Koon Chi Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 158-174). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abbreviations --- p.ii / Abstract --- p.v / Chinese Abstract --- p.viii / Table of Content --- p.x / Chapter Chapter one: --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Classification of Plant Polysaccharides --- p.2 / Chapter 1.2.1 --- Definition of Dietary Fiber --- p.3 / Chapter 1.2.2 --- Types of Soluble Dietary Fiber --- p.3 / Chapter 1.3 --- Physiological Effect of Fiber --- p.6 / Chapter 1.3.1 --- Reduction in Absorption by Viscous Polysaccharides --- p.7 / Chapter 1.3.2 --- Gastric Emptying --- p.7 / Chapter 1.3.3 --- Effect of Viscous Polysaccharides on Intraluminal Mixing --- p.8 / Chapter 1.3.4 --- Effect of Luminal Secretions on Viscosity --- p.9 / Chapter 1.4 --- Physicochemical Qualities and Hypocholesterolemic Effects --- p.9 / Chapter 1.5 --- Gastrointestinal Events and Hypocholesterolemic Effects --- p.11 / Chapter 1.5.1 --- Mouth --- p.11 / Chapter 1.5.2 --- Stomach --- p.12 / Chapter 1.5.3 --- Small intestine --- p.12 / Chapter 1.5.4 --- Large intestine --- p.13 / Chapter 1.6 --- Proposed Mechanisms for Hypocholesterolemic Effects --- p.13 / Chapter 1.6.1 --- Altered Bile Acid Absorption and Metabolism --- p.14 / Chapter 1.6.2 --- Modified Lipid Absorption and Metabolism --- p.15 / Chapter 1.6.3 --- Effects of SCFA on Lipid Metabolism --- p.15 / Chapter 1.6.4 --- Changed Hormone Concentrations --- p.16 / Chapter Chapter Two: --- Materials and Methods --- p.17 / Chapter 2.1 --- Materials --- p.17 / Chapter 2.1.1 --- Fungus --- p.17 / Chapter 2.1.2 --- Animals --- p.17 / Chapter 2.1.2.1 --- Golden Syrian Hamster --- p.17 / Chapter 2.1.2.2 --- Rabbit --- p.18 / Chapter 2.1.3 --- Characterization of Auricularia Polytricha --- p.18 / Chapter 2.1.4 --- Chromatographic materials --- p.22 / Chapter 2.1.5 --- "Determination of Plasma TC，HDL-C, LDL-C，TG，AST and ALT" --- p.24 / Chapter 2.1.6 --- HMG-CoA Reductase Activity Assay --- p.26 / Chapter 2.1.7 --- "Quantitative Determination of Liver Cholesterol, Acidic and Neutral Sterol" --- p.27 / Chapter 2.1.8 --- Animal Diets --- p.29 / Chapter 2.1.8.1 --- Hamster Diets --- p.29 / Chapter 2.1.8.2 --- Rabbit Diets --- p.29 / Chapter 2.2 --- Methods --- p.33 / Chapter 2.2.1. --- Extraction of Water-Soluble AP Polysaccharide (APP) --- p.33 / Chapter 2.2.2. --- Characterization of Auricularia Polytricha --- p.34 / Chapter 2.2.2.1 --- Determination of carbohydrate content of AP Polysaccharide --- p.34 / Chapter 2.2.2.2 --- Determination of uronic acid content of AP Polysaccharide --- p.34 / Chapter 2.2.2.3 --- Determination of protein content of AP Polysaccharide by BCA protein assay --- p.35 / Chapter 2.2.2.4 --- Determination of component sugar units of AP Polysaccharide --- p.35 / Chapter 2.2.2.5 --- Fractionation of AP Polysaccharide --- p.36 / Chapter 2.2.2.6 --- Determination of monosaccharides of AP Polysaccharide by HPLC --- p.37 / Chapter 2.2.3 --- "Determination of plasma TC, HDL-C, LDL-C,TG,AST and ALT" --- p.39 / Chapter 2.2.3.1 --- Plasma Total Cholesterol --- p.39 / Chapter 2.2.3.2 --- Plasma HDL-Cholesterol --- p.40 / Chapter 2.2.3.3 --- Plasma LDL-Cholesterol --- p.40 / Chapter 2.2.3.4 --- Plasma Triglyceride --- p.41 / Chapter 2.2.3.5 --- Plasma Aspartate Aminotransferase --- p.41 / Chapter 2.2.3.6 --- Plasma Alanine Aminotransferase --- p.42 / Chapter 2.2.4 --- HMG-CoA Reductase Activity Assay --- p.42 / Chapter 2.2.4.1 --- Preparation of Hepatic Microsome --- p.42 / Chapter 2.2.4.2 --- HMG-CoA Activity Assay --- p.43 / Chapter 2.2.5 --- Quantitative Determination of Liver Cholesterol --- p.44 / Chapter 2.2.5.1 --- Cholesterol Extraction and its Silylation --- p.44 / Chapter 2.2.5.2 --- GLC Analysis of TMS-Ether Derivative of Cholesterol --- p.45 / Chapter 2.2.6 --- Quantitative Determination of Neutral and Acidic Sterols --- p.45 / Chapter 2.2.6.1 --- Separation of Neutral and Acidic Sterols --- p.45 / Chapter 2.2.6.2 --- Conversion of Neutral Sterols to its TMS-Ether Derivative --- p.46 / Chapter 2.2.6.3 --- Conversion of Acidic Sterols to its TMS-Ether Derivatives --- p.46 / Chapter 2.2.6.4 --- GLC Analysis of Neutral and Acidic Sterols --- p.47 / Chapter 2.2.7 --- Study of Atherosclerosis of Rabbit --- p.48 / Chapter 2.2.7.1 --- Sudan III staining of the thoracic aorta --- p.48 / Chapter 2.2.7.2 --- Measurement of atheroma formation in the aorta --- p.49 / Chapter 2.2.8 --- Animal Experiments --- p.51 / Chapter 2.2.8.1 --- Protective Effect of APP in Hyperlipidemic Study (Exp. 1) --- p.51 / Chapter 2.2.8.2 --- Therapeutic Effect of APP in Hyperlipidemic Study (Exp. 2) --- p.52 / Chapter 2.2.8.3 --- Dose Response of APP in Hyperlipidemic Study (Exp. 3) --- p.52 / Chapter 2.2.8.4 --- Hypolipidemic Effect of Short Chain Fatty Acid (Exp. 4) --- p.53 / Chapter 2.2.8.5 --- Effect of APP and SCFA on HMG-CoA Reductase Activity (Exp5） --- p.53 / Chapter 2.2.8.6 --- Hypolipidemic and Anti-atherosclerotic Effect of APP (Exp. 6) ´Ø… --- p.54 / Chapter 2.3 --- Statistical analysis --- p.54 / Chapter Chapter Three: --- Fractionation and Characterization of Auricularia Polytricha Polysaccharide --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Fungal polysaccharides from Auricularia Polytricha --- p.55 / Chapter 3.3 --- Results --- p.57 / Chapter 3.3.1 --- Extraction and Fractionation of Auricularia Polytricha --- p.57 / Chapter 3.3.2 --- Determination of Carbohydrates Content --- p.58 / Chapter 3.3.3 --- Determination of Protein Content --- p.61 / Chapter 3.3.4 --- Determination of Uronic Acid Content --- p.61 / Chapter 3.3.5 --- Determination of component sugars of AP Polysaccharide --- p.65 / Chapter 3.3.6 --- Fractionation