by Wong Wing-yee, Felice. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 144-166). / Abstract also in Chinese. / Acknowledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.iv / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiv / List of Appendices --- p.xvi / Chapter Chapter I --- Literature Review / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Background of Cytochrome P450 --- p.3 / Chapter 2.1 --- Discovery --- p.3 / Chapter 2.2 --- Tissue Distribution --- p.3 / Chapter 2.3 --- Structure and Functions --- p.7 / Chapter 2.4 --- Nomenclature of the P450 Superfamily --- p.10 / Chapter 3. --- Cytochrome P450 2E1 (CYP2E1) --- p.11 / Chapter 3.1 --- Discovery --- p.11 / Chapter 3.2 --- Tissue Distribution --- p.12 / Chapter 3.3 --- Substrates and Inducers --- p.13 / Chapter 3.4 --- Toxicological Role of CYP2E1 --- p.15 / Chapter 4. --- CYP2E1-knockout Mouse Model --- p.17 / Chapter Chapter II --- Carbon Tetrachloride (CC14) Study / Chapter 1. --- Introduction --- p.19 / Chapter 1.1 --- General Properties and Usage of CC14 --- p.19 / Chapter 1.2 --- Toxicological Aspects of CC14 --- p.19 / Chapter 1.3 --- Mechanism of CCl4-induced Hepatotoxicity --- p.20 / Chapter 1.4 --- Role of CYP2E1 in CCl4-induced Hepatotoxicity --- p.23 / Chapter 1.5 --- Objectives of the Study --- p.27 / Chapter 2. --- Materials and Methods --- p.29 / Chapter 2.1 --- Chemicals and Materials --- p.29 / Chapter 2.2 --- Animals --- p.29 / Chapter 2.3 --- Acute CC14 Treatment --- p.29 / Chapter 2.4 --- Preparation of Microsomal Fractions --- p.30 / Chapter 2.5 --- Determination of Microsomal Protein Concentration --- p.31 / Chapter 2.6 --- Determination of Serum Aminotransferase Activities --- p.31 / Chapter 2.7 --- Liver Histology --- p.32 / Chapter 2.8 --- Hepatic Microsomal CYP2E1 Activity -p-nitrophenol Assay --- p.34 / Chapter 2.9 --- SDS-PAGE and Western Blot Analysis --- p.35 / Chapter 2.10 --- Detection of Lipid Peroxidation in vitro and in vivo --- p.35 / Chapter 2.10.1 --- In vitro Lipid Peroxidation - 2-Thiobarbituric Acid (TBA) assay --- p.35 / Chapter 2.10.2 --- In vivo Lipid Peroxidation - Microsomal Conjugated Dienes Detection --- p.36 / Chapter 2.11 --- Hepatic Lipid Fatty Acid Composition Analysis --- p.39 / Chapter 2.11.1 --- Lipid Extraction --- p.39 / Chapter 2.11.2 --- Thin Layer Chromatography --- p.39 / Chapter 2.11.3 --- Methylation --- p.40 / Chapter 2.11.4 --- Gas Chromatography --- p.40 / Chapter 2.12 --- Statistical Analysis --- p.41 / Chapter 3. --- Results --- p.42 / Chapter 3.1 --- "Mortality, Liver Weight and Liver Color" --- p.42 / Chapter 3.2 --- Hepatotoxicity --- p.42 / Chapter 3.2.1 --- Serum ALT and AST activities --- p.42 / Chapter 3.2.2 --- Liver Histology --- p.45 / Chapter 3.3 --- CYP2E1-catalysed PNP Activities and CYP2E1 Protein Levels --- p.49 / Chapter 3.3.1 --- CYP2El-catalyzed PNP Activities --- p.49 / Chapter 3.3.2 --- CYP2E1 Protein Levels --- p.52 / Chapter 3.4 --- Lipid Peroxidation --- p.52 / Chapter 3.4.1 --- In vitro Lipid Peroxidation --- p.52 / Chapter 3.4.2 --- In vivo Lipid Peroxidation --- p.54 / Chapter 3.5 --- Hepatic Lipid Fatty Acid Composition --- p.56 / Chapter 3.5.1 --- Fatty Acid Composition in Hepatic Phospholipid --- p.56 / Chapter 3.5.2 --- Fatty Acid Composition in Hepatic Microsomal Phospholipid --- p.59 / Chapter 3.5.3 --- Fatty Acid Composition in Hepatic Triglyceride --- p.61 / Chapter 4. --- Discussion --- p.63 / Chapter 4.1 --- CYP2E1 is Required in CCl4-mediated Hepatotoxicity --- p.63 / Chapter 4.2 --- CYP2E1 is Degraded following CC14 Exposure --- p.65 / Chapter 4.3 --- CYP2E1 is Required in CCl4-induced Lipid Peroxidation --- p.67 / Chapter 4.4 --- CYP2E1 is Required in CCl4-induced Hepatic Phospholipid Depletion --- p.70 / Chapter 4.5 --- CYP2E1 is Required in CCl4-induced Hepatic Triglyceride Accumulation --- p.72 / Chapter 5. --- Conclusion --- p.76 / Chapter Chapter III --- Chronic Ethanol Consumption Study / Chapter 1. --- Introduction --- p.77 / Chapter 1.1 --- Multiple Metabolic Pathways for Ethanol Metabolism --- p.77 / Chapter 1.2 --- Metabolism of Ethanol by the Microsomal Ethanol Oxidizing System --- p.79 / Chapter 1.3 --- Role of CYP2E1 in Ethanol Metabolism --- p.82 / Chapter 1.4 --- Role of CYP2E1 in Alcoholic Liver Disease and Associated Oxidative Stress --- p.84 / Chapter 1.5 --- Objectives of the Study --- p.89 / Chapter 2. --- Materials and Methods --- p.90 / Chapter 2.1 --- Chemicals and Materials --- p.90 / Chapter 2.2 --- Animals --- p.90 / Chapter 2.3 --- Chronic Ethanol Treatment --- p.90 / Chapter 2.3.1 --- Ethanol Diet Composition --- p.90 / Chapter 2.3.2 --- Ethanol Feeding --- p.90 / Chapter 2.4 --- Monitoring of Blood Ethanol Levels --- p.96 / Chapter 2.5 --- Preparation of Microsomal Fractions --- p.96 / Chapter 2.6 --- Determination of Microsomal Protein Concentration --- p.97 / Chapter 2.7 --- Determination of Serum Aminotransferase Activities --- p.98 / Chapter 2.8 --- Liver Histology --- p.98 / Chapter 2.9 --- SDS-PAGE and Western Blot Analysis --- p.99 / Chapter 2.10 --- Hepatic Fatty Acid Composition Analysis --- p.100 / Chapter 2.10.1 --- Lipid Extraction --- p.100 / Chapter 2.10.2 --- Thin Layer Chromatography --- p.101 / Chapter 2.10.3 --- Methylation --- p.101 / Chapter 2.10.4 --- Gas Chromatography --- p.102 / Chapter 2.11 --- Statistical Analysis --- p.103 / Chapter 3. --- Results --- p.104 / Chapter 3.1 --- Average Food Consumption --- p.104 / Chapter 3.2 --- Average Ethanol Consumption