Yeung Sau-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 139-172). / Abstracts in English and Chinese. / Title page --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iv / List of Figures --- p.viii / List of Graphs --- p.x / List of Tables --- p.xi / Abbreviations --- p.xiv / Abstract --- p.xv / Chinese Abstract --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / 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.5 / Chapter 1.1.3 --- Maturity onset diabetes of the young (MODY) --- p.6 / Chapter 1.1.4 --- Gestational diabetes --- p.7 / Chapter 1.2 --- Effect of Diabetes on Pregnancy --- p.9 / Chapter 1.3 --- Suggested Causes of Diabetic Embryopathy --- p.10 / Chapter 1.3.1 --- Glucose --- p.10 / Chapter 1.3.2 --- Ketone bodies --- p.11 / Chapter 1.3.3 --- Somatomedin inhibitors --- p.12 / Chapter 1.3.4 --- TNF-α --- p.12 / Chapter 1.3.5 --- Oxidative stress --- p.13 / Chapter 1.4 --- Animal Model of Diabetes --- p.15 / Chapter 1.4.1 --- Chemically-induced --- p.15 / Chapter 1.4.2 --- Mutants --- p.17 / Chapter 1.5 --- Gene-teratogen Interaction under Diabetic Pregnancy --- p.19 / Chapter 1.6 --- Strategy of the Thesis --- p.21 / Chapter Chapter 2 --- General Materials and Methods --- p.24 / Chapter 2.1 --- Mouse Maintenance and Mating Method --- p.25 / Chapter 2.2 --- Induction of Diabetes --- p.25 / Chapter 2.3 --- Preparation of All-trans Retinoic Acid --- p.26 / Chapter 2.4 --- Dissection of Embryos --- p.26 / Chapter 2.5 --- DNA Extraction from Yolk Sac for Genotyping --- p.27 / Chapter 2.6 --- Genotyping of Embryos --- p.28 / Chapter 2.7 --- Preparation of RNA Probes for In Situ Hybridization --- p.29 / Chapter 2.7.1 --- Mini-scale preparation of plasmid DNA --- p.29 / Chapter 2.7.2 --- Linearization of plasmid DNA --- p.30 / Chapter 2.7.3 --- In vitro transcription --- p.31 / Chapter 2.8 --- Whole Mount In Situ Hybridization --- p.33 / Chapter 2.8.1 --- Fixation and dehydration of embryos --- p.33 / Chapter 2.8.2 --- Hybridization --- p.33 / Chapter 2.8.3 --- Post-hybridization wash --- p.34 / Chapter 2.8.4 --- Antibody wash and color development --- p.35 / Chapter 2.8.5 --- Embryo powder preparation --- p.36 / Chapter 2.8.6 --- Pre-absorption of antibody --- p.35 / Chapter 2.9 --- Whole Mount TUNEL Staining --- p.36 / Chapter Chapter 3 --- "Maternal Diabetes, Sp2H and RA Interaction" --- p.39 / Chapter 3.1 --- Introduction --- p.40 / Chapter 3.1.1 --- Neural tube defects --- p.41 / Chapter 3.1.2 --- Retinoic acid as environmental factor --- p.41 / Chapter 3.1.3 --- Sp2H as genetic factor --- p.44 / Chapter 3.1.4 --- Experimental design of this chapter --- p.46 / Chapter 3.2 --- Material and Methods --- p.47 / Chapter 3.2.1 --- Sp2H mice --- p.47 / Chapter 3.2.2 --- Mating and RA injection protocol --- p.47 / Chapter 3.2.3 --- Dissection of fetuses and analysis of neural tube development --- p.48 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Maternal diabetes alone --- p.50 / Chapter 3.3.2 --- Sp2H mutation alone --- p.51 / Chapter 3.3.3 --- RA alone --- p.52 / Chapter 3.3.4 --- Maternal diabetes and RA interaction --- p.53 / Chapter 3.3.5 --- Sp2H mutation and RA interaction --- p.55 / Chapter 3.3.6 --- Sp2H mutation and maternal diabetes interaction --- p.57 / Chapter 3.3.7 --- "Maternal diabetes, Sp2H mutation and RA interaction" --- p.59 / Chapter 3.4 --- Discussion --- p.62 / Chapter 3.4.1 --- Maternal diabetes alone does not cause neural tube defects --- p.62 / Chapter 3.4.2 --- RA induces neural tube defects --- p.63 / Chapter 3.4.3 --- Interaction of maternal diabetes with RA in increasing the susceptibility to neural tube defects --- p.64 / Chapter 3.4.4 --- Embryos with Sp2H allele show increased susceptibility to neural tube defects when triggered by maternal diabetes and RA --- p.67 / Chapter Chapter 4 --- Molecular and Cellular Bases of Interaction --- p.71 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.1.1 --- Mechanism of diabetic embryopathy --- p.72 / Chapter 4.1.2 --- Mechanism of Sp2H mutation in development of neural tube defects --- p.74 / Chapter 4.1.3 --- Mechanism of RA teratogenicity --- p.75 / Chapter 4.1.4 --- "Possible common pathways shared by maternal diabetes, RA and Sp2H mutation" --- p.76 / Chapter 4.1.5 --- Experimental design of this chapter --- p.78 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.2.1 --- Sample collection for studying Pax3 expression in Sp2H/+ And +/+ embryos in response to maternal diabetes or RA by whole mount in situ hybridization --- p.80 / Chapter 4.2.2 --- "Sample collection for studying the level of apoptosis in response to the interaction of maternal diabetes, Sp2H mutation and RA by whole mount TUNEL staining" --- p.82 / Chapter 4.3 --- Results --- p.86 / Chapter 4.3.1 --- Expression levels of Pax3 mRNA detected by whole mount in situ hybridization / Chapter 4.3.1.1 --- Expression of Pax3 in Sp2H/+/- and +/+ embryos --- p.86 / Chapter 4.3.1.2 --- Effect of maternal diabetes on Pax3 expression in Sp2H/+ and +/+ embryos --- p.87 / Chapter 4.3.1.3 --- Effect of RA on Pax3 expression in Sp2H /+ and +/+ embryos --- p.88 / Chapter 4.3.2 --- Level of apoptosis detected by whole mount TUNEL --- p.89 / Chapter 4.3.2.1 --- Effect of Sp2H allele on apoptosis --- p.94 / Chapter 4.3.2.2 --- Effect of maternal diabetes on apoptosis in Sp2H/+ and +/+ embryos --- p.95 / Chapter 4.3.2.3 --- Effect of RA on apoptosis in Sp2H/+ and +/+ embryos --- p.96 / Chapter 4.3.2.4 --- Effect of maternal diabetes and RA on apoptosis in Sp2H/+ and +/+ embryos --- p.97 / Chapter 4.4 --- Discussion --- p.99 / Chapter 4.4.1 --- Underexpression of Pax3 and increases in apoptosis under maternal diabetes --- p.99 / Chapter 4.4.2 --- "RA does not down regulate Pαx3, but increases apoptosis" --- p.102 / Chapter 4.4.3 --- Interaction of maternal diabetes and RA in increasing apoptosis --- p.104 / Chapter Chapter 5 --- "Maternal Diabetes, NOD and RA Interaction" --- p.108 / Chapter 5.1 --- Introduction --- p.109 / Chapter 5.1.1 --- Diabetic embryopathy in NOD mice --- p.109 / Chapter 5.1.2 --- Experimental design of this chapter --- p.110 / Chapter 5.2 --- Materials and Methods --- p.112 / Chapter 5.2.1 --- NOD mice --- p.112 / Chapter 5.2.2 --- Mating and RA Injection Protocol --- p.112 / Chapter 5.2.3 --- Sample Collection for the Study of Pax3 Expression --- p.113 / Chapter 5.3 --- Results --- p.115 / Chapter 5.3.1 --- Maternal diabetic alone --- p.116 / Chapter 5.3.2 --- NOD mutation alone --- p.117 / Chapter 5.3.3 --- RA alone --- p.118 / Chapter 5.3.4 --- Maternal diabetes and RA interaction --- p.119 / Chapter 5.3.5 --- NOD mutation and RA interaction --- p.121 / Chapter 5.3.6 --- NOD mutation and maternal diabetes interaction --- p.123 / Chapter 5.3.7 --- "Maternal diabetes, NOD mutation and RA interaction" --- p.125 / Chapter 5.3.8 --- Expression of Pax3 in embryos with different copies of NOD alleles --- p.128 / Chapter 5.4 --- Discussion --- p.130 / Chapter 5.4.1 --- Maternal diabetes interacts with NOD mutation to increase susceptibility to neural tube defects --- p.130 / Chapter 5.4.2 --- Interaction of maternal diabetes with NOD mutation is greatly exacerbated when exposed to RA --- p.131 / Chapter 5.4.3 --- Pax3 is not involved in the interaction --- p.133 / Chapter Chapter 6 --- Conclusion and Future Perspectives --- p.134 / References --- p.139 / Figures / Graphs