of AP Polysaccharide --- p.67 / Chapter 3.3.7 --- Determination of monosaccharide components of AP Polysaccharide by HPLC --- p.72 / Chapter 3.4 --- Discussion --- p.79 / Chapter Chapter Four: --- "Protective, Therapeutic and Dose Effect of Auricularia Polytricha Polysaccharide (APP) on Hyperlipidemia" --- p.83 / Chapter 4.1 --- Introduction --- p.83 / Chapter 4.2 --- Results (Exp. 1) --- p.86 / Chapter 4.2.1 --- Body Weight and Food Intake --- p.86 / Chapter 4.2.2 --- Effect of APP Supplementation on Hepatic Cholesterol --- p.86 / Chapter 4.2.3 --- "Effect of APP Supplementation on Plasma TC, HDL-C and TG" --- p.87 / Chapter 4.2.4 --- Effect of APP Supplementation on Fecal Output of Neutral Sterols --- p.94 / Chapter 4.2.5 --- Effect of APP Supplementation on Fecal Output of Acidic Sterols --- p.94 / Chapter 4.3 --- Discussion (Exp. 1) --- p.99 / Chapter 4.4 --- Results (Exp. 2) --- p.102 / Chapter 4.4.1 --- Body Weight and Food Intake --- p.102 / Chapter 4.4.2 --- Effect of APP Supplementation on Hepatic Cholesterol --- p.102 / Chapter 4.4.3 --- Effect of APP Supplementation on Plasma TC and TG --- p.103 / Chapter 4.4.4 --- Effect of APP Supplementation on Plasma HDL-C and LDL-C --- p.104 / Chapter 4.5 --- Discussion (Exp. 2) --- p.109 / Chapter 4.6 --- Results (Exp. 3) --- p.111 / Chapter 4.6.1 --- Body Weight and Food Intake --- p.111 / Chapter 4.6.2 --- Dose Response of APP Supplementation on Hepatic Cholesterol --- p.111 / Chapter 4.6.3 --- Dose Response of APP Supplementation on Plasma TG --- p.112 / Chapter 4.6.4 --- Dose Response of APP Supplementation on Plasma HDL-C and LDL-C --- p.112 / Chapter 4.6.5 --- Dose Response of APP Supplementation on ALT and AST Activity --- p.113 / Chapter 4.6.6 --- Dose Response of APP Supplementation on Fecal Output of Neutral and Acidic Sterols --- p.113 / Chapter 4.7 --- Discussion --- p.121 / Chapter Chapter Five: --- Hypolipidemic Effect of Short Chain Fatty Acids --- p.123 / Chapter 5.1 --- "Introduction (Exp. 4,5)" --- p.123 / Chapter 5.2 --- "Results (Exp. 4,5)" --- p.125 / Chapter 5.2.1 --- Body Weight and Food Intake --- p.125 / Chapter 5.2.2 --- Effect of SCFA Supplementation on Hepatic Cholesterol --- p.125 / Chapter 5.2.3 --- "Effect of SCFA Supplementation on Plasma TG, HDL-C and LDL-C" --- p.128 / Chapter 5.2.4 --- Effect of SCFA Supplementation on AST and ALT Activity --- p.128 / Chapter 5.2.5 --- Effect of SCFA supplementation on HMG-CoA Reductase Activity --- p.133 / Chapter 5.3 --- "Discussion (Exp. 4,5)" --- p.135 / Chapter Chapter Six: --- Hypolipidemic and Antiatherosclerotic Effect of APP --- p.137 / Chapter 6.1 --- Introduction (Exp. 6) --- p.137 / Chapter 6.2 --- Results (Exp. 6) --- p.139 / Chapter 6.2.1 --- Body Weight and Food Intake --- p.139 / Chapter 6.2.2 --- Effect of APP Supplementation on Hepatic Cholesterol --- p.139 / Chapter 6.2.3 --- "Effect of APP Supplementation on Plasma TG, HDL- and LDL-C" --- p.141 / Chapter 6.2.3 --- Effect of APP Supplementation on AST and ALT Activity --- p.142 / Chapter 6.2.5 --- Effect of APP supplementation on HMG-CoA Reductase Activity --- p.146 / Chapter 6.2.6 --- Effect of APP supplementation on the Formation of Atheroma --- p.146 / Chapter 6.3 --- Discussion (Exp. 6) --- p.151 / Chapter Chapter Seven: --- General Discussion and Future Perspectives --- p.153 / References --- p.158

Sit Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 135-150). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abbreviations --- p.ii / Abstract --- p.v / Chinese Abstract --- p.vii / Table of Content --- p.ix / Chapter Chapter one: --- General Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Definition of Dietary Fiber --- p.1 / Chapter 1.3 --- Classification of Dietary Fiber --- p.2 / Chapter 1.4 --- Hypocholesterolemic Effects of Soluble Dietary Fibers --- p.3 / Chapter 1.5 --- Proposed Mechanisms for Hypocholesterolemic Effects --- p.4 / Chapter 1.5.1 --- Alter Eating Pattern --- p.4 / Chapter 1.5.2 --- Delay Gastric Emptying --- p.4 / Chapter 1.5.3 --- Modify Lipid Digestion and Absorption --- p.5 / Chapter 1.5.4 --- Effects of SCFA on Lipid Metabolism --- p.6 / Chapter 1.5.5 --- Enhance Bile Acid Excretion --- p.7 / Chapter 1.6 --- Auricularia polytricha --- p.8 / Chapter Chapter Two: --- Chemical Analysis of Auricularia polytrica --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.2 --- Materials and Methods --- p.12 / Chapter 2.2.1 --- Extraction and Fractionation of Auricularia polytricha --- p.12 / Chapter 2.2.2 --- Determination of Carbohydrate Content --- p.12 / Chapter 2.2.3 --- Determination of Protein Content --- p.13 / Chapter 2.2.4 --- Determination of Uronic Acid Content --- p.13 / Chapter 2.2.5 --- Determination of Molecular Weight by Gel Filtration Chromatography --- p.14 / Chapter 2.2.6 --- Determination of Monosaccharide Components by HPLC --- p.15 / Chapter 2.3 --- Results --- p.18 / Chapter 2.3.1 --- Yield of Auricularia polytricha polysaccharides --- p.18 / Chapter 2.3.2 --- Carbohydrate Content of APPs --- p.18 / Chapter 2.3.3 --- Protein Content of APPs --- p.18 / Chapter 2.3.4 --- Uronic Acid Content of APPs --- p.19 / Chapter 2.3.5 --- Molecular Weight of APPs --- p.22 / Chapter 2.3.6 --- Monosaccharide Components of APPs --- p.27 / Chapter 2.4 --- Discussion --- p.33 / Chapter Chapter Three: --- Hypolipidemic Effects of APPs --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials and Methods --- p.38 / Chapter 3.2.1 --- Golden Syrian Hamster --- p.38 / Chapter 3.2.2 --- Animal Experiments --- p.40 / Chapter 3.2.2.1 --- Protective Effect and Dose Response of APPs (Exp. 1) --- p.40 / Chapter 3.2.2.2 --- Therapeutic Effect of APPs (High-cholesterol Diet) (Exp. 2) --- p.40 / Chapter 