Chan, Wing Lung. / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 111-130). / Abstracts in English and Chinese. / Title --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iii / List of Tables --- p.viii / List of Figures --- p.xi / List of Graphs --- p.xii / Abbreviations --- p.xiv / Abstract --- p.xv / Abstract (Chinese) --- p.xvii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Diabetes Mellitus --- p.2 / Chapter 1.1.1 --- Type 1 diabetes mellitus --- p.3 / Chapter 1.1.2 --- Type 2 diabetes mellitus --- p.4 / Chapter 1.1.3 --- Gestational diabetes mellitus --- p.5 / Chapter 1.2 --- Diabetic Pregnancy --- p.6 / Chapter 1.2.1 --- Incidence of congenital malformations in diabetic pregnancy --- p.6 / Chapter 1.2.2 --- Long term complications in the infant of diabetic mother --- p.7 / Chapter 1.3 --- Hyperglycemia --- p.7 / Chapter 1.4 --- Oxidative Stress --- p.8 / Chapter 1.4.1 --- Oxidative stress and antioxidant enzymes --- p.8 / Chapter 1.4.2 --- Cellular function of oxidative stress --- p.9 / Chapter 1.4.3 --- Adverse effects of excess oxidative stress during embryogenesis --- p.9 / Chapter 1.5 --- Retinoic Acid --- p.10 / Chapter 1.5.1 --- Function of RA during embryonic development --- p.10 / Chapter 1.5.2 --- RA synthesis and degradation --- p.10 / Chapter 1.5.3 --- Mechanisms of retinoic acid signaling : --- p.12 / Chapter 1.5.4 --- Developmental genes regulated by RA --- p.12 / Chapter 1.6 --- Strategy of the Thesis --- p.14 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Animals --- p.17 / Chapter 2.2 --- Induction of Diabetes --- p.17 / Chapter 2.3 --- Mating Methods --- p.18 / Chapter 2.3.1 --- Mice --- p.18 / Chapter 2.3.2 --- Rats --- p.18 / Chapter 2.4 --- Whole Mount In Situ Hybridization --- p.19 / Chapter 2.4.1 --- Synthesis of DNA plasmids and riboprobes --- p.19 / Chapter 2.4.1.1 --- Mini-scale preparation of plasmid DNA --- p.19 / Chapter 2.4.1.2 --- Linearization of DNA plasmid --- p.20 / Chapter 2.4.1.3 --- In vitro transcription and labeling --- p.21 / Chapter 2.4.2 --- Fixation and dehydration of embryos --- p.22 / Chapter 2.4.3 --- Hybridization with RNA probes --- p.23 / Chapter 2.4.4 --- Post-hybridization wash --- p.24 / Chapter 2.4.4.1 --- Pre-absorption of anti-DIG antibody --- p.25 / Chapter 2.4.4.2 --- Embryo powder preparation --- p.25 / Chapter 2.4.5 --- Post antibody wash and signal development --- p.25 / Chapter 2.5 --- Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.26 / Chapter 2.5.1 --- Sample collection and storage --- p.26 / Chapter 2.5.2 --- Total RNA extraction --- p.27 / Chapter 2.5.3 --- Reverse transcription --- p.28 / Chapter 2.5.4 --- Quantitative real-time PCR --- p.28 / Chapter 2.5.5 --- Preparation of cDNA standards for real-time PCR --- p.29 / Chapter 2.6 --- RA-responsive Cell Line --- p.29 / Chapter 2.6.1 --- Cell culture --- p.30 / Chapter 2.6.2 --- Seeding 96-well plate with RA-responsive cells --- p.31 / Chapter 2.6.3 --- Applying samples to 96-well plate coated with RA-responsive cells --- p.31 / Chapter 2.6.4 --- β-galactosidase staining --- p.32 / Chapter 2.7 --- Separation of Protein Isoforms by Isoelectric Focusing (IEF) --- p.33 / Chapter 2.7.1 --- Preparing protein samples for IEF --- p.33 / Chapter 2.7.2 --- Isoelectric focusing --- p.33 / Chapter 2.7.3 --- IEF native gel staining --- p.34 / Chapter 2.7.4 --- Locating three retinaldehyde dehydrogenase (Raldh) isoforms --- p.35 / Chapter 2.8 --- In Vitro RA Synthesizing Reaction --- p.36 / Chapter Chapter 3: --- Effect of Maternal Diabetes on Retinoic Acid Synthesis in the Mouse Embryo / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Experimental Design --- p.41 / Chapter 3.3 --- Materials and Methods --- p.42 / Chapter 3.3.1 --- Sample collection --- p.42 / Chapter 3.3.1.1 --- Criteria