX et NutriNet-Santé), nous avons mis en évidence différents facteurs influençant le statut en vitamine D, parmi lesquels certains sont modifiables et pourraient ainsi faire l’objet de recommandations de santé publique (exposition solaire modérée, activité physique, maintenance d’un poids normal). Sur la base de ces déterminants, nous avons développé le score VDIP permettant d’identifier de manière simple en pratique clinique des adultes à risque d’insuffisance en vitamine D. Enfin, l’étude des connaissances liées à la vitamine D dans un large groupe d’adultes issus de la population générale a mis en évidence une certaine confusion par rapport aux sources de vitamine D et à ses effets santé établis ou non.Dans une seconde partie (aspects étiologiques, étude cas-témoin nichée dans la cohorte SU.VI.MAX), nous avons mis en évidence qu’un statut plus élevé en vitamine D (parmi des concentrations plasmatiques en 25(OH)D faibles à modérées telles qu’observées dans cette étude) pourrait être associé à une diminution de risque de cancer du sein, de cancer de la prostate et de cancers liés au tabac. Ces associations seraient toutefois modifiées par différents facteurs individuels comme le statut pondéral ou la consommation d’alcool (cancer du sein en particulier). L’étude des différents polymorphismes génétiques a permis de confirmer les résultats observés avec le statut en vitamine D.Dans un contexte où la vitamine D suscite une attention grandissante, les résultats de cette thèse contribuent à mieux connaître les facteurs influençant le statut en vitamine D, à fournir un aperçu détaillé des connaissances/croyances concernant la vitamine D en population générale et fournissent de nouveaux éléments pour une meilleure compréhension du rôle de la vitamine D dans l’étiologie des cancers. Ces travaux pourraient ainsi participer, à terme, à l’optimisation des recommandations de santé publique pour l’amélioration du statut en vitamine D de la population et pour la prévention primaire des cancers. / Two main parts structured this PhD thesis. In the first part (descriptive work, SU.VI.MAX and NutriNet-Santé cohorts), we identified factors that influenced vitamin D status, some of which being modifiable (e.g. moderate sun exposure, physical activity, normal weight) and as such constitute interesting targets for public health policies. Using these determinants, we developed the VDIP score in order to simply identify adults at risk of vitamin d insufficiency. Finally, the study dealing with vitamin D-related knowledge showed that the primary sources of vitamin D-related information were the physicians and the media and that some confusion existed regarding the sources and the proven/unproven health effects of vitamin D.In a second part (etiological work, nested case-control study, SU.VI.MAX cohort), we showed that a higher vitamin D status (within the low-to-moderate range of plasma 25(OH)D concentrations observed in this study) may be associated with a decreased risk of breast, prostate and tobacco-related cancers. These associations were modified by individual factors such as weight status and alcohol intake (in particular breast cancer). The studied genetic polymorphisms confirmed the associations observed with vitamin D status.In a context where vitamin D arouses considerable interest, these results may contribute to a better understanding of the factors that influence vitamin D status, to a better insight of what people know and believe regarding vitamin D and to a better understanding of the role of vitamin D in the etiology of cancer. These PhD findings could eventually contribute to the optimization of public health nutritional recommendations for the improvement of vitamin D status in the population and for cancer primary prevention.