3.2.2.3 --- Therapeutic Effect of APPII (Normal Diet) (Exp. 3) --- p.41 / Chapter 3.2.2.4 --- Effect of APPs on HMG-CoA Reductase and AC AT Activity (Exp. 4) --- p.42 / Chapter 3.2.3 --- Determination of Plasma AST and ALT --- p.42 / Chapter 3.2.4 --- "Determination of Plasma TC, LDL-C, HDL-C and TG" --- p.43 / Chapter 3.2.5 --- Quantitative Determination of Hepatic and Heart Cholesterol --- p.43 / Chapter 3.2.6 --- Quantitative Determination of Perirenal Adipose Tissue Triglyceride --- p.44 / Chapter 3.2.7 --- Statistical analysis --- p.45 / Chapter 3.3 --- Results (Exp. 1) --- p.47 / Chapter 3.3.1 --- Food Intake and Growth --- p.47 / Chapter 3.3.2 --- Effect of APPs on Plasma AST and ALT --- p.47 / Chapter 3.3.3 --- "Effect of APPs on Plasma TC, LDL-C, HDL-C and TG" --- p.53 / Chapter 3.3.4 --- Effect of APPs on Hepatic and Heart Cholesterol --- p.59 / Chapter 3.4 --- Discussion (Exp. 1) --- p.64 / Chapter 3.5 --- Results (Exp. 2) --- p.67 / Chapter 3.5.1 --- Food Intake and Growth --- p.67 / Chapter 3.5.2 --- Effect of APPs on Plasma AST and ALT --- p.67 / Chapter 3.5.3 --- "Effect of APPs on Plasma TC, LDL-C, HDL-C and TG" --- p.67 / Chapter 3.5.4 --- Effect of APPs on Hepatic and Heart Cholesterol --- p.71 / Chapter 3.6 --- Discussion (Exp. 2) --- p.74 / Chapter 3.7 --- Results (Exp. 3) --- p.76 / Chapter 3.7.1 --- Food Intake and Growth --- p.76 / Chapter 3.3.2 --- Effect of APPII on Plasma AST and ALT --- p.76 / Chapter 3.7.3 --- "Effect of APPII on Plasma TC, LDL-C, HDL-C and TG" --- p.76 / Chapter 3.7.4 --- Effect of APPII on Hepatic and Heart Cholesterol --- p.80 / Chapter 3.8 --- Discussion (Exp. 3) --- p.83 / Chapter Chapter Four: --- Influences of APPs on Cholesterol Homeostasis --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2. --- Materials and Methods --- p.87 / Chapter 4.2.1 --- HMG-CoA Reductase Activity Assay --- p.87 / Chapter 4.2.1.1 --- Preparation of Hepatic Microsome --- p.87 / Chapter 4.2.1.2 --- HMG-CoA Reductase Activity Assay --- p.87 / Chapter 4.2.2 --- ACAT Activity Assay --- p.88 / Chapter 4.2.2.1 --- Preparation of Hepatic and Intestinal Microsome --- p.89 / Chapter 4.2.2.2 --- ACAT Activity Assay --- p.89 / Chapter 4.2.3 --- Quantitative Determination of Neutral and Acidic Sterols --- p.90 / Chapter 4.2.3.1 --- Extraction of Neutral and Acidic Sterols --- p.90 / Chapter 4.2.3.2 --- Conversion of Neutral Sterols to its TMS-Ether Derivative --- p.91 / Chapter 4.2.3.3 --- Conversion of Acidic Sterols to its TMS-Ether Derivatives --- p.91 / Chapter 4.2.3.4 --- GLC Analysis of Neutral and Acidic Sterols --- p.92 / Chapter 4.3 --- Statistic Analysis --- p.93 / Chapter 4.4 --- Results (Exp. 4) --- p.94 / Chapter 4.4.1 --- Effect of APPs on Hepatic HMG-CoA Reductase Activity --- p.94 / Chapter 4.4.2 --- Effect of APPs on Hepatic and Intestinal AC AT Activity --- p.94 / Chapter 4.4.3 --- Effect of APPs on Fecal Excretion (Exp. 1 & 4) --- p.98 / Chapter 4.5 --- Discussion (Exp. 4) --- p.105 / Chapter Chapter Five: --- Hypolipidemic and Antiatherosclerotic Effect of APPII in Rabbit --- p.110 / Chapter 5.1 --- Introduction --- p.110 / Chapter 5.2 --- Materials and Methods --- p.113 / Chapter 5.2.1 --- New Zealant White Rabbit --- p.113 / Chapter 5.2.2 --- Hypolipidemic and Anitatherosclerosis Effect of APPII (Exp. 5) --- p.113 / Chapter 5.2.3 --- Measurement of Atheroma Formation --- p.115 / Chapter 5.3 --- Results (Exp. 5) --- p.117 / Chapter 5.3.1 --- Food Intake and Growth --- p.117 / Chapter 5.3.2 --- Effect of APPII on Plasma AST and ALT --- p.117 / Chapter 5.3.3 --- "Effect of APPII on Plasma TC, LDL-C, HDL-C and TG" --- p.117 / Chapter 5.3.4 --- Effect of APPII on Hepatic and Heart Cholesterol --- p.125 / Chapter 5.3.5 --- Effect of APPII on Perirenal Adipose Tissue Triglycerige Composition --- p.125 / Chapter 5.3.6 --- Effect of APPII on the Formation of Atheroma --- p.125 / Chapter 5.4 --- Discussion (Exp. 5) --- p.130 / Chapter Chapter Six: --- Conclusion --- p.132 / References --- p.135

Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a negative regulator of the low-density lipoprotein receptor, and PCSK9 inhibition has become an important cholesterol-lowering therapeutic strategy. PCSK9 also associates with LDL particles, and evidence suggests that the activity of PCSK9 may be regulated by LDL binding. We have investigated the biochemistry of the interaction between PCSK9 and lipoproteins. Through mutagenesis and in-vitro binding assays, we found conserved motifs in the PCSK9 N-terminus that play a role in LDL binding. Through secondary structure studies using circular dichroism and computational modelling, we determined that the N-terminal region of the PCSK9 prodomain undergoes an environment-dependent structural shift that affects the ability of PCSK9 to bind LDL. We also found that the commonly found loss-of-function polymorphism R46L is capable of modulating this structural shift. Importantly, we found a surface-exposed region of the PCSK9 prodomain that maps a cluster of gain-of-function mutations (L108R, S127R, and D129G) that severely disrupt LDL binding. Through gel shift assays and density gradient centrifugation, we observed that PCSK9 shows remodeling-dependent ability to bind different classes of lipoprotein particles in vitro, binding strongly to LDL and IDL but showing barely detectable association to VLDL. Further, in human plasma, we found that lipoprotein-bound populations of PCSK9 shifted in response to differences in lipoprotein profiles between normolipidemic and hypercholesterolemic or hypertriglyceridemic subjects. Overall, elucidation of how lipoproteins regulate PCSK9 activity will reveal new targets for designing cholesterol-lowering therapeutics.