Lee, Man Yuen. / "March 2008." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 103-128). / Abstracts in English and Chinese. / Title Page --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iii / List of Figures --- p.vii / List of Graphs --- p.viii / List of Tables --- p.x / Abbreviations --- p.xii / Abstract --- p.xiii / Abstract (Chinese) --- p.xv / 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 --- p.5 / Chapter 1.2 --- Diabetic pregnancy --- p.6 / Chapter 1.3 --- Etiology of diabetes-induced malformations --- p.9 / Chapter 1.3.1 --- Hyperglycaemia --- p.10 / Chapter 1.3.2 --- Hyperketonaemia and somatomedin inhibitors --- p.10 / Chapter 1.3.3 --- Oxidative stress --- p.11 / Chapter 1.3.4 --- Deficiency of myo-inositol and arachidonic acid --- p.12 / Chapter 1.4 --- Vitamin A --- p.13 / Chapter 1.5 --- Retinoic acid --- p.14 / Chapter 1.5.1 --- RA signaling on embryo development --- p.15 / Chapter 1.5.2 --- RA teratogenicity --- p.15 / Chapter 1.5.3 --- RA regulation --- p.17 / Chapter 1.6 --- RA and maternal diabetes-induced caudal regression share similar pathogenic mechanisms --- p.19 / Chapter 1.7 --- Strategy of the thesis --- p.21 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Animal --- p.25 / Chapter 2.2 --- Induction of diabetes --- p.25 / Chapter 2.3 --- Preparation of retinoic acid for mouse injection --- p.26 / Chapter 2.4 --- RA responsive cell line --- p.26 / Chapter 2.4.1 --- Cell culture --- p.27 / Chapter 2.4.2 --- Seeding and adding sample to 96-well plate --- p.28 / Chapter 2.4.3 --- Staining of cells --- p.28 / Chapter 2.5 --- Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) --- p.29 / Chapter 2.5.1 --- Collection and storage of tissues --- p.29 / Chapter 2.5.2 --- Total RNA extraction --- p.29 / Chapter 2.5.3 --- Reverse transcription --- p.30 / Chapter 2.5.4 --- Polymerase chain reaction --- p.30 / Chapter 2.5.5 --- Preparation of cDNA standard --- p.31 / Chapter 2.5.6 --- Mini-scale preparation of plasmid DNA --- p.32 / Chapter Chapter 3: --- Effects of Maternal Diabetes on RA Catabolism in the Tail Bud Region / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Experimental design --- p.37 / Chapter 3.3 --- Materials and methods --- p.38 / Chapter 3.3.1 --- Preparation of RA stock solution for cell culture --- p.38 / Chapter 3.3.2 --- Preparation of RA standard solutions --- p.38 / Chapter 3.3.3 --- Characterization of RA responsive cell line --- p.39 / Chapter 3.3.3.1 --- Determining optimal culture time for maximum response --- p.39 / Chapter 3.3.3.2 --- Testing toxicity of DMSO --- p.39 / Chapter 3.3.3.3 --- Detection of β-galactosidase activity by β_gal Assay Kit --- p.40 / Chapter 3.3.4 --- In vitro assay of enzymatic degradation of RA --- p.41 / Chapter 3.3.4.1 --- Testing toxicity of enzyme cofactor-reducing agent --- p.41 / Chapter 3.3.4.2 --- Collection of tail bud --- p.42 / Chapter 3.3.4.3 --- In vitro enzymatic reaction --- p.43 / Chapter 3.3.5 --- In vivo assay of enzymatic degradation of RA --- p.44 / Chapter 3.3.5.1 --- Determining the optimal time for maximum release of RA from the tail bud into the medium --- p.44 / Chapter 3.3.5.2 --- Monitoring of RA remained in the tail bud after injection of exogenous RA --- p.45 / Chapter 3.3.6 --- Statistical analysis --- p.45 / Chapter 3.4 --- Results --- p.46 / Chapter 3.4.1 --- Optimization of RA responsive cell line --- p.46 / Chapter 3.4.1.1 --- Effect of incubation time with RA on β-galactosidase expression level --- p.46 / Chapter 3.4.1.2 --- Toxicity of RA and DMSO --- p.47 / Chapter 3.4.1.3 --- Comparison of X-gal staining assay and β_gal Assay Kit for detection of β-galactosidase expression --- p.48 / Chapter 3.4.2 --- In vitro assay of RA catabolic activity in the tail bud --- p.49 / Chapter 3.4.2.1 --- Toxicity of enzyme cofactor-reducing agent --- p.49 / Chapter 3.4.2.2 --- RA catabolic activity in lysed and intact tail buds --- p.50 / Chapter 3.4.2.3 --- Effect of enzyme cofactor and inhibitor --- p.50 / Chapter 3.4.2.4 --- Comparsion of in vitro RA catabolic activity of diabetic and non-diabetic groups --- p.51 / Chapter 3.4.3 --- In vivo assay of RA catabolic activity in the tail bud --- p.52 / Chapter 3.4.3.1 --- Optimal time for maximum release of RA from the tail bud into the medium --- p.53 / Chapter 3.4.3.2 --- Comparison of RA remained in the tail bud of embryos of diabetic and non-diabetic