for selecting embryos at the same developmental stage --- p.42 / Chapter 3.3.1.2 --- Sample collection for in situ hybridization --- p.42 / Chapter 3.3.1.3 --- Sample collection for real-time quantitative RT-PCR --- p.43 / Chapter 3.3.1.4 --- Sample collection for in vitro RA synthesizing reaction --- p.44 / Chapter 3.3.2 --- Statistical analyses --- p.45 / Chapter 3.4 --- Results --- p.46 / Chapter 3.4.1 --- "Comparison of the in situ expression pattern of Raldh 1, Raldh2 and Raldh3 between embryos of diabetic and non-diabetic mice" --- p.46 / Chapter 3.4.1.1 --- In situ hybridization patterns of Raldh 1 --- p.46 / Chapter 3.4.1.2 --- In situ hybridization patterns of Raldhl --- p.46 / Chapter 3.4.1.3 --- In situ hybridization patterns of Raldh3 --- p.47 / Chapter 3.4.2 --- "Comparison of the relative expression level of Raldh 1, Raldh2 and Raldh3 between embryos of diabetic and non-diabetic mice at different developmental stages" --- p.48 / Chapter 3.4.2.1 --- Relative expression levels of Raldh 1 --- p.50 / Chapter 3.4.2.2 --- Relative expression levels of Raldh2 --- p.50 / Chapter 3.4.2.3 --- Relative expression levels of Raldh3 --- p.51 / Chapter 3.4.3 --- Comparison of the in vitro RA synthesizing activity of Raldh 1 Raldh2 and Raldh3 enzymes between embryos of diabetic and non-diabetic mice at different developmental stages --- p.52 / Chapter 3.5 --- Discussion --- p.55 / Chapter Chapter 4: --- Effect of Hyperglycemia on Retinoic Acid Synthesis / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.2 --- Experimental Design --- p.61 / Chapter 4.3 --- Materials and Methods --- p.64 / Chapter 4.3.1 --- Phlorizin treatment --- p.64 / Chapter 4.3.2 --- Whole rat embryo culture --- p.64 / Chapter 4.3.3 --- Preparation of rat serum --- p.65 / Chapter 4.3.4 --- In situ hybridization --- p.66 / Chapter 4.3.5 --- Real-time quantitative RT-PCR --- p.66 / Chapter 4.3.6 --- In vitro RA synthesizing reaction --- p.68 / Chapter 4.3.7 --- Statistical analyses --- p.68 / Chapter 4.4 --- Results --- p.70 / Chapter 4.4.1 --- "Comparison of the relative expression level of Raldh 1, Raldh2 and Raldh3 between embryos of diabetic and non-diabetic mice injected with phlorizin or suspension vehicle as control" --- p.70 / Chapter 4.4.2 --- Comparison of the in vitro RA synthesizing activity of different isoforms of Raldh enzymes between embryos of diabetic and non-diabetic mice injected with phlorizin or suspension vehicle as control --- p.73 / Chapter 4.4.3 --- In situ expression pattern of Raldh2 in rat embryos cultured in medium containing varying concentrations of D-glucose --- p.77 / Chapter 4.4.4 --- Relative expression levels of Raldh2 in rat embryos cultured in medium supplemented with varying concentrations of D-glucose --- p.78 / Chapter 4.4.5 --- In vitro RA synthesizing activity ofRaldh2 in rat embryos cultured in medium supplemented with varying concentrations of D-glucose --- p.79 / Chapter 4.5 --- Discussion : --- p.82 / Chapter Chapter 5: --- In Vitro Supplementation with RA Rescued Rat Embryos from Hyperglycemia-induced Congenital Malformations / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Experimental Design --- p.88 / Chapter 5.3 --- Materials and Methods --- p.89 / Chapter 5.3.1 --- Preparation of RA --- p.89 / Chapter 5.3.2 --- Supplementation of RA to rat embryos in culture --- p.89 / Chapter 5.3.3 --- Morphological scoring system --- p.90 / Chapter 5.3.4 --- Statistical analyses --- p.90 / Chapter 5.4 --- Results --- p.92 / Chapter 5.4.1 --- Supplementation with RA rescued embryos from hyperglyce- miainduced malformations --- p.92 / Chapter 5.5 --- Discussion --- p.101 / Chapter Chapter 6: --- Conclusion and Future Perspectives / Chapter 6.1 --- Conclusion and Future Perspectives --- p.106 / References --- p.111