INTRODUÇÃO: O consumo moderado de vinho tinto está inversamente associado ao desenvolvimento de doença cardiovascular. Efeitos do vinho tinto no sistema nervoso simpático e na reatividade vascular não estão totalmente esclarecidos. MÉTODOS: Foi avaliada a atividade simpática do nervo muscular e a dilatação mediada pelo fluxo na artéria braquial por ultrassom em 10 indivíduos hipercolesterolêmicos, 9 hipertensos e 7 controles antes e após o consumo de vinho tinto durante 15 dias. Medidas hemodinâmicas foram realizadas com Finometer: pressão arterial sistólica, diastólica, freqüência cardíaca, débito cardíaco e resistência vascular sistêmica foram calculados continuamente durante a microneurografia. Para avaliação da atividade simpática, utilizou-se punção de nervo fibular - esta foi medida como bursts/min durante período basal, teste do gelo e exercício estático com 30% da contração voluntária máxima. RESULTADOS: Após 15 dias de vinho tinto, a atividade simpática aumentou de forma significante em hipertensos e hipercolesterolêmicos (p < 0,05); porém, a dilatação mediada pelo fluxo aumentou somente em hipercolesterolêmicos (p < 0,05). As pressões arteriais sistólica e diastólica não apresentaram mudança significante. Não houve mudança na freqüência cardíaca. Houve aumento do débito cardíaco em controles, diminuição da resistência arterial sistêmica durante o gelo em controles no período basal. CONCLUSÃO: O consumo de vinho tinto aumentou a atividade simpática em hipertensos e hipercolesterolêmicos; porém, somente estes experimentaram melhoria da função endotelial, o que sugere diferentes mecanismos na regulação da função endotelial. / Introduction: Moderate red wine intake is inversely associated with development of cardiovascular disease. Red wine effects in sympathetic activity and vascular reactivity are not fully understood. Methods: Muscle sympathetic nerve activity and flow mediated dilatation of brachial artery by ultrasound were evaluated in 10 hypercholesterolemic, 9 hypertensive, and 7 controls subjects before and after red wine intake during 15 days. Hemodynamic measures were done with Finometer: arterial blood pressure, heart rate, cardiac output, and systemic vascular resistance were assessed during mycroneurography. The sympathetic activity was evaluated during baseline, cold test and isometric exercise. Results: After 15 days of red wine intake, sympathetic activity increased significantly in hypertensives and hypercholesterolemics (p < 0.05). On the other hand, flow mediated dilation increased after red wine only in hypercholesterolemics (p < 0.05). Systolic and diastolic blood pressure as well as heart rate did not change significantly. Cardiac output increased in controls and systemic vascular resistance decreased during cold test in controls. Conclusion: There were similar increases in sympathetic activity in hypertensive and hypercholesterolemic subjects; however, endothelial function was restored only in the latter group, which suggests different mechanisms regulating endothelial function.

A isoenzima citocromo P450 3A5 (CYP3A5) tem um papel importante na biotransformação de compostos endógenos e xenobióticos. Polimorfismos de nucleotídeo único (SNPs) do gene CYP3A5 foram associados com variações na expressão gênica e atividade enzimática que podem modificar o metabolismo de vários fármacos. Com a finalidade de investigar a influência de SNPs do CYP3A5 sobre a expressão de RNAm e a resposta a atorvastatina, foram selecionados 99 indivíduos normolipidêmicos (NL) e 139 hipercolesterolêmicos (HC). Os pacientes HC foram tratados com atorvastatina (10 mg/dia/4 semanas). Amostras de sangue periférico foram coletadas antes e após o tratamento para análise do perfil lipídico sérico, e extração de DNA genômico e RNA total. Os SNPs CYP3A5*3C e CYP3A5*6 foram analisados por PCR-RFLP e o CYP3A5*1D por sequenciamento de DNA. A expressão de RNAm do CYP3A5 em células mononucleares do sangue periférico (CMSP) foi analisada por PCR em tempo real. No grupo HC, os alelos CYP3A5*3C e CYP3A5*1D foram mais frequentes em brancos (*3C: 84,9% e *1D 84,8%) que em afro-descendentes (*3C: 47,8% e *1D: 55,2%, p<0,01). Os alelos *3C e *1D estavam em desequilíbrio de ligação. A frequência do alelo CYP3A5*6 foi maior em negros (6,8%) que em brancos (0,6%, p=0,002). Não houve associação entre os SNPs do CYP3A5 e o perfil lipídico basal nos grupos NL e HC. Os HC brancos portadores do haplótipo CYP3A5*3A/*3A (alelos *3C e *1D combinados) apresentaram menor redução de colesterol total e LDL-c após o tratamento com atorvastatina do que os portadores dos haplótipos não-CYP3A5*3A/*3A (p<0,05). A expressão de RNAm de CYP3A5 em CMSP aumentou em resposta a atorvastatina (p<0,05) somente nos portadores de haplótipo CYP3A5*3A/*3A. Esses resultados são sugestivos de que os SNPs CYP3A5*3C e CYP3A5*1D são associados com a expressão gênica do CYP3A5 e a resposta de redução de colesterol a atorvastatina. / The cytochrome P450 isoenzyme 3A5 (CYP3A5) has an important role on biotransformation of endogenous compounds and xenobiotics. Single nucleotide polymorphisms (SNPs) of the CYP3A5 gene have been associated with variations on gene expression and enzyme activity that can modify the metabolism of several drugs. In order to evaluate the influence of CYP3A5 SNPs on RNAm expression and response to atorvastatin, 99 mormolipidemic (NL) and 139 hypercholesterolemic (HC) individuals have been enrolled in this study. HC patients were treated with atorvastatin (10 mg/day/four weeks). Blood samples were collected before and after treatment for serum lipids analyses, genomic DNA and total RNA extraction. CYP3A5*3C, and CYP3A5*6 SNPs were analyzed by PCR-RFLP. CYP3A5*1D was analyzed by direct sequencing. CYP3A5 RNAm expression in peripheral blood mononuclear cells (PBMC) was evaluated by Real Time PCR. In HC group, the frequencies of CYP3A5*3C and CYP3A5*1D alleles were higher in white individuals (*3C: 84.9% and *1D 84.8%) that in blacks (*3C: 47.8% and *1D: 55.2%, p<0.01). CYP3A5*3C and *1D alleles were in linkage desequilibrium. CYP3A5*6 allelle frequency was higher in blacks (6.8%) that in white individuals (0.6%, p=0.002). There was no association between CYP3A5 SNPs and baseline lipid profile in NL and HC groups. White HC carriers of CYP3A5*3A/*3A haplotype (*3C and *1D combined alleles) have lower total cholesterol and LDL-c response to atorvastatin than non-CYP3A5*3A/*3A carriers (p<0.05). CYP3A5 RNAm expression on PBMC increased in response to atorvastatin (p<0.05) only in CYP3A5*3A/*3A haplotype carriers. These results are suggestive that CYP3A5*3C and CYP3A5*1D SNPs are associated with CYP3A5 gene expression and cholesterol-lowering response to atorvastatin.