A disfunção erétil (DE) é uma doença relacionada com a sinalização deficiente de óxido nítrico (NO). O NO é produzido a partir da L-arginina pelas enzimas óxido nítrico sintase neuronal (nNOS), endotelial (eNOS) e induzida (iNOS). A dimetilarginina assimétrica (ADMA) é um inibidor endógeno dos três subtipos existentes de NOS, e é metabolizada principalmente pelas enzimas dimetilarginina dimetilaminohidrolase 1 e 2 (DDAH1 e DDAH2). Vários estudos têm associado alterações em genes, expressão ou atividade das enzimas DDAH com distúrbios em que a sinalização de NO é prejudicada. O objetivo deste estudo foi avaliar se o nível de resposta ao tratamento da DE com sildenafil pode estar associado a polimorfismos dos genes DDAH1 (rs1554597 e rs18582) e DDAH2 (rs805304 e rs805305) e, também, aos haplótipos formados por estes polimorfismos. Foram selecionados 70 pacientes com DE pós-prostatectomia (DEPP) e 70 pacientes com DE clínica (DEC). A função erétil dos voluntários foi avaliada através do questionário Índice Internacional de Função Erétil (IIEF). Para avaliação da resposta ao sildenafil, foram calculadas a diferença entre as pontuações pré e pós-tratamento (?IIEF) e a percentagem atingida da máxima resposta possível (?IIEF%) de cada paciente. Também, os pacientes de cada grupo foram divididos em bons e maus respondedores ao sildenafil de acordo com a mediana dos valores de ?IIEF%. Os genótipos dos rs1554597, rs805304 e rs805305 foram obtidos pela técnica de reação em cadeia da polimerase (PCR) seguida de digestão enzimática, e do rs18582 pela técnica de PCR alelo específica. O software PHASE 2.1 foi utilizado para estimar os haplótipos em cada grupo. Os resultados mostraram que o alelo variante A do rs18582 apresentou tendência para associação com piores respostas ao sildenafil no grupo DEC (P=0,058). No grupo DEPP, portadores dos alelos variantes A do rs805304 e G do rs805305 foram associados a melhores respostas ao sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; e score IIEF pós-tratamento, P=0,014). Não foram encontradas outras associações significativas. Estes resultados mostram que os marcadores genéticos rs805304 e rs805305 do DDAH2 podem influenciar as respostas ao sildenafil em pacientes com DE. / Erectile dysfunction (ED) is a disease related to deficient nitric oxide (NO) signaling. NO is produced from L-arginine by three isoforms of the enzyme nitric oxide synthase: neuronal (nNOS), endothelial (eNOS) and induced (iNOS). Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the three existing NOS subtypes, and is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 and 2 (DDAH1 and DDAH2) enzymes. Several studies have associated changes in genes, expression or activity of DDAH enzymes with disorders in which NO signaling is impaired. The aim of this study was to evaluate whether the response to ED treatment with sildenafil may be associated with polymorphisms of the DDAH1 (rs1554597 and rs18582) and DDAH2 genes (rs805304 and rs805305), as well as the haplotypes formed by these polymorphisms. We selected 70 patients with postprostatectomy ED (PPED) and 70 patients with clinical ED (CED). The erectile function of the volunteers was assessed using the International Index for Erectile Function (IIEF) questionnaire. To evaluate the response to sildenafil, the difference between the pre- and post-treatment scores (?IIEF) and the percentage reached from the maximum possible response (?IIEF%) of each patient were calculated. Also, patients from each group were divided into good and bad responders to sildenafil according to the median values of ?IIEF%. The genotypes of rs1554597, rs805304 and rs805305 were obtained by the polymerase chain reaction (PCR) technique followed by enzymatic digestion, and rs18582 by the allele-specific PCR technique. The PHASE 2.1 software was used to estimate the haplotypes in each group. The results showed that the variant A allele of rs18582 showed a tendency to be associated with a greater chance of worse responses to sildenafil in the DEC group (P=0,058). In the DEPP group, carriers of the variant alleles A of rs805304 and G of rs805305 were associated with better responses to sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; and post-treatment IIEF score, P=0,014). No other significant associations were found. These results show that the genetic markers rs805304 and rs805305 of DDAH2 may influence the responses to sildenafil in patients with ED.

