Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 141 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Grace S.N. Lau. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 335-362). / List of Abbreviations --- p.i / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction and Study Objectives / Chapter 1.1 --- Metabolic activation - role in drug toxicity and carcinogenesis --- p.5 / Chapter 1.2 --- Hepatocellular carcinoma --- p.12 / Chapter 1.2.1 --- Epidemiology --- p.12 / Chapter 1.2.2 --- Aetiological factors --- p.17 / Chapter 1.2.2.1 --- Hepatitis B virus infection --- p.17 / Chapter 1.2.2.2 --- Cirrhosis --- p.24 / Chapter 1.2.2.3 --- Aflatoxins --- p.25 / Chapter 1.2.2.4 --- Other factors --- p.26 / Chapter 1.2.2.5 --- Summary --- p.29 / Chapter 1.3 --- Study objectives --- p.30 / Chapter Chapter 2 --- The Metabolism of Paracetamol in Healthy Subjects andin Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1. --- History of paracetamol --- p.34 / Chapter 2.1.2 --- Pharmacology of paracetamol --- p.37 / Chapter 2.1.3 --- "Absorption, Distribution, Metabolism and Excretion" --- p.38 / Chapter 2.1.3.1 --- Absorption --- p.38 / Chapter 2.1.3.2 --- Distribution --- p.41 / Chapter 2.1.3.3 --- Metabolism --- p.42 / Chapter 2.1.3.4 --- Excretion --- p.57 / Chapter 2.1.4 --- Toxicity and Overdosage --- p.59 / Chapter 2.2 --- Estimation of paracetamol and its metabolites in plasma and urine by high performance liquid chromatography --- p.72 / Chapter 2.2.1 --- Introduction --- p.72 / Chapter 2.2.2 --- Analytical method --- p.76 / Chapter 2.2.2.1 --- Materials --- p.76 / Chapter 2.2.2.2 --- Instrumentation --- p.77 / Chapter 2.2.2.3 --- Collection and storage of samples --- p.79 / Chapter 2.2.2.4 --- Chromatographic conditions --- p.79 / Chapter 2.2.3 --- Urine assay --- p.79 / Chapter 2.2.3.1 --- Preparation of standards and test samples for urine assay --- p.79 / Chapter 2.2.3.2 --- Calculation of results for urine assay --- p.80 / Chapter 2.2.3.3 --- Results of urine assay --- p.81 / Chapter 2.2.3.4 --- Validation of urine assay --- p.81 / Chapter 2.2.4 --- Plasma assay --- p.83 / Chapter 2.2.4.1 --- Preparation of standards and test samples for plasma assay --- p.83 / Chapter 2.2.4.2 --- Calculation of results for plasma assay --- p.91 / Chapter 2.2.4.3 --- Results of plasma assay --- p.91 / Chapter 2.2.4.4 --- Validation of plasma assay --- p.93 / Chapter 2.2.5 --- Summary --- p.99 / Chapter 2.3 --- The pharmacokinetics of paracetamol in healthy subjects --- p.103 / Chapter 2.3.1 --- Introduction --- p.103 / Chapter 2.3.2 --- Study protocol --- p.103 / Chapter 2.3.3 --- Methods --- p.103 / Chapter 2.3.3.1 --- Subjects --- p.103 / Chapter 2.3.3.2 --- Drug administration and sampling --- p.104 / Chapter 2.3.3.3 --- Drug analysis --- p.108 / Chapter 2.3.3.4 --- Calculations --- p.108 / Chapter 2.3.4 --- Pharmacokinetic analysis --- p.109 / Chapter 2.3.5 --- Statistical analysis --- p.113 / Chapter 2.3.6 --- Results --- p.114 / Chapter 2.3.6.1 --- Plasma Results --- p.114 / Chapter 2.3.6.2 --- Urine Results --- p.118 / Chapter 2.3.6.3 --- Pharmacokinetic Results --- p.125 / Chapter 2.3.6.4 --- Statistical Results --- p.134 / Chapter 2.3.7 --- Discussion --- p.145 / Chapter 2.4 --- "The pharmacokinetics of paracetamol in healthy subjects, patients with liver disease and hepatocellular carcinoma" --- p.155 / Chapter 2.4.1 --- Introduction --- p.155 / Chapter 2.4.2 --- Study protocol --- p.156 / Chapter 2.4.3 --- Methods --- p.156 / Chapter 2.4.3.1 --- Subjects --- p.156 / Chapter 2.4.3.2 --- Drug administration and sampling --- p.157 / Chapter 2.4.3.3 --- Drug analysis --- p.160 / Chapter 2.4.3.4 --- Calculations --- p.160 / Chapter 2.4.4 --- Pharmacokinetic analysis --- p.161 / Chapter 2.4.6 --- Results --- p.162 / Chapter 2.4.6.1 --- Plasma Results --- p.162 / Chapter 2.4.6.2 --- Urine Results --- p.162 / Chapter 2.4.6.3 --- Pharmacokinetic Results --- p.179 / Chapter 2.4.7 --- Discussion --- p.194 / Chapter 2.4.8 --- Summary --- p.203 / Chapter Chapter 3 --- Metabolic Activation of Aflatoxin B1 in Healthy Subjects and in Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 3.1 --- General introduction --- p.206 / Chapter 3.1.1 --- Chemical structures and properties --- p.207 / Chapter 3.1.2 --- Contamination of food by aflatoxins --- p.209 / Chapter 3.1.3 --- Metabolism of aflatoxins --- p.210 / Chapter 3.1.4 --- Human diseases possibly related to exposure to aflatoxins --- p.226 / Chapter 3.1.4.1 --- Acute aflatoxicosis --- p.226 / Chapter 3.1.4.2 --- Reye's syndrome --- p.227 / Chapter 3.1.4.3 --- Kwashiorkor --- p.228 / Chapter 3.1.4.4 --- Impaired