A hipercolesterolemia familiar (HF) é uma doença autossômica dominante causada por mutações no gene do receptor de LDL (LDLR) (cromossomo 19p13.1 - p13.3), que alteram parcialmente ou totalmente a função do LDLR. A HF é também uma das doenças genéticas mais comuns com freqüências estimadas de heterozigotos e homozigotos de 1/500 e 1/1.000.000, respectivamente. Manifesta-se com altos níveis de LDL colesterol, arco corneal, xantomas tendíneos e sintomas prematuros de doença coronariana.. A grande heterogeneidade observada na manifestação clínica desta doença pode ser explicada, ao menos parcialmente, pelo amplo espectro de mutações no gene do LDLR. O presente estudo teve por objetivo a caracterização molecular do gene LDLR em pacientes com HF do Rio Grande do Sul (RS), Brasil. Para isso, foram obtidas amostras de DNA de 40 indivíduos provenientes de cinco macrorregiões do Estado, representando seis diferentes populações de ascendência européia, para a realização do seqüenciamento direto do gene do LDLR, com posterior análise por meio das ferramentas de bioinformática. Quinze mutações pontuais foram identificadas no gene do LDLR, a saber: c.408C>T (D115D), c.1616C>T (P518L), c.1773C>T (N570N) e c.2243A>G (D727G) na região codificadora, IVS6+36G>A, IVS6+171G>A, IVS11+56C>T, IVS11- 69G>T, IVS11-55A>C, IVS15-136A>G, IVS16+46C>T e IVS17-42A>G na região intrônica, e *52G>A, *105T>G e *141G>A na região 3\'-UTR. Destas, oito ainda não foram descritas na literatura (três situadas nos exons, quatro nos introns e uma na região 3\'-UTR). A mutação*52G>A foi previamente identificada em pacientes com HF da região Sudeste do Brasil, sugerindo que possa exercer um importante efeito na patogênese da HF em pacientes brasileiros. Em relação às macrorregiões do RS, os portugueses, italianos e espanhóis apresentaram o maior número de mutações dentre os grupos étnicos analisados. Assim, os resultados obtidos confirmam que existe um amplo de espectro de mutações no gene do LDLR. As mutações nas regiões intrônicas precisam ser investigadas sobre seu efeito potencial no desenvolvimento de HF. Considerando que este é o primeiro estudo que teve por objetivo a caracterização molecular de pacientes com HF no RS, novos estudos que visem a elucidação das bases moleculares da HF devem ser realizados, a fim de obter uma melhor caracterização genética desta doença no Brasil. / Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene (chromosome 19p13.1 - p13.3), which alter partially or totally the LDLR function. FH is also one of the most common inherited disorders with frequencies of heterozygotes and homozygotes estimated to be 1/500 and 1/1.000.000, respectively. Affected individuals display high levels of LDL cholesterol, arcus corneae, tendon xanthomas and premature symptomatic coronary heart disease. The extensive heterogeneity observed in the clinical manifestation of this disorder may be explained, at least partially, by the broad spectrum of mutations identified in the LDLR gene. The present study had as the main goal the molecular characterization of the LDLR gene in patients with FH from Rio Grande do Sul (RS) State, Brazil. For this, DNA samples were obtained from 40 individuals living in five macroregions of RS, representing six different isolated populations of European ascendancy. The LDLR gene was subjected to the direct sequencing with further analysis through bioinformatics tools. Fifteen punctual mutations were identified in the LDLR gene, namely: c.408C>T (D115D), c.1616C>T (P518L), c.1773C>T (N570N) and c.2243A>G (D727G) in the coding region, IVS6+36G>A, IVS6+171G>A, IVS11+56C>T, IVS11-69G>T, IVS11-55A>C, IVS15-136A>G, IVS16+46C>T and IVS17-42A>G in the intronic region, and *52G>A, *105T>G and *141G>A in the 3\'-UTR region. Of these, eight were not yet described in the literature (three situated in exons, four in introns and one in 3\'- UTR region). The *52G>A mutation was previously identified in FH patients from Southeast Brazil, suggesting that it can exert an important effect in the pathogenesis of FH in Brazilian patients. In relation to the macroregions of Rio Grande do Sul, Portuguese, Italian and Spanish subjects carried the highest number of mutations among the ethnic groups analyzed. Thus, the results obtained confirm the existence of a broad spectrum of mutations in the LDLR gene. The mutations in intronic regions need to be investigated in relation to its potential effect in the development of FH. Taking into account that this is the first study that had as the goal the molecular characterization of FH patients in RS, further studies aimed at elucidating the molecular bases of FH should be performed, in order to obtain the better characterization of this disease in Brazil.

Familial hypercholesterolemia is a genetic disease affecting about 10 million people around the world. Those who carry the disease have a very high risk of developing cardiovascular diseases and commonly encounter myocardial infarction at the early age of 40. Therefore, a diagnosis and immediate treatment are very important for these patients. Despite many combinations of available drugs, there are many patients who still cannot reach the desired cholesterol levels. Mipomersen is a new lipid-lowering drug which inhibits the synthesis of apolipoprotein B, a common component of lipoproteins such as low-density lipoprotein. Inhibition of this protein leads to reduced production of these lipoproteins and reduces the risk of cardiovascular diseases. The drug is currently only indicated for treating patients with homozygous familial hypercholesterolemia.  Unfortunately, there have been many reports of adverse events in patients using mipomersen which has proven problematic.         The aim of this thesis is to analyze the efficacy and safety of mipomersen when treating patients with familial hypercholesterolemia. This has been done by searching for five clinical trials in the database Web of Science. The studies were required to include patients with familial hypercholesterolemia, use mipomersen as the study drug and analyze its effect and safety.   The studies showed that mipomersen has a very good effect in decreasing low-density lipoproteins as well as other lipoproteins in comparison to placebo. Many of the patients who were treated with mipomersen displayed several adverse events and the most common were injection-site reaction and influenza-like symptoms. Elevated levels of aminotransaminase and increased fat deposit in the liver were also common. Based on the five clinical trials analyzed in this thesis, mipomersen is an effective lipid-lowering drug which reduces low density lipoprotein cholesterol, apolipoprotein B and lipoprotein (a) in patients with familial hypercholesterolemia. Elevations in alanine aminotransferase and aspartate aminotransferase are common in patients treated with mipomersen. This could indicate a negative impact on the liver. To be more certain of its safety profile, more research could be needed. There are however, new treatments that combines statins and a proprotein convertase subtilisin/kexin 9 inhibitor, which could be the future of lipid-lowering treatments and mipomersen would then likely be substituted.