for Ethanol Liquid Diet Feeding Group --- p.104 / Chapter 3.3 --- Body Weight Gain --- p.104 / Chapter 3.4 --- Blood Ethanol Levels --- p.108 / Chapter 3.5 --- "Mortality, Liver Weight and Liver Color" --- p.108 / Chapter 3.6 --- Serum ALT and AST Activities --- p.110 / Chapter 3.7 --- Liver Histology --- p.114 / Chapter 3.8 --- Western Blot Analysis --- p.119 / Chapter 3.9 --- Hepatic Lipid Fatty Acid Composition --- p.119 / Chapter 3.9.1 --- Fatty Acid Composition in Hepatic Phospholipid --- p.119 / Chapter 3.9.2 --- Fatty Acid Composition in Hepatic Triglyceride --- p.123 / Chapter 4. --- Discussion --- p.126 / Chapter 4.1 --- Nutrients Displacement after Chronic Ethanol Consumption --- p.126 / Chapter 4.2 --- Varied Blood Ethanol Levels after Chronic Ethanol Consumption --- p.127 / Chapter 4.3 --- Increase in CYP2E1 Levels after Chronic Feeding of Ethanolin WT mice --- p.127 / Chapter 4.4 --- Lack of Evidence Indicating the Development of Ethanol- Induced Liver Injury --- p.129 / Chapter 4.4.1 --- No Elevations in Serum ALT and AST Activities --- p.129 / Chapter 4.4.2 --- Normal Liver Histology --- p.130 / Chapter 4.4.3 --- Lack of Triglyceride Accumulation --- p.131 / Chapter 4.4.4 --- Elevations in Hepatic PL --- p.132 / Chapter 4.5 --- Possible Reasons for the Absence of Liver Damage after Chronic Ethanol Consumption in our Mouse Model --- p.134 / Chapter 5. --- Conclusion --- p.137 / Chapter Chapter IV --- Concluding Remarks / Chapter 1. --- A Comparison between Acute CC14 Study and Chronic Ethanol Consumption Study --- p.139 / Chapter 1.1 --- Regulation of CYP2E1 Expression --- p.139 / Chapter 1.2 --- Free Radical Production Involved in CC14- and Chronic Ethanol Consumption-Mediated Liver Injury --- p.140 / Chapter 1.3 --- An Overall Comparison between CC14 study and Chronic Ethanol Consumption Study --- p.140 / Chapter 2. --- Future Studies --- p.142 / Chapter 2.1 --- Acute CC14 Study --- p.142 / Chapter 2.1.1 --- Calcium Homeostasis Studies --- p.142 / Chapter 2.1.2 --- Spin Trapping Studies --- p.142 / Chapter 2.2 --- Chronic Ethanol Study --- p.142 / Chapter 2.2.1 --- "Generation of a Heterozygous ""Ethanol-Sensitive"" Mouse Strain (SV/129/ter x C57BL/6)" --- p.143 / Chapter 3. --- Concluding Remarks --- p.143 / References --- p.144 / Appendix --- p.167

INTRODUÇÃO: A síndrome de Williams-Beuren (SWB) resulta de uma deleção de aproximadamente 1.5 Mb na região 7q11.23. A haploinsuficiência ocasiona alterações do desenvolvimento neurológico assim como malformações em múltiplos sistemas. OBJETIVOS: Testar utilidade de marcadores polimórficos para o diagnóstico da síndrome, determinar a proporção de pacientes com microdeleção, comparar características clínicas entre pacientes com e sem microdeleção e investigar origem parental. MÉTODOS: Selecionamos 32 pacientes com avaliação clínica para SWB atendidas no ICr. Critérios de inclusão: presença de dismorfismo craniofacial acompanhado ou não de alterações cardiovasculares, comportamento característico ou hiperacusia. Para a genotipagem do trio - afetado, mãe e pai, utilizamos os marcadores D7S1870, Eln 17/éxon18 e Hei. Análise em gel de poliacrilamida à 7% com imagens digitalizadas. RESULTADOS: Os marcadores D7S1870, Hei e Eln17/éxon18 foram 78% informativos e 22% não informativos. O marcador mais informativo foi o D7S1870 69%, seguido do Hei 55% e ELN 17/éxon18 em 43%. Houve microdeleção em 56% e ausência em 22%. A origem parental da deleção foi 9 materna e 8 paterna. As alterações craniofaciais e cardiovasculares não tiveram diferenças estatisticamente significantes entre portadores e não portadores da microdeleção. O comportamento amigável resultou numa diferença estatística muito significante (p=0,006) onde 88% tinham e 28% não tinham microdeleção. A hiperacusia teve diferença estatística significante (p=0,020) presente em 55% dos pacientes com microdeleção. CONCLUSÃO: Os dados obtidos demonstraram que os marcadores utilizados são úteis no diagnóstico da SWB e acessível para utilização em serviço público. / INTRODUCTION: Williams-Beuren syndrome (WBS) results of ~1.5 Mb commonly deleted region chromosome 7q11.23 in 90-95% of all clinically typical cases. The clinical manifestations can be variable and is a developmental disorder with multisystem manifestations caused by haploinsufficiency for contiguous genes in this region. OBJECTIVE: Polimorphic markers were tested to determine the proportion of patients with and without microdeletion, to compare the clinical features and to establish the parental origin of the deletion. METHODS: 32 probands with WBS ascertained according to well-established diagnostic criteria. Genotyping using polimorphic markers D7S1870, Eln 17/éxon18 and Hei was performed on DNA from the patients and their available parents. RESULTS: The three markers were informative in 78% and non informative in 22%. The best marker was D7S1870 with 69%, followed by Hei in 55% and ELN 17/éxon18 in 43%.The microdeletion was present in 56% and absent in 22%. Craniofacial and cardiovascular alterations did not have significant statistical differences between probands with or without microdeletion. Two following characteristics (friendly personality and hyperacusia) were more frequent in the deleted group and these differences were statistical significant (p=0,006 and 0,02 respectively). CONCLUSIONS: Polimorphic markers used here demonstrated its viability and utility for the confirmation diagnosis of SWB in a public service.