immune function --- p.229 / Chapter 3.1.4.5 --- Hepatocellular carcinoma --- p.230 / Chapter 3.1.5 --- Biochemical and molecular epidemiology of aflatoxins --- p.232 / Chapter 3.2 --- Development of an ELISA method to monitor AFB1 exposure in human serum --- p.237 / Chapter 3.2.1 --- Introduction --- p.237 / Chapter 3.2.2 --- Preparation of all the components necessary for analysing AFB1-albumin adducts by ELISA --- p.243 / Chapter 3.2.2.1 --- Materials --- p.243 / Chapter 3.2.2.2 --- Preparation of rabbit AFB1 antiserum --- p.244 / Chapter 3.2.2.3 --- Preparation of the rat monoclonal antibody --- p.244 / Chapter 3.2.2.4 --- Concentration of cell culture supernatant by ammonium sulphate precipitation --- p.246 / Chapter 3.2.2.5 --- Preparation of the BSA-AFB1 conjugate --- p.248 / Chapter 3.2.2.6 --- Preparation of the immunoaffinity gel --- p.250 / Chapter 3.2.2.7 --- Preparation of the ELISA plates --- p.251 / Chapter 3.2.3 --- General procedures used in the analysis of AFB1- albumin adducts --- p.252 / Chapter 3.2.3.1 --- Competitive ELISA binding assay --- p.253 / Chapter 3.2.3.2 --- Sep-pak C18 cartridge --- p.254 / Chapter 3.2.3.3 --- Immunoaffinity column --- p.255 / Chapter 3.2.3.4 --- Evaporation process --- p.255 / Chapter 3.2.3.5 --- HPLC --- p.256 / Chapter 3.2.3.6 --- Radioactive counting --- p.256 / Chapter 3.2.3.7 --- Albumin isolation --- p.257 / Chapter 3.2.3.8 --- Digestion of albumin --- p.257 / Chapter 3.2.3.9 --- Animal procedures --- p.258 / Chapter 3.2.4 --- Validations --- p.259 / Chapter 3.2.4.1 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.259 / Chapter 3 2.4.2 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.259 / Chapter 3 2.4.3 --- Elution characteristics and capacity of the immunoaffinity column --- p.261 / Chapter 3.2.4.4 --- Comparison of immunoaffinity gels prepared with different affinity gels --- p.261 / Chapter 3.2.4.5 --- Immunoaffinity column experiment of AFB1-lysine --- p.263 / Chapter 3.2.4.6 --- HPLC Analysis of fractions from immunoaffinity column --- p.263 / Chapter 3.2.4.8 --- HPLC analysis of fractions from Sep- Pak C18 cartridge --- p.264 / Chapter 3.2.4.9 --- Digestion of serum albumin by proteinase K --- p.264 / Chapter 3.2.4.10 --- Effect of ethanol in samples to be loaded onto Sep-Pak C18 cartridge --- p.265 / Chapter 3.2.4.11 --- Effect of drying in a vacuum concentrator on recovery of radioactivity of 3H-AFB1 --- p.266 / Chapter 3.2.4.12 --- Evaluation of the overall procedure for the analysis of serum albumin adducts of AFB1 --- p.267 / Chapter 3.2.4.13 --- HPLC analysis of samples obtained after digestion and all clean-up procedures --- p.268 / Chapter 3.2.5 --- Results and discussion --- p.268 / Chapter 3.2.5.1 --- BSA-AFB1 conjugate --- p.268 / Chapter 3.2.5.2 --- Treatment of experimental animals with 3H-AFB1 --- p.270 / Chapter 3.2.5.3 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.272 / Chapter 3.2.5.4 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.275 / Chapter 3.2.5.5 --- Sep-Pak C18 cartridge - elution characteristics and capacity --- p.279 / Chapter 3.2.5.6 --- Elution characteristics of immunoaffinity columns --- p.282 / Chapter 3.2.5.7 --- Immunoaffinity column experiment of AFB1-lysine --- p.290 / Chapter 3.2.5.8 --- Digestion of serum albumin by proteinase K --- p.295 / Chapter 3.2.5.9 --- Effect of ethanol in samples to be applied onto Sep-Pak C18 cartridges --- p.297 / Chapter 3.2.5.10 --- Recovery of radioactivity after dryingin a vacuum concentrator --- p.300 / Chapter 3.2.5.11 --- Recovery of the overall clean-up procedure for the analysis of serum albumin adducts of AFB1 --- p.300 / Chapter 3.2.5.12 --- HPLC analysis of samples obtained after all clean-up procedures --- p.305 / Chapter 3.2.5.13 --- The use of rabbit anti-AFB1 anti-serum and rat anti-AFB1 monoclonal antibody --- p.308 / Chapter 3.2.6 --- Summary --- p.309 / Chapter 3.3 --- Monitoring of AFBralbumin adducts in plasma of patients with liver disease and hepatocellular carcinoma --- p.311 / Chapter 3.3.1 --- Introduction --- p.311 / Chapter 3.3.2 --- Material and methods --- p.314 / Chapter 3.3.2.1 --- Subject --- p.314 / Chapter 3.3.2.2 --- Sample collections --- p.315 / Chapter 3.3.2.4 --- Assay for AFB1-albumin adducts --- p.315 / Chapter 3.3.2.5 --- Statistical analysis --- p.318 / Chapter 3.3.3 --- Results and discussion --- p.318 / Chapter Chapter 4 --- Summary and Ideas for Further Studies --- p.330 / Acknowledgements --- p.333 / References --- p.335 / Appendices --- p.364