Lee Chung-hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 113-133). / Abstracts in English and Chinese. / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- History of soybean --- p.1 / Chapter 1.2 --- Health benefits of soybean --- p.2 / Chapter 1.3 --- Introduction to flavonoids --- p.3 / Chapter 1.4 --- Bioavailability of flavonoids --- p.5 / Chapter 1.5 --- Chemistry of isoflavones --- p.6 / Chapter 1.6 --- Estrogenic property of isoflavones --- p.8 / Chapter 1.7 --- Nutritional significance of isoflavones and their glycosides --- p.8 / Chapter 1.7.1 --- Anticarcinogenic activity --- p.9 / Chapter 1.7.2 --- Antioxidative activity --- p.10 / Chapter 1.7.3 --- Cardioprotective activity --- p.13 / Chapter 1.7.4 --- Osteoprotective activity --- p.14 / Chapter 1.7.5 --- Neuroprotective activity --- p.15 / Chapter 1.7.6 --- Antiangiogenic activity --- p.16 / Chapter Chapter 2 --- Composition of Soybean Isoflavones / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Objective --- p.19 / Chapter 2.3 --- Materials and Methods --- p.20 / Chapter 2.3.1 --- Extraction and isolation --- p.20 / Chapter 2.3.1.1 --- Preparation of soybean butanol extract --- p.20 / Chapter 2.3.1.2 --- Preparation of isoflavones and their glycosides from soybean butanol extract --- p.20 / Chapter 2.3.2 --- HPLC analysis --- p.21 / Chapter 2.3.2.1 --- Sample preparation for the HPLC analysis --- p.21 / Chapter 2.3.2.2 --- HPLC analysis --- p.22 / Chapter 2.3.2.3 --- Quantification of the flavonoids and their glycosides --- p.24 / Chapter 2.4 --- Results --- p.25 / Chapter 2.4.1 --- Structural identification --- p.25 / Chapter 2.4.1.1 --- Compound 1 --- p.25 / Chapter 2.4.1.2 --- Compound 2 --- p.26 / Chapter 2.4.1.3 --- Compound 3 --- p.26 / Chapter 2.4.1.4 --- Compound 4 --- p.27 / Chapter 2.4.1.5 --- Compound 5 --- p.27 / Chapter 2.4.1.6 --- Compound 6 --- p.28 / Chapter 2.4.1.7 --- Compound 7 --- p.28 / Chapter 2.4.1.8 --- Compound 8 --- p.29 / Chapter 2.4.2 --- Quantification of isoflavones in traditional Chinese foods --- p.29 / Chapter 2.5 --- Discussion --- p.32 / Chapter Chapter 3 --- Hypocholesterolemic Effects of Soymilkin Hamsters / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.1.1 --- Lipoproteins and their functions --- p.35 / Chapter 3.1.2 --- Risk factors of cardiovascular disease --- p.36 / Chapter 3.1.3 --- Hamster as an animal model of cholesterol metabolism --- p.38 / Chapter 3.2 --- Objective --- p.39 / Chapter 3.3 --- Materials and Methods --- p.40 / Chapter 3.3.1 --- Preparation of soymilk --- p.40 / Chapter 3.3.2 --- Animals --- p.40 / Chapter 3.3.2.1 --- Experiment one - Hypocholesterolemic effect of soymilk in hamsters --- p.40 / Chapter 3.3.2.1 --- Experiment two 一 The effect of fluid cross-over between soymilk and cow´ة s milk on serum cholesterol in hamsters --- p.41 / Chapter 3.3.3 --- Serum lipid and lipoprotein determinations --- p.42 / Chapter 3.3.4 --- Determination of cholesterol in organs --- p.42 / Chapter 3.3.5 --- Statistics --- p.43 / Chapter 3.4 --- Results --- p.46 / Chapter 3.4.1 --- Experiment one-Hypocholesterolemic effect of soymilk in hamsters --- p.46 / Chapter 3.4.1.1 --- Growth and food intake --- p.46 / Chapter 3.4.1.2 --- "Effect of SM and CM on TG, TC and HDL-C" --- p.46 / Chapter 3.4.1.3 --- Effect of SM and CM on non-HDL-C and ratio of non-HDL-C to HDL-C --- p.46 / Chapter 3.4.1.4 --- Effect of SM and CM on concentration of hepatic cholesterol --- p.47 / Chapter 3.4.1.5 --- "Effect of SM and CM on brain, heart and kidney cholesterol" --- p.47 / Chapter 3.4.2 --- Experiment two - The effect of fluid cross-over between soymilk and cow´ةs milk on serum cholesterol in hamsters --- p.52 / Chapter 3.4.2.1 --- Growth and food intake --- p.52 / Chapter 3.4.2.2 --- Effect of fluid cross-over on serum TC --- p.52 / Chapter 3.5 --- Discussion --- p.55 / Chapter Chapter 4 --- Antioxidant Activities of Soybean Isoflavones and Their Glycosides / Chapter 4.1 --- Introduction --- p.58 / Chapter 4.1.1 --- Role of low density lipoprotein oxidation in the development of atherosclerosis --- p.59 / Chapter 4.1.2 --- LDL oxidation --- p.61 / Chapter 4.1.3 --- Thiobarbituric acid reactive substances (TBARS) as an index of LDL oxidation --- p.62 / Chapter 4.1.4 --- "The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power""" --- p.65 / Chapter 4.1.5 --- "1,1-diphenyl-2-picrylhydrazyl (DPPH) as a measure of free radical scavenging capacity" --- p.65 / Chapter 4.1.6 --- Antioxidant and LDL oxidation --- p.65 / Chapter 4.2 --- Objective --- p.67 / Chapter 4.3 --- Materials and Methods --- p.68 / Chapter 4.3.1 --- Preparation of samples --- p.68 / Chapter 4.3.2 --- Isolation of LDL from human serum --- p.68 / Chapter 4.3.3 --- LDL oxidation --- p.69 / Chapter 4.3.4 --- TBARS assay --- p.69 / Chapter 4.3.5 --- FRAP assay --- p.70 / Chapter 4.3.6 --- DPPH assay --- p.71 / Chapter 4.3.7 --- Statistics --- p.72 / Chapter 4.4 --- Results --- p.73 / Chapter 4.4.1 --- Effects of seven individual soybean isoflavones and their glycosides on LDL oxidation --- p.73 / Chapter 4.4.2 --- The antioxidant power of individual soybean isoflavones and their glycosides in the FRAP assay --- p.73 / Chapter 4.4.3 --- Activity of individual soybean isoflavones and their glycosides as radical scavenging antioxidants --- p.74 / Chapter 4.5 --- Discussion --- p.78 / Chapter Chapter 5 --- Hypocholesterolemic Effects of Soybean Isoflavones in Ovariectomized Golden Syrian Hamsters / Chapter 5.1 --- Introduction --- p.83 / Chapter 5.1.1 --- Coronary heart disease in women --- p.83 / Chapter 5.1.2 --- Menopause as a risk factor in CHD --- p.84 / Chapter 5.1.3 --- Dietary soy in treating postmenopausal hypercholesterolemia --- p.85 / Chapter 5.2 --- Objective --- p.87 / Chapter 5.3 --- Materials and Methods --- p.88 / Chapter 5.3.1 --- Preparation of soymilk --- p.88 / Chapter 5.3.2 --- Preparation of soybean extract --- p.88 / Chapter 5.3.3 --- Animals --- p.89 / Chapter 5.3.4 --- Serum lipid determinations --- p.90 / Chapter 5.3.5 --- Determination of tissue cholesterol content --- p.90 / Chapter 5.3.6 --- Extraction of neutral and acidic sterols from fecal samples --- p.90 / Chapter 5.3.6.1 --- Determination of neutral sterols --- p.91 / Chapter 5.3.6.2 --- Determination of acidic sterols --- p.92 / Chapter 5.3.6.3 --- GLC analysis of neutral and acidic sterols --- p.92 / Chapter 5.3.7 --- Statistics --- p.93 / Chapter 5.4 --- Results --- p.96 / Chapter 5.4.1 --- Growth and food intake --- p.96 / Chapter 5.4.2 --- Effect of ovariectomy on serum TC --- p.96 / Chapter 5.4.3 --- "Effect of soymilk and soybean extract on serum TC,TG and HDL-C" --- p.96 / Chapter 5.4.4 --- Effect of soymilk and soybean extract on non-HDL-C and ratio of non- HDL-C to HDL-C --- p.97 / Chapter 5.4.5 --- Effect of soymilk and soybean extract on concentration of hepatic cholesterol --- p.97 / Chapter 5.4.6 --- Effect of soymilk and soybean extract on heart and kidney cholesterol --- p.97 / Chapter 5.4.7 --- Effect of soymilk and soybean extract on fecal neutral and acidic sterols --- p.103 / Chapter 5.5 --- Discussion --- p.106 / Chapter Chapter 6 --- Conclusion --- p.110 / References --- p.113