Based on previous studies, the increasing prevalence of hypertension may be associated with factors such as obesity, dietary salt and fat intake. This study examined the common biochemical and anthropometric markers that are associated with blood pressure elevation, increasing metabolic and haemodynamic derangement in subjects in Hong Kong, and related those phenotypic markers to some genetic polymorphisms relevant to hypertension. / Five hundred and thirty nine Hong Kong Chinese subjects were examined. They were aged from 20 to 60 years, and were hypertensive or normotensive siblings from families with a hypertensive proband, and normotensive controls without a family history of hypertension. The interrelationships between pathophysiological changes and various neurohormones considered relevant to the development of hypertension were investigated. Fasting blood and 24 hour urine samples were collected. Plasma insulin, plasma leptin, plasma renin activity (PRA), serum angiotensin converting enzyme (ACE) activity, aldosterone, 24 hour urine noradrenaline, adrenaline, dopamine and kallikrein were measured. A robust assay for the measurement of urine free cortisol and cortisone and 6beta-hydroxycortisol by an LC-MS/MS method was developed and validated. The ratio between urine free cortisol and cortisone was used as an estimate of the activity of 11beta-hydroxysteroid dehydrogenase type II (11betaHSD2) for cortisol metabolism. These parameters were related to polymorphisms in three genes, the angiotensinogen (AGT) gene M23 5T, the dopamine D1 receptor (DD1R) gene A-48G and the dopamine D2 receptor (DD2R) gene Taq1 A polymorphisms. Analysis of variance was employed for the parameters in the three groups of subjects and for an age-matched sibling pair analysis (using 1 normotensive and 1 hypertensive sibling from each family). Comparisons between parameters were also made after dividing the whole population into 3 groups according to the tertiles of blood pressure. (1) Central (higher waist to hip ratio and waist circumferences) and general (greater body mass index and weight) obesity were found in both hypertensive patients and the normotensive siblings compared to the control subjects. These obesity indices showed strong positive relationships with increased insulin resistance and blood pressure. The obesity indices were also independently associated with systolic and diastolic blood pressure, with central obesity showing the stronger associations. (2) Hypertensives had more adverse lipid profiles, insulin resistance and higher fasting plasma glucose levels. This suggested that the blood pressure elevation in the hypertensives may be mediated through obesity and insulin resistance. (3) Both the hypertensive and normotensive members of sibling pairs had lower noradrenaline and cortisol excretion and higher activity of 11betaHSD2 compared to the normotensive controls. The result showed positive relationships between noradrenaline and increased obesity, insulin resistance and blood pressure, while the relationship between adrenaline and blood pressure was inversed. (4) Lower plasma ACE activity and aldosterone were found in the hypertensives and their siblings than in the normotensive controls. There was a reduction in PRA across the blood pressure tertiles as blood pressure increased. In addition to the higher 11betaHSD2 activity, a negative relationship between aldosterone and blood pressure in hypertensive siblings was observed. These findings may indicate the protective mechanism of these systems in this population. In subjects with the different polymorphisms of AGT M235T, there were no differences in the PRA, serum ACE activity or aldosterone, but lower urine cortisol and kallikrien were found in relation to increasing numbers of the T allele. There was a weak association between the AGT M235T polymorphism and hypertension. (5) Despite the strong correlation of dopamine excretion between hypertensive and normotensive siblings within families, lower dopamine levels were found in the normotensive siblings. A consistent positive relationship was found between urine dopamine and sodium excretion, which supports the concept of the natriuretic