Submitted by Filipe dos Santos (fsantos@pucsp.br) on 2016-11-22T12:45:04Z No. of bitstreams: 1 Rodrigo de Oliveira Andrade.pdf: 1064494 bytes, checksum: 538b5c2fe9f0ed25b6af19d72a15810f (MD5) / Made available in DSpace on 2016-11-22T12:45:04Z (GMT). No. of bitstreams: 1 Rodrigo de Oliveira Andrade.pdf: 1064494 bytes, checksum: 538b5c2fe9f0ed25b6af19d72a15810f (MD5) Previous issue date: 2016-08-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The 19th century constitutes a period of strong incidence of researches on cell biology. With the advent of new microscopy techniques, a more comprehensive study of cells behaviour has allowed a better understanding of the conditions that could favour or even determine the appearance of tumours. From then on, several scientists have proposed conceptual, medical and institutional pathways of research and treatment against the disease. The aim of this work is to analyse the panorama of studies and theories on cancer etiology proposed between the latest decades of 19th century and the first two decades of 20th century. We intend to understand how the concepts related to cancer mechanisms have been built and reinterpreted over those decades, and how has this knowledge arrived in Brazil. In this regard, Brazilian doctor Alfredo Leal Pimenta Bueno’s work is going to be used as a case study. Pimenta Bueno presented his ideas on the biochemical phenomena that could trigger cancer in a series of scientific articles published in the Brazilian scientific journal O Brasil Médico between 1926 and 1928. In this dissertation, we show that cancer has transformed into an entity that was more and more discussed and studied in the Europe over the 19th century, and that its construction as a medical problem in Brazil during the first two decades of the 20th century occurred in a scenario in which the attention was still focused on diseases with a greater social impact, such as tuberculosis. At the same time, we have verified that Pimenta Bueno was one of the few Brazilian scientists who investigated cancer etiology, constructing his theory based on the writings of several scientists, appealing to them whenever necessary in order to justify his points of view. In his articles, Pimenta Bueno concludes that cancer could be caused by different agents — all of them would act in the way of causing an irritation, resulting in an over hydration inside cells, which would return to their embryonic stage and, finally, reacquire their proliferation capacity / O século XIX constitui um período de forte incidência de pesquisas no campo da biologia celular. Com o advento de novas técnicas de microscopia, o estudo detalhado do comportamento das células possibilitou um melhor entendimento das condições que favorecem ou determinam o surgimento dos tumores. A partir de então, diversos cientistas propuseram caminhos conceituais, médicos ou institucionais de pesquisa e tratamento para a enfermidade. O objetivo deste trabalho é analisar o panorama de estudos e teorias sobre a etiologia do câncer em fins do século XIX e início do século XX. Pretendemos compreender como os conceitos relacionados aos mecanismos da doença foram construídos e reinterpretados ao longo daquelas décadas e como esse conhecimento chegou ao Brasil. Para isso, usaremos como estudo de caso os trabalhos do médico mineiro Alfredo Leal Pimenta Bueno, que apresentou suas ideias sobre os fenômenos bioquímicos que desencadeariam o câncer em uma série de artigos na revista O Brasil Médico entre 1926 e 1928. Nesta dissertação, mostramos que o câncer se transformou durante o século XIX em uma entidade cada vez mais discutida e estudada na Europa, e que sua construção como problema médico no Brasil nas primeiras décadas do século XX se deu em meio a um cenário em que a preocupação maior ainda se centrava em doenças de maior impacto social, como a tuberculose. Ao mesmo tempo, verificamos que Pimenta Bueno foi um dos poucos cientistas brasileiros a investigar a etiologia do câncer, construindo seu discurso a partir dos escritos de diversos cientistas, recorrendo a esses autores sempre que preciso a fim de justificar seus pontos de vista. Pimenta Bueno conclui que o câncer teria as mais variadas causas, todas idênticas pelo só fato de que agiam produzindo uma irritação, de que resultaria uma super hidratação celular, sua volta ao estágio embrionário, e, finalmente, o reestabelecimento de sua capacidade de proliferação

Orientador: Lucilene Arilho Ribeiro-Bicudo / Resumo: As cardiopatias congênitas (CCs) podem ser definidas como qualquer anormalidade na estrutura e/ou na função cardiocirculatória presente ao nascimento. Constituem as malformações congênitas mais comuns entre recém-nascidos vivos, podendo se apresentar de duas formas: isoladas (ou não sindrômicas) e sindrômicas. De caráter multifatorial, o surgimento de CCs envolve fatores ambientais, genéticos e epigenéticos. A etiologia genética de CCs ainda é pouco conhecida. Entre as causas genéticas conhecidas, podemos destacar: aneuploidias, alterações na estrutura dos cromossomos, desequilíbrios citogenômicos (perdas e ganhos genômicos ou variações no número de cópias genômicas – CNVs), mutações pontuais, variações em um único nucleotídeo, entre outras. Dentre essas, as CNVs contribuem com aproximadamente 10% na etiologia genética de CCs não sindrômicas, e cerca de 20% entre as sindrômicas. O objetivo deste trabalho foi investigar possíveis desequilíbrios citogenômicos em pacientes diagnosticados com CCs sindrômicas e não sindrômicas idiopáticas. Foram recrutados 31 pacientes, sendo 13 sindrômicos e 18 não sindrômicos. Todos foram submetidos à avaliação genético-clínica. As amostras foram coletadas a partir do sangue periférico, e realizou-se o cariótipo convencional para todos os sindrômicos. A análise por MLPA foi realizada em 27 pacientes. O DNA genômico dos pacientes sindrômicos selecionados foi submetido a duas plataformas de CMA (array-CGH/SNP arrays): SNP-array 850K HumanCytoSNP (... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Congenital heart defects (CHD) can be defined as any abnormality in the structure and /or cardiocirculatory function present at birth. Congenital malformations are more common among live newborns. CHD can be presented at two forms: isolated (or non syndromic) and syndromic ones. Of multifactorial spectrum, the emergence of CHD involves environmental, genetic and epigenetic factors. The genetic etiology of CHD is still poorly understood. Among the known genetic causes, we can highlight: aneuploidies, changes in chromosome structure, cytogenetic imbalances (losses and genomic gains, can be also called copy number variations - CNVs), point mutations, variants in a single nucleotide, among others. Among these, CNVs contribute to approximately 10% in the genetic etiology of non-syndromic CHD, and about 20% among syndromic ones. The aim of this study was to investigate possible cytogenetic imbalances in patients diagnosed with idiopathic syndromic and non-syndromic CHD. Thirty one patients were recruited, of which 13 syndromic and 18 non-syndromic. Each patient were submitted to genetic-clinical evaluation. Only patients with an undefined syndromic condition were included in cytogenetic investigations. Samples were collected from the peripheral blood, and the conventional karyotype was performed for all syndromic patients. MLPA analysis was performed in 27 patients. The genomic DNA of the selected patients was analyzed into two CMA (array-CGH / SNP arrays) platforms: SNP-array 850K... (Complete abstract click electronic access below) / Mestre

O ácido gama-aminobutírico (GABA), principal neurotransmissor inibitório, está envolvido no mecanismo intrínseco do início da puberdade. Os efeitos inibitórios do GABA sobre a secreção de GnRH (hormônio hipotalâmico estimulador da secreção das gonadotrofinas) são mediados pelo receptor tipo A (GABAA) que é composto por diferentes subunidades organizadas de forma heteropentamérica. A subunidade a1, codificada pelo gene GABRA1 localizado no locus 5q34-35, é a mais implicada na atividade inibitória do GABA. A puberdade precoce dependente de gonadotrofinas (PPDG) predomina no sexo feminino, sendo na maioria dos casos idiopática. Recentemente, defeitos moleculares das subunidades dos receptores de GABA têm sido identificados em pacientes com anormalidades eletroencefalográficas específicas. Neste estudo, investigamos a presença de mutações funcionais ou polimorfismos do GABRA1 em meninas com a forma idiopática de PPDG e avaliamos as anormalidades EEG neste grupo. Trinta e uma meninas com diagnóstico clínico e hormonal da forma idiopática da PPDG, sendo 6 casos familiais (19,4%) e 25 casos esporádicos (80,6%), e 73 controles não relacionados foram selecionados. Todas as pacientes com PPDG apresentaram ressonância magnética de sistema nervoso central normal. Vinte e três meninas foram submetidas a estudo eletroencefalográfico (EEG). O DNA genômico foi extraído do sangue periférico de todas as pacientes e controles. A região codificadora do GABRA1 foi amplificada utilizando-se oligonucleotídeos intrônicos específicos, seguida por purificação enzimática e seqüenciamento automático. Dois polimorfismos conhecidos do GABRA1 foram também estudados pelo programa GeneScan e pela técnica de digestão enzimática (enzima TaiI). O seqüenciamento automático do GABRA1 não revelou mutações funcionais. Identificamos 7 polimorfismos no GABRA1: duas variantes exônicas 156T>C e 1323G>A, localizados no éxons 4 e 11, respectivamente, e 5 polimorfismos intrônicos - IVS2-712(GT)n, no íntron 2, caracterizado por número variável de repetições GT; IVS3+12A>T, no íntron 3; IVS8+45T>G no íntron 8; IVS9+76T>G no íntron 9 e IVS10+15G>A, no íntron 10. Estes polimorfismos não alteram o uso do sítio de splice original. Não houve diferença estatisticamente significante entre a distribuição genotípica e a freqüência alélica dos 2 polimorfismos exônicos e do polimorfismo IVS2-712(GT) encontrados no grupo de pacientes e no grupo controle. O EEG revelou anormalidades em 6 de 23 meninas (4 sem epilepsia). A distribuição genotípica e a freqüência alélica dos polimorfismos do GABRA1 não difereriram significativamente entre as pacientes com PPDG sem e com anormalidades eletroencefalográficas. Nós concluímos que mutações funcionais ou polimorfismos no GABRA1 não estão envolvidos na etiologia da forma idiopática da PPDG e não estão associadas às anormalidades eletroencefalográficas encontradas. Adicionalmente, a presença de alterações eletroencefalográficas em pacientes com PPDG sem epilepsia sugere que a análise eletroencefalográfica deva ser incluída na investigação da PPDG / The gamma-aminobutyric acid (GABA), a dominant inhibitory neurotransmitter, is involved in the intrinsic mechanism of the onset of the puberty. Their inhibitory effects on the GnRH (hypothalamic gonadotropin release hormone) secretion are mediated by type A receptor (GABAA), composed by different subunits which are organized in a heteropentameric form. The alpha-1 subunit, encode by GABRA1 gene located at locus 5q34- 35, is the most implicated in the inhibitory activity of GABA. The gonadotropin-dependent precocious puberty (GDPP) is predominant in females, being idiopathic in the majority of the cases. Recently, molecular defects of the GABA receptor subunits have been identified in patients with specific electroencephalographic (EEG) abnormalities. In