Guan, Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 123-145). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABSTRACT --- p.II / LIST OF ABBREVIATIONS --- p.VII / TABLE OF CONTENT --- p.IIX / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Distribution and Origins --- p.1 / Chapter 1.3 --- History of Use --- p.2 / Chapter 1.4 --- Chemical Structure --- p.3 / Chapter 1.5 --- Physiologic Properties --- p.5 / Chapter 1.6 --- Absorption and Metabolism --- p.7 / Chapter 1.6.1 --- Concentration of Isoflavones in Plasma --- p.10 / Chapter 1.6.2 --- Urinary Excretion --- p.10 / Chapter 1.7 --- Healthy Effects --- p.11 / Chapter 1.7.1 --- Menopausal Symptom --- p.11 / Chapter 1.7.2 --- Cardiovascular Disease --- p.12 / Chapter 1.7.3 --- Osteoporosis --- p.13 / Chapter 1.7.4 --- Tumors --- p.14 / Chapter 1.7.4.1 --- Breast Cancer --- p.14 / Chapter 1.7.4.2 --- Prostate Cancer --- p.15 / Chapter 1.7.5 --- Alcohol Addiction --- p.16 / Chapter 1.7.6 --- Potential Adverse Effects --- p.16 / Chapter 1.8 --- Summary --- p.17 / Chapter Chapter 2 --- Determination of Isoflavones in Soybean and Gegen --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.1.1 --- Classification and Structure of Phytoestrogens --- p.19 / Chapter 2.1.2 --- Isoflavones in Soybeans and Gegen --- p.21 / Chapter 2.1.3 --- Methods of Determination --- p.26 / Chapter 2.1.3.1 --- Isolation and Purification of Isoflavones --- p.26 / Chapter 2.1.3.2 --- Analytical Methods for Isoflavones in Soybeans and Gegen --- p.26 / Chapter 2.2 --- Objective --- p.28 / Chapter 2.3 --- Materials and Methods --- p.29 / Chapter 2.3.1 --- Extraction and Isolation of Soybean and Gegen Isoflavone Extracts --- p.29 / Chapter 2.3.2 --- HPLC Analysis --- p.29 / Chapter 2.3.2.1 --- Sample Preparation for the HPLC Analysis --- p.29 / Chapter 2.3.2.2 --- HPLC Analysis --- p.30 / Chapter 2.3.3.3 --- Qualitative Analysis of the Isoflavones and Their Glycosides in Soybean and Gegen --- p.30 / Chapter 2.4 --- Results --- p.31 / Chapter 2.4.1 --- Isoflavone Identification of Soybean Extract --- p.31 / Chapter 2.4.2 --- Isoflavone Identification of Gegen Extract --- p.33 / Chapter 2.5 --- Discussion --- p.35 / Chapter Chapter 3 --- Hypocholesterolemic Effects of Soybean and Gegen Isoflavone Extracts in Ovariectomized，Intact Male and Castrated Golden Syrian Hamsters --- p.38 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Objective --- p.41 / Chapter 3.3 --- Materials and Methods --- p.42 / Chapter 3.3.1 --- Preparation of Soybean and Gegen Isoflavone Extracts --- p.42 / Chapter 3.3.2 --- Animals and Diets --- p.42 / Chapter 3.3.3 --- Serum Lipid and Lipoprotein Determinations --- p.45 / Chapter 3.3.4 --- Determination of Cholesterol Concentration in the Organs --- p.45 / Chapter 3.3.5 --- Statistics --- p.46 / Chapter 3.4 --- Results --- p.48 / Chapter 3.4.1 --- Food Intake and Body and Relative Organ Weights --- p.48 / Chapter 3.4.2 --- Effects of Soybean and Gegen Isoflavone Extracts on Serum and Organ Cholesterol in Ovariectomized and Intact Male and Castrated Hamsters --- p.56 / Chapter 3.5 --- Discussion --- p.61 / Chapter Chapter 4 --- Possible Developmental and Reproductive Toxicity of Soybean Isoflavones on SD Rats --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Objective --- p.70 / Chapter 4.3 --- Materials and Methods --- p.71 / Chapter 4.3.1 --- Diet --- p.71 / Chapter 4.3.2 --- Animals --- p.73 / Chapter 4.3.3 --- Study Design --- p.73 / Chapter 4.3.4 --- Measurement of Reproductive Hormones --- p.74 / Chapter 4.3.5 --- Measurement of Sperm Number --- p.74 / Chapter 4.3.6 --- Statistics --- p.75 / Chapter 4.4 --- Results --- p.77 / Chapter 4.4.1. --- Food Intake and Food Efficiency Ratio --- p.77 / Chapter 4.4.2 --- Growth Trend --- p.79 / Chapter 4.4.3 --- Organ Weight --- p.82 / Chapter 4.4.3.1 --- Absolute Organ Weight --- p.82 / Chapter 4.4.3.2 --- Relative Organ Weight --- p.84 / Chapter 4.4.4 --- Reproductive Hormone Levels --- p.86 / Chapter 4.4.5 --- Epididymis Parameters of Male Rats --- p.88 / Chapter 4.5 --- Discussion --- p.90 / Chapter Chapter 5 --- Possible Developmental and Reproductive Toxicity of Gegen Isoflavones on SD Rats --- p.97 / Chapter 5.1 --- Introduction --- p.97 / Chapter 5.2 --- Objective --- p.99 / Chapter 5.3 --- Materials and Methods --- p.100 / Chapter 5.3.1 --- Animals and Diets --- p.100 / Chapter 5.3.2 --- Study Design --- p.100 / Chapter 5.3.3 --- Statistics --- p.101 / Chapter 5.4 --- Results --- p.103 / Chapter 5.4.1 --- Food Consumption and Food Efficiency Ratio --- p.103 / Chapter 5.4.2 --- Growth Trend --- p.105 / Chapter 5.4.3 --- Organ Weights --- p.108 / Chapter 5.4.3.1 --- Absolute Organ Weights --- p.108 / Chapter 5.4.3.2 --- Relative Organ Weight --- p.110 / Chapter 5.4.4 --- Reproductive Hormone Levels --- p.112 / Chapter 5.4.5 --- Epididymis Parameters of Male Rats --- p.114 / Chapter 5.5 --- Discussion --- p.116 / Chapter Chapter 6 --- Conclusions --- p.121 / References --- p.123

A frequência de Hipercolesterolemia Familial (HF) ainda é desconhecida no Brasil, principalmente pela ausência de estudos com caracterização genotípica associada à fenotípica. Os dados epidemiológicos existentes se baseiam apenas no fenótipos e carecem do diagnóstico molecular confirmatório. O objetivo do presente estudo foi identificar as principais causas genéticas da HF em pacientes diagnosticados fenotipicamente através de um painel exômico com 61 genes a fim de contribuir para um sistema de confirmação do diagnostico molecular em uma amostra da população brasileira. Para isso foram incluídos 141 pacientes, não aparentados, portadores de HF atendidos pelo setor de dislipidemias do Instituto Dante Pazzanese de Cardiologia, Laboratório de Analises Clinicas da Faculdade de Ciências Farmacêuticas da Universidade Federal do Rio Grande do Norte e do Programa Hipercol Brasil do Instituto do Coração. As amostras de sangue periférico foram obtidas para determinações fenotípicas laboratoriais e extração de DNA genômico. A biblioteca de DNA foi construída utilizando o kit Nextera® Rapid Capture Enrichment Custom enriquecendo os éxons de 61 genes que direta ou indiretamente estão relacionados com metabolismo do colesterol. O ultrassequenciamento foi realizado utilizando kit MiSeq Reagent (300 a 500 