effect of dopamine. There was no phenotypic difference found in any of the biochemical parameters in relation to the DDIR A-48G and DD2R Taq1 A polymorphisms, but there were weak associations with blood pressure and these polymorphisms in the sibling study. / The normotensive siblings had metabolic abnormalities similar to but less severe than the hypertensive probands, which suggests that the genetic effects and interacting effect of shared lifestyle and environmental factors with their hypertensive family member may be influential on the healthy siblings. Adaptive changes were seen in some of the blood pressure regulating systems in both the hypertensive probands and the normotensive siblings. The major factors predisposing to the hypertension in these subjects appeared to be obesity and insulin resistance and the adaptive changes were insufficient to compensate for these in the hypertensive subjects. / Chu Ten Wah Tanya. / "March 2006." / Adviser: Brian Tom Linson. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1547. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 304-341). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Ko Kin Ming Jeffery. / Thesis submitted in: July 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 99-111). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xv / List of Abbreviations --- p.xvi / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Malignant Lymphoma --- p.1 / Chapter 1.2 --- Non-Hodgkin's Lymphoma --- p.1 / Chapter 1.3 --- Diffuse Large B-cell Lymphoma --- p.2 / Chapter 1.3.1 --- General Features of Diffuse Large B-cell Lymphoma --- p.2 / Chapter 1.3.2 --- Morphologic variants of Diffuse Large B-cell Lymphoma --- p.4 / Chapter 1.3.2.1 --- Centroblastic vairant --- p.5 / Chapter 1.3.2.2 --- Immunoblastic variant --- p.5 / Chapter 1.3.2.3 --- Anaplastic variant --- p.6 / Chapter 1.3.3 --- Immunophenotype of Diffuse Large B-cell Lymphoma --- p.6 / Chapter 1.3.3.1 --- Lineage-associated antigens --- p.6 / Chapter 1.3.3.1.1 --- B-cell lineage antigens --- p.6 / Chapter 1.3.3.1.2 --- T-cell lineage antigens --- p.7 / Chapter 1.3.3.2 --- Antigen involved in regulation of cell proliferation and apoptosis --- p.8 / Chapter 1.3.3.2.1 --- Proliferation markers --- p.8 / Chapter 1.3.3.2.2 --- Cell cycle regulators --- p.8 / Chapter 1.3.3.2.3 --- Protein controlling apoptosis --- p.10 / Chapter 1.3.4 --- Subtypes of Diffuse Large B-cell Lymphoma --- p.10 / Chapter 1.3.4.1 --- Classification method of DLBCL subtypes --- p.11 / Chapter 1.3.4.1.1 --- DNA microarray --- p.11 / Chapter 1.3.4.1.2 --- Immunohistochemistry pattern --- p.14 / Chapter 1.3.4.1.2.1 --- CD10 --- p.16 / Chapter 1.3.4.1.2.2 --- Bcl-6 --- p.16 / Chapter 1.3.4.1.2.3 --- CD138 --- p.17 / Chapter 1.3.4.1.2.4 --- MUM1/IRF4 --- p.17 / Chapter 1.3.4.2 --- Prognosis of 、DLBCL subtypes --- p.19 / Chapter 1.4 --- Interleukin 10 --- p.22 / Chapter 1.4.1 --- The IL-10 gene --- p.23 / Chapter 1.4.2 --- IL-10 promoter --- p.23 / Chapter 1.5 --- IL-10 receptor --- p.24 / Chapter 1.6 --- Cellular Signaling Pathways Regulated by IL-10 --- p.25 / Chapter 1.6.1 --- Jak/Stat Pathway --- p.25 / Chapter 1.6.2 --- Inhibition of NF B pathway --- p.26 / Chapter 1.7 --- Function of IL-10 --- p.27 / Chapter 1.7.1 --- Effects of IL-10 on immune cells in vitro --- p.27 / Chapter 1.7.2 --- Effects of IL-10 on B-cells --- p.28 / Chapter 1.8 --- IL-10 and IL-10 receptor in malignant diseases --- p.29 / Chapter 1.8.1 --- Melanoma --- p.29 / Chapter 1.8.2 --- Carcinoma --- p.30 / Chapter 1.8.3 --- Lymphoma --- p.30 / Chapter 1.9 --- Single Nucleotide Polymorphism (SNP) --- p.33 / Chapter 1.9.1 --- SNPs in cancer research --- p.34 / Chapter 1.9.1.1 --- Susceptibility to cancer and SNPs --- p.35 / Chapter 1.9.1.2 --- Outcome and SNPs --- p.35 / Chapter 1.10 --- SNP in the IL-10 promoter --- p.36 / Chapter 1.11 --- IL-10 promoter SNP in DLBCL --- p.37 / Chapter Chapter 2: --- Aims of Study --- p.39 / Chapter Chapter 3: --- Materials and Methods --- p.41 / Chapter 3.1 --- Sample Recruitment --- p.41 / Chapter 3.2 --- DNA preparation for Single Nucleotide Polymorphism (SNP) analysis --- p.41 / Chapter 3.2.1 --- Isolation of Peripheral Blood Mononuclear Cell (PBMC) from buffy coat from blood of normal control group --- p.41 / Chapter 3.2.2 --- Preparation for NHL and DLBCL samples from paraffin-embedded sections for DNA extraction --- p.42 / Chapter 3.2.3 --- DNA extraction for SNP analysis --- p.42 / Chapter 3.3 --- SNP analysis by Restriction Fragment Length Polymorphism (RFLP) --- p.43 / Chapter 3.3.1 --- Amplification of target site by PCR --- p.43 / Chapter 3.3.2 --- SNP analysis --- p.45 / Chapter 3.4 --- Determination of haplotypic frequency --- p.50 / Chapter 3.5 --- Classification of DLBCL by immunohistochemistry --- p.50 / Chapter 3.5.1 --- Staining pattern of CD10 --- p.53 / Chapter 3.5.2 --- Staining pattern of Bcl-6 --- p.54 / Chapter 3.5.3 --- Staining pattern of CD138 --- p.55 / Chapter 3.5.4 --- Staining pattern of MUM1/IRF4 --- p.56 / Chapter 3.6 --- Statistical Analysis --- p.57 / Chapter Chapter 4: --- Results --- p.58 / Chapter 4.1 --- SNPs of IL-10 promoter in normal controls --- p.58 / Chapter 4.1.1 --- Allelic Frequencies and genotype distributions --- p.58 / Chapter 4.1.2 --- Haplotypic Frequencies of normal controls --- p.58 / Chapter 4.2 --- SNP of the IL-10 promoter in non-Hodgkin's lymphomas --- p.59 / Chapter 4.2.1 --- Allelic frequencies and genotype distributions --- p.59 / Chapter 4.2.2 --- Haplolypic frequencies --- p.61 / Chapter 4.4 --- SNPs of the IL-10 promoter in DLBCL --- p.62 / Chapter 4.4.1 --- Allelic frequencies and genotype distributions --- p.62 / Chapter 4.4.2 --- Haplotypic frequencies --- p.64 / Chapter 4.5 --- SNP of the IL-10 promoter in different subtypes of DLBCL --- p.65 / Chapter 4.5.1 --- Classification of DLBCL by immunohistochemistry --- p.65 / Chapter 4.5.2 --- SNP of the IL-10 promoter in Germinal Center DLBCL (GC-DLBCL) --- p.67 / Chapter 4.5.2.1 --- Allelic frequencies and genotype distributions --- p.67 / Chapter 4.5.1.2 --- Haplotypic frequencies --- p.69 / Chapter 4.5.2 --- SNP of the IL-10 promoter in Activated Germinal Center DLBCL (AGC-DLBCL) --- p.70 / Chapter 4.5.2.1 --- Allelic frequencies and genotype distributions --- p.70 / Chapter 4.5.2.2 --- Haplotypic frequencies --- p.72 / Chapter 4.5.3 --- SNP of the IL-10 promoter in Activated non-Germinal Center DLBCL (ANGC-DLBCL) --- p.73 / Chapter 4.5.3.1 --- Allelic frequencies and genotype distributions --- p.73 / Chapter 4.5.3.2 --- Haplotypic frequencies --- p.75 / Chapter 4.5.4 --- SNP of the IL-10 promoter in Unclassified DLBCL (UC-DLBCL). --- p.76 / Chapter 4.5.4.1 --- Allelic frequencies and genotype distributions --- p.76 / Chapter 4.5.4.2 --- Haplotypic frequencies --- p.78 / Chapter 4.6 --- Summary of SNP of the IL-10 promoter in DLBCL subtypes --- p.79 / Chapter 4.7 --- Overall survival analysis --- p.80 / Chapter 4.7.1 --- Clinical data of DLBCL --- p.80 / Chapter 4.7.2 --- Cox Proportional Hazards Regression Analysis in DLBCL --- p.81 / Chapter Chapter 5: --- Discussion --- p.88 / Chapter 5.1 --- SNP for low IL-10 production in Hong Kong population --- p.88 / Chapter 5.2 --- NHL in low IL-10 production population --- p.90 / Chapter 5.2.1 --- The relationship between IL-10 and NHL --- p.90 / Chapter 5.2.2 --- Allelic frequencies and haplotype of the IL-10 promoter in NHL --- p.90 / Chapter 5.3 --- Classification of DLBCL --- p.91 / Chapter 5.3.1 --- Current prognostic analysis --- p.91 / Chapter 5.3.2 --- DLBCL subtypes distribution in Hong Kong is different from Caucasian --- p.92 / Chapter 5.4 --- IL-10 and DLBCL --- p.93 / Chapter 5.5 --- SNP of IL-10 promoter in DLBCL subtypes --- p.94 / Chapter 5.5.1 --- Allelic frequencies and haplotype of DLBCL subtypes --- p.94 / Chapter 5.5.2 --- Rare haplotypes were discovered in DLBCL --- p.94 / Chapter 5.6 --- Overall survival Analysis --- p.95 / Chapter 5.6.1 --- Univariate Cox Proportional Hazards Regression Analysis --- p.95 / Chapter 5.6.2 --- Bivariate Cox Proportional Hazards Regression Analysis --- p.96 / Chapter Chapter 6: --- Conclusion --- p.97 / References --- p.99