this study, we investigated the presence of functional mutations or polymorphisms of the GABRA1 in girls with the idiopathic form of the GDPP and evaluated EEG abnormalities in this group. Thirty-one girls with clinical and hormonal diagnosis of GDPP idiopathic form, being 6 familial cases (19.4%) and 25 sporadic cases (80.6%), and 73 unrelated controls were selected. All patients with GDPP had normal magnetic resonance of central nervous system. Twenty-three girls were submitted to electroencephalographic study. Genomic DNA was extracted from peripheral blood of all patients and controls. The entire coding region of the GABRA1 was amplified using specific intronic oligonucleotides, followed by enzymatic purification and automatic sequencing. Two known polymorphisms of the GABRA1 were also studied by GeneScan software and digestion with restriction endonuclease TaiI. The automatic sequencing of the GABRA1 did not reveal any functional mutations. We identified 7 polymorphisms in the GABRA1: two silent exonic variants 156T>C e 1323G>A, located at exons 4 e 11, respectively, e 5 polymorphisms - IVS2-712(GT)n, at intron 2, characterized by a variable number of repeat GT; IVS3+12A>T, at intron 3; IVS8+45T>G at intron 8; IVS9+76T>G at intron 9 and IVS10+15G>A, at íntron 10. These polymorphisms did not alter the use of original splicing site. No significant statistical difference of the genotypic distribution and allele frequency of the exonic polymorphisms (156T>C and 1323G>A) and IVS2-712(GT)n between unrelated patients and control group was obtained. Electroencephalographic tracings were abnormal in 6 of 23 girls (4 without epilepsy). No significant statistical difference of the genotype distribution and allele frequence were found between patients without and with EEG abnormalities. We conclude that functional mutations or polymorphisms in the GABRA1 are not involved in the etiology of idiopathic GDPP in this study, and they are not associated with electroencephalografic abnormalities. In addition, EEG abnormalities present in girls with GDPP without epilepsy, suggest that EEG analysis should be included in the investigation of the precocious puberty.

by Sung Ying-Man, Mandy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 112-125). / Abstracts in English and Chinese. / Acknowledgments --- p.I / List of Abbreviations --- p.II / Abstract --- p.IV / 摘要 --- p.VII / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemiology --- p.1 / Chapter 1.2 --- Aetiological factors --- p.4 / Chapter 1.2.1 --- Hepatitis B infection --- p.4 / Chapter 1.2.2 --- Aflatoxin exposure --- p.5 / Chapter 1.2.3 --- Alcohol consumption --- p.5 / Chapter 1.2.4 --- Genetic risk factors --- p.6 / Chapter 1.3 --- Aims of the study --- p.7 / Chapter Chapter 2 --- Polymorphisms of candidate genes in Interleukin- signalling pathway in HCC --- p.6 / Chapter 2.1 --- Introduction --- p.9 / Chapter 2.2 --- Materials and Methods --- p.15 / Samples and Genomic DNA isolation --- p.15 / PCR-PFLP --- p.15 / dHPLC --- p.16 / Direct Sequencing --- p.17 / Stattistical Analysis --- p.17 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Known IL6 polymorphisms --- p.25 / Chapter 2.3.2 --- IL-6R and gp130 polymorphisms --- p.28 / Chapter 2.3.3 --- Stat-3 polymorphisms --- p.29 / Chapter 2.3.4 --- SOCS-1 polymophisms --- p.32 / Chapter 2.3.5 --- Mutation screening of IL-6 gene --- p.34 / Chapter 2.3.6 --- Mutation screening of SOCS-1 gene --- p.39 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- Interleukin-6 --- p.40 / Chapter 2.4.2 --- Gp130 and IL6-R --- p.43 / Chapter 2.4.3 --- STAT-3 --- p.44 / Chapter 2.4.4 --- SOCS-l --- p.46 / Chapter Chapter 3 --- Methylation status of SOCS-1 gene in Chinese HCC patients / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Methods and Materials --- p.53 / Tissue Sampling --- p.53 / Methylation specific PCR (MSP) --- p.53 / Chapter 3.3 --- Results --- p.55 / Chapter 3.4 --- Discussion --- p.56 / Chapter Chapter 4 --- Polymorphisms of enzyme encoding genesin steroidogenesis in Chinese / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.1.1 --- Steroid 5a reductases (SRD5A) --- p.62 / Chapter 4.1.1a --- Steroid 5a reductase type II (SRD5A2) --- p.63 / Chapter 4.1.1b --- Steroid 5a reductase type I (SRD5A1) --- p.65 / Chapter 4.1.2 --- Cytochrome P450al7 (CYP17) --- p.67 / Chapter 4.1.3 --- "Cytochrome P450, family 1，subfamily A polypeptide1 (CYP1A1)" --- p.69 / Chapter 4.1.4 --- "Cytochrome P450, subfamily IIIA (niphedipine oxidase) polypeptide 4 (CYP3A4)" --- p.71 / Chapter 4.2 --- Materials and Methods --- p.74 / Samples and Genomic DNA isolation --- p.74 / PCR-PFLP --- p.74 / Direct Sequencing --- p.74 / Statistical Analysis --- p.74 / Chapter 4.3 --- Results --- p.77 / Chapter 4.3.1 --- SRD5A2 --- p.77 / Chapter 4.3.2 --- Linkage Disequilibrium in SRD5A2 gene --- p.83 / Chapter 4.3.2 --- SRD5A1 --- p.84 / Chapter 4.3.3 --- CYP17 --- p.87 / Chapter 4.3.4 --- CYP1A1 --- p.89 / Chapter 4.3.5 --- CYP3A4 --- p.92 / Chapter 4.3.6 --- Logistic regression --- p.95 / Chapter 4.4 --- Discussion --- p.96 / Chapter 4.4.1 --- SRD5A2 --- p.96 / Chapter 4.4.2 --- SRD5A1 --- p.99 / Chapter 4.4.3 --- CYP17 --- p.101 / Chapter 4.4.4 --- CYP1A1 --- p.103 / Chapter 4.4.5 --- CYP3A4 --- p.106 / Chapter 4.4.6 --- Logistic Regression --- p.107 / Chapter Chapter 5 --- Conclusions and Future Prospect --- p.108 / Chapter 5.1 --- Conclusions --- p.108 / Chapter 5.2 --- Future works and prospect --- p.111 / References --- p.113