Cheung, Tsz Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 97-116). / Abstracts in English and Chinese. / Thesis/Assessment Committee Members --- p.ii / Declaration for Plagiarism and Copyright --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.viii / Table of Contents --- p.ix / List of Abbreviations --- p.xii / List of Figures --- p.xv / List of Tables --- p.xvi / Chapter CHAPTER TWO: --- Introduction / Chapter 1.1 --- Background Information / Chapter 1.1.1 --- Breast Cancer --- p.1 / Chapter 1.1.2 --- General Statistics of Breast Cancer Worldwide and in Hong Kong --- p.1 / Chapter 1.1.3 --- Risk Factors for Breast Cancer --- p.2 / Chapter 1.1.4 --- Breast Cancer Treatment and Side Effects --- p.2 / Chapter 1.1.5 --- Types of Breast Cancer --- p.3 / Chapter 1.2 --- Estrogen and Estrogen Receptor / Chapter 1.2.1 --- Estrogen --- p.4 / Chapter 1.2.2 --- Estrogen Receptor --- p.5 / Chapter 1.2.3 --- Estrogen Receptor mediated Gene Transcription --- p.5 / Chapter 1.2.4 --- Estrogen Receptor Alpha and Estrogen Receptor Beta --- p.6 / Chapter 1.2.5 --- Estrogen Receptor Positive Breast Cancer and Treatment --- p.7 / Chapter 1.3 --- Estrogen metabolism and Cytochrome P450 family 1 (CYP1) members / Chapter 1.3.1 --- Estrogen Metabolism in Human --- p.9 / Chapter 1.3.2 --- CYP1A1 and CYP1B1 --- p.9 / Chapter 1.3.3 --- Estrogen Metabolism in Breast --- p.10 / Chapter 1.3.4 --- Carcinogenesis of Estrogens and Estrogen Metabolites --- p.13 / Chapter 1.3.5 --- The Importance of CYP1B1 in Carcinogenesis --- p.15 / Chapter 1.4 --- Aryl Hydrocarbon Receptor / Chapter 1.4.1 --- General Information of Aryl Hydrocarbon Receptor --- p.16 / Chapter 1.4.2 --- Signaling/Regulation Pathways of Aryl Hydrocarbon Receptor --- p.17 / Chapter 1.4.3 --- Crosstalk with Estrogen Receptor --- p.17 / Chapter 1.5 --- Introduction of Herba Scutellaria Barbata and its active ingredient Pheophorbide a --- p.19 / Chapter 1.6 --- Hyposthesis and Objectives --- p.21 / Chapter CHAPTER TWO: --- Direct Cytotoxic/Cytostatic Effect of Pheophorbide a / Chapter 2.1 --- Backgrounds --- p.22 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Chemicals --- p.24 / Chapter 2.2.2 --- Cell Lines --- p.26 / Chapter 2.2.3 --- "Cell Culture Mediums, Buffers and Consumables" / Chapter 2.2.3.1 --- Roswell Park Memorial Institute Tissue Culture Medium1640 (RPMI1640) --- p.26 / Chapter 2.2.3.2 --- RPMI 1640 (Phenol Red-free) --- p.26 / Chapter 2.2.3.3 --- Serum supplement - Fetal Bovine Serum (FBS) --- p.27 / Chapter 2.2.3.4 --- Serum supplement - Charcoal/Dextran Stripped FBS --- p.27 / Chapter 2.2.3.5 --- Antibiotics - Penicillin-Streptomycin (P/S) --- p.27 / Chapter 2.2.3.6 --- Trypsin (0.25%) with EDTA --- p.27 / Chapter 2.2.3.7 --- Trypsin (2.5%) (Phenol Red-free) with EDTA --- p.28 / Chapter 2.2.3.8 --- Dulbeccóةs Phosphate-Buffered Saline (D-PBS) --- p.28 / Chapter 2.2.3.9 --- Tissue Culture Flasks and Multi-well Plate --- p.28 / Chapter 2.2.3.10 --- Trypan Blue Solution --- p.29 / Chapter 2.2.4 --- Reagents for Direct Cytotoxity Test / Chapter 2.2.4.1 --- MTT Assay --- p.29 / Chapter 2.2.4.2 --- Tritiated Thymidine Incorporation Assay --- p.29 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Cell Culture --- p.30 / Chapter 2.3.2 --- Direct Cytotoxicity/Cytostatic Test / Chapter 2.3.2.1 --- MTT Assay --- p.31 / Chapter 2.3.2.2 --- Tritiated Thymidine Incorporation Assay --- p.32 / Chapter 2.3.3 --- Statistical Analysis --- p.32 / Chapter 2.4 --- Results / Chapter 2.4.1 --- The Cytotoxic Effect of Pheophorbide a --- p.34 / Chapter 2.4.2 --- The Combine Effect of Pheophorbide a with 17-β Estradiol and Tamoxifen Citrate --- p.34 / Chapter 2.5 --- Discussions --- p.48 / Chapter CHAPTER THREE: --- Mechanistic Study of Pheophorbide a / Chapter 3.1 --- Backgrounds --- p.53 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Real time PCR / Chapter 3.2.1.1 --- General Chemicals and Equipments --- p.54 / Chapter 3.2.1.2 --- RNA isolation --- p.55 / Chapter 3.2.1.3 --- Reverse Transcription --- p.55 / Chapter 3.2.1.4 --- Real Time PCR --- p.56 / Chapter 3.2.2 --- Western Blotting / Chapter 3.2.2.1 --- Microsome Isolation --- p.58 / Chapter 3.2.2.2 --- Measurement of Protein Concentration --- p.58 / Chapter 3.2.2.3 --- Western Blotting --- p.58 / Chapter 3.2.3 --- Estrogen Metabolism Assay / Chapter 3.2.3.1 --- Chemicals --- p.59 / Chapter 3.2.3.2 --- Estrogen Metabolites Extraction --- p.60 / Chapter 3.2.3.3 --- Liquid Chromatography/Mass Spectrometry --- p.60 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Real time PCR / Chapter 3.3.1.1 --- Cell Culture --- p.61 / Chapter 3.3.1.2 --- RNA Isolation and Reverse Transcription --- p.61 / Chapter 3.3.1.3 --- Real Time PCR --- p.62 / Chapter 3.3.2 --- Western Blotting / Chapter 3.3.2.1 --- Cell Culture --- p.63 / Chapter 3.3.2.2 --- Microsome Isolation --- p.63 / Chapter 3.3.2.3 --- Measurement of Protein Concentration --- p.64 / Chapter 3.3.2.4 --- Western Blotting --- p.64 / Chapter 3.3.3 --- Estrogen Metabolism Assay / Chapter 3.3.3.1 --- Preparation of Calibration Standard --- p.65 / Chapter 3.3.3.2 --- Cell Culture --- p.66 / Chapter 3.3.3.3 --- Estrogen Metabolites Extraction --- p.66 / Chapter 3.3.3.4 --- Liquid Chromatography/Mass Spectrometry --- p.67 / Chapter 3.3.4 --- Statistical Analysis --- p.68 / Chapter 3.4 --- Results --- p.69 / Chapter 3.5 --- Discussions --- p.80 / Chapter CHAPTER FOUR: --- Overall Conclusion and Future Directions / Chapter 4.1 --- Significance of the Study --- p.87 / Chapter 4.2 --- Overall Conclusion --- p.87 / Chapter 4.3 --- Limitation and Difficulties of the Study --- p.89 / Chapter 4.4 --- Future Directions --- p.89 / Appendices / "Appendix I The Melting Curve of real time PCR for β-actin, CYP1A1 and CYP1B1" --- p.92 / Appendix II The Calibration Curve of BSA for Protein Concentration Measurement --- p.93 / Appendix III The Representative Peak of Estradiol Metabolite Standards with corresponding Retention Time --- p.94 / Appendix IV The Calibration Curve of Different Estrogen Metabolites for LC/MS --- p.95 / Appendix V The Accuracy and Precision of Quality Control of Estradiol Metabolites --- p.96 / Bibliography --- p.97

Lui, Ming Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 117-129). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.III / Abstract in Chinese --- p.V / List of Abbreviations --- p.VII / List of Figures --- p.X / List of Tables --- p.XII / Table of Contents --- p.XIII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Overview on type 2 diabetes --- p.1 / Chapter 1.1.1 --- Definition of diabetes --- p.1 / Chapter 1.1.2 --- Diagnostic criteria --- p.2 / Chapter 1.1.3 --- Prevalence and societal impact --- p.2 / Chapter 1.1.4 --- Risks factors for type 2 diabetes --- p.4 / Chapter 1.1.4.1 --- Metabolic syndrome --- p.4 / Chapter 1.1.4.2 --- Genetics of type 2 diabetes --- p.6 / Chapter 1.1.4.3 --- "Environmental risk factors, lifestyle and energy imbalance" --- p.8 / Chapter 1.1.5 --- Pathophysiology of type 2 diabetes --- p.9 / Chapter 1.1.5.1 --- Insulin secretion and signaling --- p.9 / Chapter 1.1.5.1.1 --- Insulin Secretion --- p.9 / Chapter 1.1.5.1.2 --- Insulin signaling --- p.11 / Chapter 1.1.5.2 --- Natural history of type 2 diabetes --- p.12 / Chapter 1.1.5.3 --- Insulin resistance --- p.13 / Chapter 1.1.5.4 --- Impairment in insulin secretion --- p.15 / Chapter 1.1.5.5 --- Endocannabinoid system: A new target for energy balance and metabolism --- p.16 / Chapter 1.1.5.6 --- Effects of diabetes mellitus and its complications --- p.16 / Chapter 1.2 --- Biology of prostacyclin synthase (PTGIS) --- p.18 / Chapter 1.2.1 --- Molecular information of PTGIS --- p.18 / Chapter 1.2.2 --- Transcriptional control of PTGIS --- p.19 / Chapter 1.2.3 --- Protein structure of PGIS --- p.21 / Chapter 1.2.4 --- Sub-cellular localization and tissue distribution --- p.22 / Chapter 1.2.5 --- Function of PGIS --- p.25 / Chapter 1.2.5.1 --- Function of PGI2 in blood vessels --- p.26 / Chapter 1.2.5.2 --- Role of PGh in embryo development --- p.26 / Chapter 1.2.5.3 --- Role of PGI2 in apoptosis --- p.27 / Chapter 1.2.5.4 --- Targeted knock-out mice phenotype --- p.27 / Chapter 1.2.6 --- Relationship between PTGIS and diseases --- p.28 / Chapter 1.2.6.1 --- Genetic association --- p.28 / Chapter 1.2.6.2 --- Inactivation and tyrosine nitration of PGIS by peroxynitrite --- p.29 / Chapter 1.3 --- Biology of peroxisome proliferator-activated receptor delta (PPARD) --- p.30 / Chapter 1.3.1 --- Molecular information of PPARD --- p.30 / Chapter 1.3.2 --- Transcriptional control of PPARD --- p.31 / Chapter 1.3.3 --- Translational control and protein structure --- p.32 / Chapter 1.3.4 --- Sub-cellular localization and tissue expression --- p.35 / Chapter 1.3.5 --- Function of PPARδ --- p.37 / Chapter 1.3.5.1 --- Mechanisms of action --- p.37 / Chapter 1.3.5.2 --- Ligands for PPARδ --- p.38 / Chapter 1.3.5.3 --- PPARδ in lipoprotein metabolism --- p.39 / Chapter 1.3.5.4 --- PPARδ action in adipose tissue --- p.39 / Chapter 1.3.5.5 --- PPARδ action in skeletal and cardiac muscle --- p.40 / Chapter 1.3.5.6 --- PPARδ action in liver --- p.42 / Chapter 1.3.5.7 --- PPARδ and endocannabinoid system --- p.42 / Chapter 1.3.5.8 --- PPARδ action in inflammation --- p.43 / Chapter 1.3.5.9 --- Targeted knock-out mice phenotype --- p.44 / Chapter 1.3.5.10 --- Disease association --- p.44 / Chapter 