A disfunção erétil (DE) é uma doença relacionada com a sinalização deficiente de óxido nítrico (NO). O NO é produzido a partir da L-arginina pelas enzimas óxido nítrico sintase neuronal (nNOS), endotelial (eNOS) e induzida (iNOS). A dimetilarginina assimétrica (ADMA) é um inibidor endógeno dos três subtipos existentes de NOS, e é metabolizada principalmente pelas enzimas dimetilarginina dimetilaminohidrolase 1 e 2 (DDAH1 e DDAH2). Vários estudos têm associado alterações em genes, expressão ou atividade das enzimas DDAH com distúrbios em que a sinalização de NO é prejudicada. O objetivo deste estudo foi avaliar se o nível de resposta ao tratamento da DE com sildenafil pode estar associado a polimorfismos dos genes DDAH1 (rs1554597 e rs18582) e DDAH2 (rs805304 e rs805305) e, também, aos haplótipos formados por estes polimorfismos. Foram selecionados 70 pacientes com DE pós-prostatectomia (DEPP) e 70 pacientes com DE clínica (DEC). A função erétil dos voluntários foi avaliada através do questionário Índice Internacional de Função Erétil (IIEF). Para avaliação da resposta ao sildenafil, foram calculadas a diferença entre as pontuações pré e pós-tratamento (?IIEF) e a percentagem atingida da máxima resposta possível (?IIEF%) de cada paciente. Também, os pacientes de cada grupo foram divididos em bons e maus respondedores ao sildenafil de acordo com a mediana dos valores de ?IIEF%. Os genótipos dos rs1554597, rs805304 e rs805305 foram obtidos pela técnica de reação em cadeia da polimerase (PCR) seguida de digestão enzimática, e do rs18582 pela técnica de PCR alelo específica. O software PHASE 2.1 foi utilizado para estimar os haplótipos em cada grupo. Os resultados mostraram que o alelo variante A do rs18582 apresentou tendência para associação com piores respostas ao sildenafil no grupo DEC (P=0,058). No grupo DEPP, portadores dos alelos variantes A do rs805304 e G do rs805305 foram associados a melhores respostas ao sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; e score IIEF pós-tratamento, P=0,014). Não foram encontradas outras associações significativas. Estes resultados mostram que os marcadores genéticos rs805304 e rs805305 do DDAH2 podem influenciar as respostas ao sildenafil em pacientes com DE. / Erectile dysfunction (ED) is a disease related to deficient nitric oxide (NO) signaling. NO is produced from L-arginine by three isoforms of the enzyme nitric oxide synthase: neuronal (nNOS), endothelial (eNOS) and induced (iNOS). Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the three existing NOS subtypes, and is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 and 2 (DDAH1 and DDAH2) enzymes. Several studies have associated changes in genes, expression or activity of DDAH enzymes with disorders in which NO signaling is impaired. The aim of this study was to evaluate whether the response to ED treatment with sildenafil may be associated with polymorphisms of the DDAH1 (rs1554597 and rs18582) and DDAH2 genes (rs805304 and rs805305), as well as the haplotypes formed by these polymorphisms. We selected 70 patients with postprostatectomy ED (PPED) and 70 patients with clinical ED (CED). The erectile function of the volunteers was assessed using the International Index for Erectile Function (IIEF) questionnaire. To evaluate the response to sildenafil, the difference between the pre- and post-treatment scores (?IIEF) and the percentage reached from the maximum possible response (?IIEF%) of each patient were calculated. Also, patients from each group were divided into good and bad responders to sildenafil according to the median values of ?IIEF%. The genotypes of rs1554597, rs805304 and rs805305 were obtained by the polymerase chain reaction (PCR) technique followed by enzymatic digestion, and rs18582 by the allele-specific PCR technique. The PHASE 2.1 software was used to estimate the haplotypes in each group. The results showed that the variant A allele of rs18582 showed a tendency to be associated with a greater chance of worse responses to sildenafil in the DEC group (P=0,058). In the DEPP group, carriers of the variant alleles A of rs805304 and G of rs805305 were associated with better responses to sildenafil (?IIEF, P=0,007; ?IIEF%, P=0,025; and post-treatment IIEF score, P=0,014). No other significant associations were found. These results show that the genetic markers rs805304 and rs805305 of DDAH2 may influence the responses to sildenafil in patients with ED.