Diabetic pregnancy is associated with increased risk of congenital malformations. Previous studies have shown that maternal diabetes can interact with the vitamin A metabolite, all-trans retinoic acid (RA), in increasing embryonic susceptibility to caudal regression and neural tube defects. The aim of this thesis is to investigate the cellular and molecular mechanisms that underlie this interaction. / First hypothesis. RA concentration in the embryo is tightly regulated by the synthesizing enzyme retinaldehyde dehydrogenase type II (RALDH2), and the degrading enzyme CYP26. Alteration in expression levels of these enzymes under maternal diabetes may affect the availability of RA and thus its teratogenicity. / In conclusion, results of this thesis provide insight into the mechanism of how maternal diabetes interacts with RA in enhancing embryonic susceptibility to congenital malformations. This is also the first report to show that maternal diabetes alters RA homeostasis. (Abstract shortened by UMI.) / Second hypothesis. The transfer of RA to the nucleus for molecular action is regulated by cytoplasmic cellular retinoic acid binding proteins CRABP-I and CRABP-II. Alteration in expression levels of these binding proteins under maternal diabetes may affect the amount of RA reaching the nucleus and thus its teratogenicity. / Third hypothesis. The action of RA is mediated via different nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR). Alteration in expression levels of these receptors under maternal diabetes may affect the efficacy of RA signal transduction and thus its teratogenicity. / Three hypotheses are proposed to explain the underlying mechanism of increased embryonic susceptibility to RA teratogenicity under maternal diabetes: / To investigate these hypotheses, expression levels of various genes in different groups were compared. Result show that there are no significant differences in mRNA expression levels of CRABP-I, CRABP-II, RARgamma, RARgamma and RXRalpha between embryos of diabetic and non-diabetic mice with or without RA treatment. In contrast, expression levels of Raldh2 and CYP26 are significantly reduced in embryos of diabetic mothers, and in embryos of non-diabetic mice cultured in vitro in hyperglycemic conditions. Moreover, embryos of diabetic mice show significantly reduced response to RA-induced up-regulation of CYP26. These findings suggest that the rate of degradation of RA is slower in embryos of diabetic mice and thus the teratogenic effect of RA is enhanced. / Leung Bo Wah. / "July 2005." / Adviser: Alisa S. W. Shum. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3779. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 158-198). / 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.

Chan Hoi-Huen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 104-123). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Publications --- p.vi / Table of contents --- p.viii / List of Figures --- p.xii / List of Tables --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Blood vessels and blood pressure --- p.1 / Chapter 1.2 --- Smooth muscle --- p.2 / Chapter 1.3 --- Endothelium --- p.3 / Chapter 1.4 --- Vasodilation and Vasoconstriction --- p.5 / Chapter 1.5 --- Calcitonin gene-related peptide (CGRP) --- p.6 / Chapter 1.5.1 --- Discovery of CGRP --- p.6 / Chapter 1.5.2 --- Localization and distribution of CGRP --- p.7 / Chapter 1.5.3 --- Structure profile of CGRP --- p.8 / Chapter 1.5.4 --- CGRP and the vascular system --- p.10 / Chapter 1.6 --- Nitric oxide --- p.11 / Chapter 1.6.1 --- Production of NO by nitric oxide synthase (NOS) --- p.12 / Chapter 1.6.2 --- Actions of NO in smooth muscle --- p.14 / Chapter 1.6.3 --- Synergism with CGRP --- p.14 / Chapter 1.7 --- Other research of CGRP in the laboratory of Professor Ronald R. Fiscus --- p.15 / Chapter 1.8 --- "Aging, diabetes mellitus, sex hormones and cardiovascular system" --- p.16 / Chapter 1.9 --- Aim of study --- p.18 / Chapter Chapter 2 --- Methods and materials --- p.19 / Chapter 2.1 --- General experimental methods --- p.19 / Chapter 2.1.1 --- Measurement of blood pressure in anaesthetized rats --- p.19 / Chapter 2.1.2 --- Tissue bath experiments --- p.20 / Chapter 2.1.2.1 --- Preparation of isolated rat aortic rings --- p.20 / Chapter 2.1.2.2 --- Measurement of contractile and relaxant responses in the rat aortic rings --- p.21 / Chapter 2.1.3 --- Culture of aortic rat vascular smooth muscle cells --- p.22 / Chapter 2.1.4 --- Immunostaining for smooth muscle α-actin in cultured smooth muscle cells --- p.23 / Chapter 2.1.5 --- Determination of nitrite levels in smooth muscle cell culture media --- p.24 / Chapter 2.1.6 --- Measurement of protein contents --- p.25 / Chapter 2.1.7 --- Reversed Transcription- Polymerase Chain Reaction (RT-PCR) --- p.26 / Chapter 2.1.7.1 --- mRNA isolation --- p.26 / Chapter 2.1.7.2 --- Reverse transcription (RT) --- p.27 / Chapter 2.1.7.3 --- Polymerase chain reaction (PCR) --- p.27 / Chapter 2.1.7.4 --- Agarose slab gel electrophoresis --- p.29 / Chapter 2.1.7.5 --- Capillary electrophoresis --- p.29 / Chapter 2.2 --- Reagents --- p.30 / Chapter Chapter 3 --- Impairment of hypotension to calcitonin gene-related peptide in female rats with streptozotocin-induced diabetes mellitus or ovariectomy --- p.40 / Chapter 3.1 --- Introduction --- p.40 / Chapter 3.2 --- Methods --- p.45 / Chapter 3.2.1 --- Animal Preparation --- p.45 / Chapter 3.2.2 --- Statistical analysis --- p.46 / Chapter 3.3 --- Results --- p.47 / Chapter 3.3.1 --- "Body weight, blood glucose and initial blood pressure" --- p.47 / Chapter 3.3.2 --- Hypotensive responses to CGRP in ovariectomized rats --- p.48 / Chapter 3.3.3 --- Hypotensive responses to CGRP in diabetic rats --- p.49 / Chapter 3.3.4 --- Hypotensive responses to CGRP in rats with diabetes and ovariectomy --- p.50 / Chapter 3.4 --- Discussion --- p.50 / Chapter 3.5 --- Conclusions --- p.56 / Chapter Chapter 