1.4 --- Functional relationship of PGIS and PPARδ: possible role in energy metabolism --- p.46 / Chapter 1.5 --- Methods for studying genetics of type 2 diabetes and linkage analysis results --- p.47 / Chapter 1.5.1 --- Genome-wide scan --- p.47 / Chapter 1.5.2 --- Candidate gene approach --- p.48 / Chapter 1.6 --- Hypothesis and objectives --- p.49 / Chapter 1.7 --- Long-term significance --- p.49 / Chapter Chapter 2: --- Association Study of Prostacyclin Synthase and Peroxisome Proliferator-Activated Receptor Delta Gene Polymorphisms with Type2 Diabetes and Related Metabolic Traits / Chapter 2.1 --- Introduction and research design --- p.50 / Chapter 2.2 --- Study population --- p.52 / Chapter 2.2.1 --- Ethics approval --- p.52 / Chapter 2.2.2 --- Subjects --- p.52 / Chapter 2.2.3 --- Clinical assessments --- p.52 / Chapter 2.3 --- Materials and methods --- p.55 / Chapter 2.3.1 --- DNA samples --- p.55 / Chapter 2.3.2 --- Marker selection --- p.55 / Chapter 2.3.3 --- Genotyping --- p.57 / Chapter 2.3.4 --- Statistical analysis --- p.59 / Chapter 2.4 --- Results and Discussion --- p.60 / Chapter 2.4.1 --- Clinical characteristics of the study population --- p.60 / Chapter 2.4.2 --- Genotyping and LD analysis --- p.60 / Chapter 2.4.3 --- Association with type 2 diabetes and related metabolic traits --- p.61 / Chapter 2.4.3.1 --- Single SNP association with type 2 diabetes --- p.61 / Chapter 2.4.3.2 --- Single SNP association with metabolic traits --- p.64 / Chapter 2.4.3.3 --- Gene-gene interaction on type 2 diabetes --- p.74 / Chapter 2.4.3.4 --- Gene-gene interaction on metabolic traits --- p.74 / Chapter 2.5 --- Limitation and improvement --- p.79 / Chapter 2.6 --- Conclusions --- p.79 / Chapter Chapter 3: --- Functional Studies of Prostacyclin Synthase rs508757-A/G Intronic Polymorphism / Chapter 3.1 --- Introduction and research design --- p.80 / Chapter 3.2 --- Materials and methods --- p.81 / Chapter 3.2.1 --- Bioinformatics --- p.81 / Chapter 3.2.1.1 --- Cross-species alignment --- p.81 / Chapter 3.2.1.2 --- BLAST search and open reading frame prediction --- p.81 / Chapter 3.2.1.3 --- Transcription factor binding sites prediction --- p.82 / Chapter 3.2.2 --- PCR amplification from cDNA --- p.82 / Chapter 3.2.3 --- Culture of mammalian cell --- p.83 / Chapter 3.2.3.1 --- Cell line --- p.83 / Chapter 3.2.3.2 --- Medium and supplement --- p.83 / Chapter 3.2.3.3 --- Cell culture wares --- p.83 / Chapter 3.2.3.4 --- Cell culture conditions --- p.84 / Chapter 3.2.4 --- Construction of reporter vectors with rs508757 flanking sequence --- p.84 / Chapter 3.2.4.1 --- Cloning and vector preparation --- p.84 / Chapter 3.2.4.2 --- Site-directed mutagenesis --- p.84 / Chapter 3.2.5 --- Dual-luciferase reporter assay --- p.85 / Chapter 3.2.5.1 --- Transfection of VSMC --- p.85 / Chapter 3.2.5.2 --- Cell lysis and luminescence measurement --- p.86 / Chapter 3.2.6 --- Circular Dichroism --- p.87 / Chapter 3.2.6.1 --- Introduction to DNA quardruplex structure and circular dichroism --- p.87 / Chapter 3.2.6.1.1 --- DNA quardruplex --- p.87 / Chapter 3.2.6.1.2 --- Circular dichroism --- p.88 / Chapter 3.2.6.2 --- Circular dichroism measurement --- p.89 / Chapter 3.2.6.2.1 --- DNA samples --- p.89 / Chapter 3.2.6.2.2 --- CD spectroscopy --- p.89 / Chapter 3.2.7 --- Statistical analysis --- p.90 / Chapter 3.3 --- Results and Discussion --- p.91 / Chapter 3.3.1 --- Cross-species alignment --- p.91 / Chapter 3.3.2 --- BLAST search and ORF prediction --- p.92 / Chapter 3.3.3 --- PCR results on testing the presence of a new transcript --- p.93 / Chapter 3.3.4 --- Effect of rs508757 flanking sequence on SV40 promoter activity --- p.94 / Chapter 3.3.5 --- Circular dichroism experiment on rs508757 flanking sequence --- p.96 / Chapter 3.3.6 --- DNA slipping model --- p.98 / Chapter 3.3.7 --- Transcription factor binding site prediction --- p.99 / Chapter 3.4 --- Limitation and improvement --- p.107 / Chapter 3.5 --- Conclusions --- p.107 / Chapter Chapter 4: --- "General Discussion, Conclusion and Future Perspectives" / Chapter 4.1 --- General discussion --- p.108 / Chapter 4.2 --- Future perspectives --- p.115 / Chapter 4.2.1 --- "Association on type 2 diabetes and molecular interaction between transcription factors, PTGIS and PPARD" --- p.115 / Chapter 4.2.2 --- Association with diabetic nephropathy --- p.115 / Chapter 4.2.3 --- Study tissue or cell type specific actions of PGIS and PPARδ --- p.116 / Chapter 4.3 --- Conclusions to my project --- p.116 / Chapter Chapter 5: --- Bibliography --- p.117 / Appendix --- p.130

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 83-100).