Fatores de restrição são as primeiras proteínas celulares envolvidas no combate a infecções virais, são considerados uma defesa intrínseca das células, constituindo-se em uma rápida resposta frente a invasão de patógenos. Essas moléculas são bastante diversas e são capazes de interferir em algum ponto do ciclo viral, atenuando ou bloqueando a evolução da infecção. Após a descoberta da existência desses fatores, alguns estudos têm direcionado o foco para as possíveis alterações genéticas que podem influenciar a estrutura dessas proteínas e, deste modo, interferir sobre suscetibilidade e progressão de doenças infecciosas, como a infecção pelo HIV/aids. O objetivo do presente estudo foi avaliar três SNPs de três diferentes fatores de restrição (o TRIM5α – rs10838525, a APOBEC3F – rs2076101 e o CUL5 – rs7117111) e observar suas frequências em diferentes grupos étnicos, bem como a associação desses fatores com a suscetibilidade ao HIV e a progressão a aids, em um grupo de soronegativos e soropositivos. Foram selecionados 345 indivíduos HIV+ atendidos no setor de Infectologia do Hospital Nossa Senhora da Conceição e 324 indivíduos HIV– doadores de sangue do Hospital de Clínicas de Porto Alegre. Os SNPs foram identificados através da técnica de PCR TaqManTM. O teste qui-quadrado foi utilizado para a análise das frequências e por regressão logística univariada foi avaliado o OR com 95% de IC entre os modelos dominantes e recessivos. Entre os SNPs estudados apenas o rs7117111 apresentou resultado estatisticamente significativo para o genótipo GG em relação a proteção ao HIV-1 (OR 0,661, IC 95% 0,449-0,974, P=0,036) e esse mesmo genótipo, também, parece estar relacionado aos progressores rápidos, pois apresentou uma tendência nessa relação quando ajustado pela etnia (OR ajustado 2,115, IC 95% 0,990- 4,520, P=0,053). Tais achados demonstram que alterações genéticas, especificamente no gene CUL5, podem influenciar a suscetibilidade ao HIV-1 e podem, também, interferir na progressão a aids. Esses resultados geram questionamentos de grande valia para um maior entendimento da influência genética do sistema de defesa intrínseco celular no curso da infecção. / Host restriction factors are the first cellular proteins engaged in antiviral response, they are considerate an intrinsic cell defense with the aim to be a rapid answer against the invasion of pathogens. This molecules have a vary diversity in structure and each one act in a distinct stages of viral life cycle, however always with the same objective to attenuate or block the infection. After the discovery of this restriction factors, some researches focus in looking for genetic variation that can be influence in structure protein and with this way interfere in HIV susceptibility or progress to AIDS. The aim of present work was evaluate tree SNPs of tree different restrictions factors (TRIM5α – rs10838525, APOBEC3F – rs2076101 and CUL5 – rs7117111) and detect yours frequencies in different ethics group, as well as, evaluate the SNPs`s capacity in influence the susceptibility to HIV and progress to AIDS in a seronegative and seropositive groups. For this research was selected 345 samples of HIV+ individuals from the Infectology sector of Nossa Senhora da Conceição hospital and 324 HIV- samples from blood donors of Clinics Hospital of Porto Alegre. Through PCR TaqManTM assay the SNPs was genotyping. The qui-square test was used to analyze the frequencies and by unvaried logistic regression was estimate the OR with 95% CI to dominant and recessive models. Between the tree SNPs chosen only rs7117111 was statistically significant the GG genotype with the HIV-1 protection (GG, OR: 0,661, 95% CI 0,449-0,974, P=0.036) and this same genotype seems to be to related with rapid progress to AIDS, because the result shows a tendency when adjusted for ethnicity in the recessive model (adjusted OR 2,115, IC 95% 0,990-4,520, P=0,053). This finds shows the genetics alterations, specify in the CUL5 gene, can alter the susceptibility to HIV-1 and can interfere in the progress to AIDS. Theses results are also important for the understanding of the genetic alterations in the host antiviral intrinsic mechanisms anti-HIV and can bring new insights for strategies against HIV pandemic.