4 --- Severe impairment of CGRP-induced hypotension in vivo and vasorelaxation in vitro in elderly rats --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Methods --- p.64 / Chapter 4.2.1 --- Tissue preparation for vascular rings --- p.64 / Chapter 4.2.2 --- Vasorelaxation studies in vitro --- p.65 / Chapter 4.2.3 --- Animal preparation for in vivo studies --- p.65 / Chapter 4.2.4 --- Measurement of hypotensive responses to CGRP --- p.66 / Chapter 4.2.5 --- Statistical analysis --- p.66 / Chapter 4.3 --- Results --- p.67 / Chapter 4.3.1 --- Effect of age on CGRP-induced vasorelaxations in rings of thoracic aorta and caudal arteries --- p.67 / Chapter 4.3.2 --- Effect of age on acetylcholine-induced responses in aortic rings --- p.68 / Chapter 4.3.3 --- CGRP-induced hypotension in young female and male rats --- p.68 / Chapter 4.3.4 --- CGRP-induced hypotension in elderly female and male rats --- p.68 / Chapter 4.3.5 --- CGRP-induced hypotension in elderly female rats with ovariectomy --- p.69 / Chapter 4.4 --- Discussion --- p.69 / Chapter 4.5 --- Conclusions --- p.73 / Chapter Chapter 5 --- "Effects of CGRP on interleukin-Iβ-, lipopolysaccharides- and ginseng extract-induced production of nitrite oxide in vascular smooth muscle cells of elderly rats" --- p.82 / Chapter 5.1 --- Introduction --- p.82 / Chapter 5.2 --- Methods --- p.83 / Chapter 5.2.1. --- Animal model --- p.83 / Chapter 5.2.2. --- Culture of vascular smooth muscle cells --- p.84 / Chapter 5.2.3 --- Extraction of total RNA --- p.84 / Chapter 5.2.4 --- Reverse transcription and polymerase chain reaction (RT-PCR) --- p.35 / Chapter 5.2.5 --- Capillary electrophoresis with laser-induced fluorescence detector (CE-LIF) --- p.85 / Chapter 5.2.6 --- Determination of nitrite levels in smooth muscle cell culture media --- p.85 / Chapter 5.2.7 --- Measurement of protein contents --- p.86 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- "Effects of IL-Iβ, alone and in combination with CGRP, on NO production in young and elderly VSMCs" --- p.86 / Chapter 5.3.2 --- "Effects of LPS, alone and in combination with CGRP, on NO production in young and elderly VSMCs" --- p.89 / Chapter 5.3.3 --- "Effects of ginseng extract, alone and in combination with CGRP, on NO production in VSMCs" --- p.89 / Chapter 5.4 --- Discussion --- p.90 / Chapter 5.5 --- Conclusions --- p.93 / Chapter Chapter 6 --- General discussion and Conclusions --- p.100 / References --- p.104

Submitted by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-09-28T15:32:12Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Clarissa Fontoura dissertação final 2013.pdf: 4771397 bytes, checksum: c43a071b5dc04dfd4428016dfecf98f9 (MD5) / Approved for entry into archive by Ana Lúcia Torres (bfmhuap@gmail.com) on 2017-09-28T15:32:31Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Clarissa Fontoura dissertação final 2013.pdf: 4771397 bytes, checksum: c43a071b5dc04dfd4428016dfecf98f9 (MD5) / Made available in DSpace on 2017-09-28T15:32:31Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Clarissa Fontoura dissertação final 2013.pdf: 4771397 bytes, checksum: c43a071b5dc04dfd4428016dfecf98f9 (MD5) Previous issue date: 2013 / University of Iowa / A fissura labial com ou sem fissura palatina (FL/P) é uma anomalia craniofacial muito comum em humanos e pode ocorrer como característica de um quadro sindrômico ou isolada quando os indivíduos afetados não apresentam qualquer anomalia estrutural associada. A etiologia da FL/P é complexa, com a contribuição de componentes genéticos e ambientais. Diversos genes/loci candidatos a FL/P foram sugeridos nos últimos anos, contudo, discrepâncias entres os resultados são comumente encontradas. Recentemente, os genes WNT3, WNT9B, PBX1, TP63, e ARGHAP29 foram citados como possíveis genes candidatos à etiologia das FL/P devido à importante função que exercem durante o desenvolvimento craniofacial. O objetivo deste trabalho foi avaliar a associação entre polimorfismos nestes genes com o fenótipo de FL/P em uma população brasileira. Para tanto, setenta famílias, constituídas por um indivíduo afetado e seus pais não afetados, foram examinadas clinicamente e amostras de saliva foram coletadas para estudos moleculares. Um total de 20 polimorfismos distribuídos nos genes WNT3, WNT9B, PBX1, P63, e ARGHAP29 foram estudados com relação à associação com FL/P utilizando-se o método de TaqMan. O teste de desequilíbrio de transmissão (TDT) foi utilizado para detectar a associação de alelos em cada marcador nos indivíduos com FL/P, através do programa Family-Based Association Test (FBAT). O nível de significância foi determinado em P ≤ 0,05. Houve associação positiva entre FL/P para os genes ARGHAP29 (rs1048854), TP63 (rs4575879) e WNT9B (rs1530364) com FL/P. Não foi detectada associação entre alelos e genótipos de WNT3 e PBX1 com FL/P. Estes resultados sugerem que ARGHAP29, TP63 e WNT9B podem estar envolvidos na etiologia da FL/P na população estudada / Cleft lip with or without cleft palate (CL/P) is a common craniofacial anomaly in humans, and may occur as part of a syndrome or isolated, when the affected individuals do not present any associated structural anomalies. The etiology of CL/P is complex, with both genetic and environmental factors involved. Several genes /loci have been suggested in the past years although discrepancies among results are often found. Previous studies have demonstrated that WNT3, WNT9B, PBX1, TP63, and ARGHAP29 may be involved in the etiology of the CL/P due to the important function of these genes during craniofacial development. The aim of this study was to evaluate the association between polymorphisms in these genes and CL/P in a Brazilian population. Seventy families, composed by an affected individual and their unaffected parents, were examined clinically and saliva samples were collected for molecular analyses. A total of 20 polymorphisms distributed in WNT3, WNT9B, PBX1, TP63, and ARGHAP29 were investigated using the TaqMan method. The Family-Based Association Test (FBAT) and the transmission disequilibrium test (TDT) were used to verify the association between each marker allele and CL/P. The level of significance was established at P ≤ 0.05. Positive associations were detected between CL/P and three markers in ARGHAP29 (rs1048854), TP63 (rs4575879) and WNT9B (rs1530364) genes. No association was detected between CL/P and markers in WNT3 and PBX1. These results suggest that ARGHAP29, WNT9B and TP63 may be involved in the etiology of CL/P in the studied population