INTRODUÇÃO. A Imunodeficiencia comum variável (ICV) é uma doença heterogênea caracterizada pela redução dos niveis de IgG, IgA e/ou IgM e da função de anticorpo. As manifestações clínicas incluem a presença de infecções recorrentes ou crônicas, doenças inflamatórias/autoimunes e incidência aumentada de malignidades como linfomas e carcinomas, caracterizando-se, consequentemente, por um estado de ativação imune persistente e alterações do metabolismo oxidativo. Tanto a paraoxonase 1(PON1) quanto o polimorfismo do citocromo p450 (CYP) 2E1 têm importante participação nos processos oxidativos, controlando a extensão dos danos causados por alterações na concentração de oxidantes. Acredita-se que, tal qual ocorre na população normal, tanto a PON1 quanto a CYP2E1 tenham importante papel na gravidade e na sobrevida dos pacientes com ICV. OBJETIVO: estudar os polimorfismos de PON1 e CYP2E1, bem como a atividade arilesterase da PON1, e sua relação com o perfil lipídico, características clínicas, morbidade e mortalidade em pacientes com ICV. MÉTODOS/RESULTADOS: Foram avaliadas as frequências alélicas dos polimorfismos de PON1 e CYP2E1, o perfil lipídico e a atividade arilesterase da PON1 em 101 pacientes com ICV e 16 pacientes com hipogamaglobulinemia secundária (HS) e 130 controles saudáveis. Nos dois grupos de pacientes foi analisada a presença de parâmetros clínicos e laboratoriais, morbidade e gravidade da doença. Houve diferença na frequência dos genótipos de PON1-L55M entre pacientes primários e secundários, sendo o alelo 55L e o genótipo 55LM mais frequente no grupo HS. A atividade arilesterase de PON1 mostrou-se menor nos pacientes com ICV em relação ao grupo controle, com pacientes do genótipo 55MM apresentando os menores valores de atividade. Pacientes com o genótipo 55MM apresentaram doença mais grave quando comparados aos demais grupos genotípicos, sendo essa diferença causada não por uma característica deletéria deste genótipo, mas pelo papel protetor desempenhado pelo alelo 55L; pacientes portadores desse alelo apresentaram menor prevalência de manifestações graves como neoplasias, hepatomegalia, sepse e óbitos, bem como maior sobrevida daqueles que apresentavam ao menos uma cópia deste alelo. CONCLUSÃO: Este constitui o primeiro relato demonstrando maior frequência do genótipo 55LM e do alelo 55L em pacientes com hipogamaglobulinemia secundária e de menor atividade arilesterase em pacientes com ICV portadores do genótipo 55MM. Nossos resultados são sugestivos de que a presença do alelo 55L possa desempenhar papel protetor na ICV. Além disso, foi constatado que a presença de linfonodomegalia, hepatomegalia, esplenomegalia, sepse e hipertensão portal possam ser fatores preditivos tanto de um quadro de doença mais grave quanto de maior mortalidade / Introduction: Common Variable Immunodeficiency (CVID) is a heterogeneous disease characterized by reduced levels of IgG, IgA and / or IgM and antibody function. Clinical manifestations include the presence of recurrent or chronic infections, inflammatory/autoimmune diseases, and increased incidence of malignancies such as lymphomas and carcinomas, which is characterized by a state of persistent immune activation and alterations in oxidative metabolism. Both paraoxonase 1 (PON1) and cytochrome p450 (CYP) 2E1 polymorphism have an important role in oxidative processes, controlling the extent of damage caused by changes in oxidant concentration. It is believed that, as in the normal population, both PON1 and CYP2E1 play an important role in the severity and survival of patients with CVID. Objectives: to study the polymorphisms of PON1 and CYP2E1, as well as the arilesterase activity of PON1, and its relation with the lipid profile, clinical characteristics, morbidity and mortality in patients with CVID. Methods/Results: The allelic frequencies of the PON1 and CYP2E1 polymorphisms, the lipid profile and the arilesterase activity of PON1 in 101 patients with CVID and 16 patients with secondary hypogammaglobulinemia (HS) and 130 healthy controls were evaluated. The presence of clinical and laboratory parameters, morbidity and severity of the disease were analyzed in both groups of patients. There was a difference in the frequency of PON1-L55M genotypes between primary and secondary patients, being the 55L allele and the 55LM genotype more frequent in the HS group. The arilesterase activity of PON1 was lower in patients with CVID than in the control group, with patients of the 55MM genotype showing the lowest values. Patients with the 55MM genotype presented a more severe disease when compared to the other genotype groups, this difference is being caused not by a deleterious characteristic of this genotype, but by the protective role played by the 55L allele; patients with this allele had a lower prevalence of severe manifestations such as malignancies, hepatomegaly, sepsis and deaths, as well as a longer survival of those who had at least one copy of this allele. CONCLUSION: This is the first report showing a higher frequency of the 55LM genotype and the 55L allele in patients with secondary hypogammaglobulinemia and of lower arilesterase activity in patients with CVID with 55MM genotype. Our results suggest that the presence of the 55L allele may play a protective role in CVID. In addition, it was found that the presence of lymph node enlargement, hepatomegaly, splenomegaly, sepsis and portal hypertension may be predictive factors of both a more severe disease and a higher mortality rate