ciclos) na plataforma MiSeq (Illumina). Os resultados de sequenciamento foram inicialmente alinhados a uma sequência referência e analisados para eliminação de falsos positivos, segundo os parâmetros de qualidade, tais como: cobertura mínima de 30x, frequência do alelo alterado maior que 20% e diferença da distribuição das leituras entre as sequências nucleotídicas menor que 15%. Foram identificadas 472 diferentes variantes em 56 dos genes presentes no painel, sendo 45 consideradas como não descritas. Nos genes APOA1, APOA2, LIPC, RBP4 e TIMP1 não foram observadas variantes dentro dos critérios estabelecidos. Das variantes observadas 25 identificadas em 30 (21,2%) pacientes já tinha sido publicadas em relação à HF nos três principais genes (LDLR, APOB e PCSK9), confirmando o diagnóstico. Foi caracterizado genotipicamente outras dislipidemias primárias em 7 pacientes, sem diagnóstico molecular de HF, através de variantes identificadas no ultrassequenciamento em outros genes. Dos 104 pacientes que não possuíam nenhuma variante já previamente caracterizada, 69 possuíam variantes relacionados com o metabolismo do colesterol. As variantes sem patogenicidade conhecida foram avaliadas através de ferramentas de predição in silico e 22 delas possuíam características sugestivas de patogenicidade em pelo menos 4 das ferramentas utilizadas, duas delas também mostraram alterar a estrutura da proteína segundo análises de docking molecular. Foram identificadas também 223 variantes em região não transcritas (UTR). Quando realizada as análises estatística de todas as variantes identificadas, observamos associação de 13 variantes com concentrações mais elevadas de colesterol da LDL, 5 com concentrações mais elevadas de apolipoproteina B-100, 5 com concentrações mais elevadas de colesterol total, 6 com presença de arco córneo, 2 com manifestação de xantelasmas, 2 com ausência de xantomas e 3 com a presença de doença arterial coronariana. Dessas 6 variantes já haviam sido previamente descritas com HF ou algum outro fenótipo associado e 2 não tinham citação na literatura pesquisada, mas possuíam característica patogênica para a proteína segundo as ferramentas de predição in silico. Este estudo permitiu a identificação das causas genéticas da HF em pacientes brasileiros diagnosticados fenotipicamente, mostrando que a técnica escolhida permitiu caracterizar 21,2% dos pacientes. Além disso, foi possível identificar outras dislipidemias primárias e caracterizar algumas variantes que, apesar de necessitarem serem validadas, indicam uma possível associação com a HF, aumentando o esclarecimento do fenótipo com o genótipo para 74,5%. Este estudo também possibilitou a identificação de novas variantes que devem ser avaliadas para confirmar associação com a doença e utilizar para o diagnóstico propondo um novo painel poligênico. / The frequency of Familial Hypercholesterolemia (FH) is still unknown in Brazil, mainly due to the absence of studies with genotypic characterization associated with phenotype. Existing epidemiological data are based only on the phenotypes and lack the confirmatory molecular diagnosis. The aim of the present study was to identify main genetic causes of FH in patients diagnosed phenotypically through an exomic panel with 61 genes in order to contribute to a system of confirmation molecular diagnosis in a sample of the Brazilian population. To this end, 141 non-related patients with FH treated by the dyslipidemia sector of the Institute Dante Pazzanese of Cardiology, Clinical Analysis Laboratory of the Faculty of Pharmaceutical Sciences of the University Federal of Rio Grande do Norte and the Hipercol Brazil Program of the Heart Institute. Peripheral blood samples were obtained for laboratory phenotypic determinations and extraction of genomic DNA. The DNA library was constructed using the Nextera&#174; Rapid Capture Enrichment Custom kit, enriching with éxons of 61 genes that are directly or indirectly related to cholesterol metabolism. Ultrasequencing was performed using MiSeq Reagent kit (300 to 500 cycles) on the MiSeq platform (Illumina). The sequencing results were initially aligned to a reference sequence and analyzed for false positive elimination according to quality parameters such as: minimum coverage of 30x, altered allele frequency greater than 20%, and difference in the distribution of reads between sequences nucleotides less than 15%. 472 different variants were identified in 56 of the genes present in the panel, of which 45 were considered not described. In the APOA1, APOA2, LIPC, RBP4 and TIMP1 genes no variants were observed within the established criteria. In 25 of the variants observed presents in 30 (21.2%) patients had already been published in relation to FH in the three main genes (LDLR, APOB and PCSK9), confirming the diagnosis. Other primary dyslipidemias were caracterized genotypically in 7 patients, without molecular diagnosis of HF, through variants identified in ultrasequencing in other genes. Of the 104 patients who did not have any previously characterized variant, 69 had variants related to cholesterol metabolism. The variants without known pathogenicity were evaluated using in silico prediction tools and 22 of them had characteristics suggestive of pathogenicity at least 4 of the tools used, two of them also showed to alter the structure of the protein according to molecular docking analyzes. Were also identified 223 non-transcribed region (UTR) variants. Statistical analysis of all the variants identified showed association of 13 variants with higher concentrations of LDL cholesterol, 5 with higher concentrations of apolipoprotein B-100, 5 with higher concentrations of total cholesterol, 6 with presence of an arc corneal, 2 with manifestation of xanthelasms, 2 with absence of xanthomas and 3 with the presence of coronary artery disease. Of these 6 variants had previously been described with HF or some other associated phenotype and 2 had no citation in the researched literature, but had a pathogenic characteristic for the protein according to in silico prediction tools. This study allowed the identification of the genetic causes of FH in Brazilian patients diagnosed phenotypically, showing that the technique chosen allowed to characterize 21.2% of the patients. In addition, it was possible to identify other primary dyslipidemias and to characterize some variants that, although they need to be validated, indicate a possible association with HF, increasing the clarification of the phenotype with the genotype to 74.5%. This study also allowed the identification of new variants that should be evaluated to confirm association with the disease and to use for the diagnosis proposing a new polygenic panel.