INTRODUÇÃO: O carcinoma hepatocelular (CHC) é o mais frequente tipo de câncer primário do fígado e a sua incidência vem aumentando nas últimas décadas, , tornando-o hoje a terceira causa de morte por câncer no mundo. Em cerca de 70 a 80% dos pacientes, o CHC é precedido pelo desenvolvimento de cirrose hepática. Existe um consenso de que a ressecção cirúrgica do tumor é a única terapêutica efetivamente comprovada. Esta ressecção pode ser realizada tanto através de uma hepatectomia como pelo transplante hepático. Atualmente, apenas 30 a 40% dos pacientes se beneficiam dos tratamentos ditos curativos e, mesmo entre esses pacientes, a sobrevida em cinco anos continua baixa, em torno de 60 a 70%, com taxa de recorrência do tumor em torno de 50% em três anos. Alguns estudos mostraram um pior prognóstico para os pacientes com CHC cuja etiologia é a infecção por vírus B ou C. Isso nos leva à questão sobre a existência de uma diferença entre as diversas etiologias do CHC e o seu prognóstico. OBJETIVOS: Comparar o prognóstico (sobrevida global e livre de doença em cinco anos) de pacientes submetidos à hepatectomia para o tratamento do CHC com relação às diversas etiologias da hepatopatia e estudar fatores prognósticos nesse grupo de pacientes. MÉTODO: Foi realizado um levantamento de prontuários dos pacientes submetidos à hepatectomia entre 2000 e 2014 para tratamento de CHC, seguido de análise estatística desse banco de dados, visando a avaliação de parâmetros clínicos, laboratoriais e cirúrgicos. Os pacientes foram divididos em grupos de acordo com a etiologia da hepatopatia, sendo feita uma análise de sobrevida para comparação. RESULTADOS: Não houve diferença estatisticamente significante de prognóstico entre os grupos de pacientes divididos conforme a etiologia do CHC. A sobrevida global e livre de doença em cinco anos dos pacientes dessa amostra foi de 49,9% e 40,7%, respectivamente. As variáveis prognósticas estatisticamente significantes para sobrevida global foram nível sérico de alfafetoproteína (p=0,043), nível sérico de CA19.9 (p=0,028), invasão da cápsula tumoral (p=0,030), margem livre (p=0,004) e presença de complicações pós-operatórias (p < 0,001). CONCLUSÕES: Pelos dados dessa amostra, pudemos constatar que não houve diferença em relação ao prognóstico entre os grupos de pacientes das diversas etiologias de CHC. As variáveis nível sérico de alfafetoproteína e de CA 19.9, invasão da cápsula tumoral, margem livre e complicações pósoperatórias podem ser consideradas preditoras de pior prognóstico / INTRODUCTION: Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and its incidence is increasing around the world in the last decades, making HCC the third cause of death by cancer in the world. In about 70 to 80% of patients, HCC is preceded by cirrhosis of the liver. It is believed that hepatic resection is the single proven curative treatment. This resection can be done in the form of a hepatectomy or liver transplantation. Nowadays, only 30 to 40% of HCC patients can benefit from these curative treatments and, among them, survival in five years is still around 60 to 70%, with tumor recurrence rate around 50% in three years. Some studies have shown a worse prognosis for HCC patients whose etiology is viral. That brings us to the question about the existence of a difference between the various etiologies of HCC and its prognosis. OBJECTIVES: To compare the prognosis (overall and disease-free survival at five years) of patients undergoing hepatectomy for the treatment of HCC with respect to various etiologies of liver disease and to study prognostic factors in this group of patients. METHOD: We performed a review of medical records of patients undergoing hepatectomy between 2000 and 2014 for the treatment of HCC, followed by statistical analysis of this database for evaluation of clinical, laboratory and surgical parameters. Patients were divided into groups according to the etiology of liver disease followed by overall and disease-free survival analysis for comparison. RESULTS: There was no statistically significant difference in the outcomes of the groups of patients divided according to the etiology of HCC. Overall and disease-free survival at five years of patients in this sample was 49.9% and 40.7%, respectively. Statistically significant prognostic variables for overall survival were serum alpha-fetoprotein (p = 0.043), serum CA19.9 (p = 0.028), invasion of the tumor capsule (p = 0.030), resection margins (p = 0.004) and presence of postoperative complications (p < 0.001). CONCLUSIONS: From the data of this sample, we could verify that there was no prognostic differences between the groups of HCC patients of the various etiologies. The variables serum alphafetoprotein and CA 19.9, invasion of the tumor capsule, resection margins and presence of postoperative complications can be considered predictive of worse prognosis