O melanoma é uma doença complexa, associada com diversos fatores de risco genéticos e ambientais. Este o tipo mais agressivo de câncer de pele e origina-se nos melanócitos, as células da pele produtoras de pigmento nos mamíferos. Polimorfismos de base única (Single Nucleotide Polymorphisms - SNPs) presentes em genes envolvidos na pigmentação têm sido descritos envolvidos na modulação de risco para o melanoma, porém o conhecimento neste campo ainda é bastante limitado. Neste estudo, foi avaliado o efeito de quatro SNPs em quatro genes de pigmentação: TYR (rs1126809), HERC2 (rs1129038), SLC24A5 (rs1426654) e SLC45A2 (rs16891982) no aumento de risco para melanoma, usando análises de regressão logística multivariada e redução de dimensão multifatorial (MDR), em uma abordagem caso-controle. Em 255 indivíduos (120 pacientes com melanoma e 135 controles sem melanoma) provenientes do Rio Grande do Sul, Brasil, identificamos associação com o risco para melanoma em três dos quatro SNPs investigados (HERC2 rs1129038, P=0.017; SLC24A5 rs1426654, P<0.001; e SLC45A2 rs16891982, P=0.002). Além disso, a interação entre rs1426654 e rs16891982 (genótipos AA e GG, respectivamente), aumentou significamente o risco para melanoma nas análises de regressão logística multivariada e análises de MDR [OR = 6.936 (CI 95%: 1.607 – 50.294), P= 0.022]. Estes resultados contribuem para o conhecimento atual, indicando que esses SNPs contribuem para o aumento de risco de desenvolvimento de melanoma. / The melanoma is a complex disease, associated with several environmental and genetic risk factors. This is the most aggressive type of skin cancer and originates in melanocytes, the pigment producing skin cells in mammals. Single nucleotide polymorphisms (SNPs) in pigmentation genes have been describe in melanoma risk modulation but our knowledge in the field is still limited. Here, we assessed the effect of SNPs in four pigmentation genes – TYR (rs1126809), HERC2 (rs1129038), SLC24A5 (rs1426654), and SLC45A2 (rs16891982) on increase of melanoma risk using multivariate logistic regression and a multifactorial dimension reduction (MDR) analysis, in a case-control approach. In 255 individuals (120 melanoma patients and 135 controls free melanoma) from Rio Grande do Sul, Brazil, we identified an association of melanoma risk with three of the four SNPs studied (HERC2 rs1129038, P=0.017; SLC24A5 rs1426654, P<0.001; and SLC45A2 rs16891982, P=0.002). In addition, the interaction between rs1426654 and rs16891982 (AA and GG genotypes, respectively) significantly increased the risk of melanoma [OR = 6.936 (CI 95%: 1.607 – 50.294), P= 0.022] in both MRD and multivariate logistic regression analyses. Our results contribute to the current knowledge, indicating that SNPs contribute to the increase risk of melanoma.