INTRODUÇÃO. A Imunodeficiencia comum variável (ICV) é uma doença heterogênea caracterizada pela redução dos niveis de IgG, IgA e/ou IgM e da função de anticorpo. As manifestações clínicas incluem a presença de infecções recorrentes ou crônicas, doenças inflamatórias/autoimunes e incidência aumentada de malignidades como linfomas e carcinomas, caracterizando-se, consequentemente, por um estado de ativação imune persistente e alterações do metabolismo oxidativo. Tanto a paraoxonase 1(PON1) quanto o polimorfismo do citocromo p450 (CYP) 2E1 têm importante participação nos processos oxidativos, controlando a extensão dos danos causados por alterações na concentração de oxidantes. Acredita-se que, tal qual ocorre na população normal, tanto a PON1 quanto a CYP2E1 tenham importante papel na gravidade e na sobrevida dos pacientes com ICV. OBJETIVO: estudar os polimorfismos de PON1 e CYP2E1, bem como a atividade arilesterase da PON1, e sua relação com o perfil lipídico, características clínicas, morbidade e mortalidade em pacientes com ICV. MÉTODOS/RESULTADOS: Foram avaliadas as frequências alélicas dos polimorfismos de PON1 e CYP2E1, o perfil lipídico e a atividade arilesterase da PON1 em 101 pacientes com ICV e 16 pacientes com hipogamaglobulinemia secundária (HS) e 130 controles saudáveis. Nos dois grupos de pacientes foi analisada a presença de parâmetros clínicos e laboratoriais, morbidade e gravidade da doença. Houve diferença na frequência dos genótipos de PON1-L55M entre pacientes primários e secundários, sendo o alelo 55L e o genótipo 55LM mais frequente no grupo HS. A atividade arilesterase de PON1 mostrou-se menor nos pacientes com ICV em relação ao grupo controle, com pacientes do genótipo 55MM apresentando os menores valores de atividade. Pacientes com o genótipo 55MM apresentaram doença mais grave quando comparados aos demais grupos genotípicos, sendo essa diferença causada não por uma característica deletéria deste genótipo, mas pelo papel protetor desempenhado pelo alelo 55L; pacientes portadores desse alelo apresentaram menor prevalência de manifestações graves como neoplasias, hepatomegalia, sepse e óbitos, bem como maior sobrevida daqueles que apresentavam ao menos uma cópia deste alelo. CONCLUSÃO: Este constitui o primeiro relato demonstrando maior frequência do genótipo 55LM e do alelo 55L em pacientes com hipogamaglobulinemia secundária e de menor atividade arilesterase em pacientes com ICV portadores do genótipo 55MM. Nossos resultados são sugestivos de que a presença do alelo 55L possa desempenhar papel protetor na ICV. Além disso, foi constatado que a presença de linfonodomegalia, hepatomegalia, esplenomegalia, sepse e hipertensão portal possam ser fatores preditivos tanto de um quadro de doença mais grave quanto de maior mortalidade / Introduction: Common Variable Immunodeficiency (CVID) is a heterogeneous disease characterized by reduced levels of IgG, IgA and / or IgM and antibody function. Clinical manifestations include the presence of recurrent or chronic infections, inflammatory/autoimmune diseases, and increased incidence of malignancies such as lymphomas and carcinomas, which is characterized by a state of persistent immune activation and alterations in oxidative metabolism. Both paraoxonase 1 (PON1) and cytochrome p450 (CYP) 2E1 polymorphism have an important role in oxidative processes, controlling the extent of damage caused by changes in oxidant concentration. It is believed that, as in the normal population, both PON1 and CYP2E1 play an important role in the severity and survival of patients with CVID. Objectives: to study the polymorphisms of PON1 and CYP2E1, as well as the arilesterase activity of PON1, and its relation with the lipid profile, clinical characteristics, morbidity and mortality in patients with CVID. Methods/Results: The allelic frequencies of the PON1 and CYP2E1 polymorphisms, the lipid profile and the arilesterase activity of PON1 in 101 patients with CVID and 16 patients with secondary hypogammaglobulinemia (HS) and 130 healthy controls were evaluated. The presence of clinical and laboratory parameters, morbidity and severity of the disease were analyzed in both groups of patients. There was a difference in the frequency of PON1-L55M genotypes between primary and secondary patients, being the 55L allele and the 55LM genotype more frequent in the HS group. The arilesterase activity of PON1 was lower in patients with CVID than in the control group, with patients of the 55MM genotype showing the lowest values. Patients with the 55MM genotype presented a more severe disease when compared to the other genotype groups, this difference is being caused not by a deleterious characteristic of this genotype, but by the protective role played by the 55L allele; patients with this allele had a lower prevalence of severe manifestations such as malignancies, hepatomegaly, sepsis and deaths, as well as a longer survival of those who had at least one copy of this allele. CONCLUSION: This is the first report showing a higher frequency of the 55LM genotype and the 55L allele in patients with secondary hypogammaglobulinemia and of lower arilesterase activity in patients with CVID with 55MM genotype. Our results suggest that the presence of the 55L allele may play a protective role in CVID. In addition, it was found that the presence of lymph node enlargement, hepatomegaly, splenomegaly, sepsis and portal hypertension may be predictive factors of both a more severe disease and a higher mortality rate

O epitélio nasal é a primeira porção do sistema respiratório a entrar em contato com o ambiente externo. Partículas da poluição do ar, principalmente os compostos orgânicos absorvidos, podem atuar como liberadores endócrinos. O receptor aril hidrocarboneto (AhR) é um importante competidor dos receptores de estrógeno-beta (ERbeta) que regulam a transcrição do gene para enzimas de metabolização xenobióticas (enzimas do citocromo P450). O objetivo deste estudo é identificar e quantificar ERbeta, AhR, CYP1A1, CYP1A2, CYP1B1 e o perfil de muco no epitélio nasal de camundongos machos e fêmeas em diferentes fases do ciclo estral. Camundongos BALB/c machos (n=32) e fêmeas (n=84) foram expostos ao ar ambiente e ao MP2,5 concentrado a 600 ug.m-³ em um concentrador de partículas ambientais (CPAs). As fêmeas foram divididas de acordo com as fases do ciclo estral: proestro, estro e diestro. O epitélio nasal foi avaliado por RT-PCR e imuno-histoquímica para análise de expressão de ERbeta (proteína), Erbeta-1 e Erbeta-2 (gene), AhR (proteína e gene) e Cyp1a1, Cyp1a2 and Cyp1b1 (gene). A quantificação de muco neutro - Periodic Acid Schiff\'s (PAS+) e ácido - Alcian Blue (AB+) foi avaliada por morfometria. As exposições foram realizadas durante 5 dias/semana, por 45 ± 55 dias. A expressão de Erbeta-2 RNAm apresentou diferenças em resposta à exposição ao CPAs (p=0,016), bem como uma diminuição em fêmeas, quando comparadas aos camundongos machos (p=0,036). A expressão de Cyp1b1 RNAm foi significantemente menor no grupo exposto ao CPAs, em relação ao grupo exposto ao ar ambiente nas fêmeas em diestro (p=0,036). A expressão de Erbeta foi aumentada no epitélio nasal de fêmeas em estro expostas ao CPAs (p=0,005) e a expressão de AhR foi menor em fêmeas em proestro expostas ao CPAs (p=0,048). A exposição ao CPAs levou ao aumento do conteúdo de muco ácido em camundongos machos (p=0,048), o qual diminuiu em fêmeas (p=0,040), quando comparados ao grupo ar ambiente. Este estudo mostrou que houve diferentes respostas à exposição à poluição do ar no epitélio nasal entre machos e fêmeas, e que essas diferenças podem estar relacionadas com a predisposição de fêmeas apresentarem maior suscetibilidade a doenças respiratórias das vias aéreas / The nasal epithelium is the first portion of the respiratory system to reach contact with the external environment. Air pollution particles, mainly the organic compounds absorbed into them, may act as endocrine releasers. The aryl hydrocarbon (AhR) receptor is an important competitor of estrogenic receptors-beta (ERbeta) that regulate transcription of gene coding for xenobiotic-metabolizing enzymes (cytochrome P450 enzymes). The aim of this study is to identify and quantify in the nasal epithelium of male and female mice in different estrous cycle phases related with ERbeta, AhR, CYP1A1, 1A2, 1B1 and the mucus profile. Male (n=32) and female (n=84) BALB/c mice were exposed to ambient air and PM2.5 concentrated at 600 ug.m-³ in an ambient particle concentrator with a particulate matter diameter of 2.5 um (PM2.5). Females were subdivided in three estrous cycles: proestrus, estrus and diestrus. Nasal epithelium was evaluated through RT-PCR and immunohistochemistry for the expression of ERbeta (protein), Erbeta-1 and Erbeta-2 (gene expression), AhR (protein and gene expression) and Cyp1a1, Cyp1a2 and Cyp1b1 (gene expression). Morphometry was applied for evaluation of mucus profile: acid - Alcian Blue (AB+) and neutral - Periodic Acid Schiff\'s (PAS+). Exposure happened for 5 days/week, for 45 ± 55 days. There were differences in Erbeta-2 mRNA in response to exposition to CPAs (p=0.016), and a significant decrease in female compared male mice (p=0.036). Cyp1b1 mRNA was significantly smaller in the CPAs-exposed group compared with the ambient air group in diestrus female mice (p=0.036). The ERbeta expression increased in the nasal epithelium of CPAs-exposed females in the estrus cycle (p=0.005), and the AhR expression decreased in the proestrus cycle of CPAs-exposed females (p=0.048). The exposure to the CPAs led to an increase in the acidic content of mucus in male mice (p=0.048), and decreased in female mice (p=0.040), compared to the ambient air group. This study showed there were different responses in the nasal epithelia of male and female mice exposed to air pollution, which could be related to the predisposition of the females to present more susceptibility to airway respiratory diseases