mice --- p.53 / Chapter 3.5 --- Discussion --- p.55 / Chapter Chapter 4: --- Analysis of Cyp26 Expression in the Tail Bud Region / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Experimental design --- p.63 / Chapter 4.3 --- Materials and methods --- p.64 / Chapter 4.3.1 --- Sample collection --- p.64 / Chapter 4.3.2 --- Real-time quantitative RT-PCR --- p.64 / Chapter 4.3.3 --- Statistical analysis --- p.66 / Chapter 4.4 --- Results --- p.67 / Chapter 4.4.1 --- "Relative expression levels of Cyp26al, Cyp26bl and Cyp26cl" --- p.67 / Chapter 4.4.2 --- Molecular changes in Cyp26al in tail bud region after maternal RA treatment --- p.68 / Chapter 4.5 --- Discussion --- p.72 / Chapter Chapter 5: --- Comparison of Cyp26al Heterozygous and Wild-type Embryos in Diabetic and Non-diabetic Pregnancies / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.2 --- Experimental design --- p.79 / Chapter 5.3 --- Materials and methods --- p.81 / Chapter 5.3.1 --- Animal --- p.81 / Chapter 5.3.2 --- DNA genotyping --- p.81 / Chapter 5.3.3 --- Measurement of RA remained in the tail bud after RA treatment --- p.82 / Chapter 5.3.4 --- Analysis of extent of caudal regression --- p.83 / Chapter 5.3.5 --- Real-time quantitative RT-PCR --- p.83 / Chapter 5.3.6 --- Statistical analysis --- p.84 / Chapter 5.4 --- Results --- p.85 / Chapter 5.4.1 --- Comparsion of pregnancy outcome of mating with ICR or Cyp26al+/- males --- p.85 / Chapter 5.4.2 --- Expression levels of Cyp26al determined by real-time quantitative RT-PCR --- p.85 / Chapter 5.4.3 --- Determination of RA catabolic activity --- p.86 / Chapter 5.4.4 --- Extent of caudal regression --- p.90 / Chapter 5.5 --- Discussion --- p.93 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.96 / References --- p.102 / Figures / Graphs

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Diabetic pregnancy is associated with increased risk of congenital malformations. Previous studies have shown that maternal diabetes can interact with the vitamin A metabolite, all-trans retinoic acid (RA), in increasing embryonic susceptibility to caudal regression and neural tube defects. The aim of this thesis is to investigate the cellular and molecular mechanisms that underlie this interaction. / First hypothesis. RA concentration in the embryo is tightly regulated by the synthesizing enzyme retinaldehyde dehydrogenase type II (RALDH2), and the degrading enzyme CYP26. Alteration in expression levels of these enzymes under maternal diabetes may affect the availability of RA and thus its teratogenicity. / In conclusion, results of this thesis provide insight into the mechanism of how maternal diabetes interacts with RA in enhancing embryonic susceptibility to congenital malformations. This is also the first report to show that maternal diabetes alters RA homeostasis. (Abstract shortened by UMI.) / Second hypothesis. The transfer of RA to the nucleus for molecular action is regulated by cytoplasmic cellular retinoic acid binding proteins CRABP-I and CRABP-II. Alteration in expression levels of these binding proteins under maternal diabetes may affect the amount of RA reaching the nucleus and thus its teratogenicity. / Third hypothesis. The action of RA is mediated via different nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR). Alteration in expression levels of these receptors under maternal diabetes may affect the efficacy of RA signal transduction and thus its teratogenicity. / Three hypotheses are proposed to explain the underlying mechanism of increased embryonic susceptibility to RA teratogenicity under maternal diabetes: / To investigate these hypotheses, expression levels of various genes in different groups were compared. Result show that there are no significant differences in mRNA expression levels of CRABP-I, CRABP-II, RARgamma, RARgamma and RXRalpha between embryos of diabetic and non-diabetic mice with or without RA treatment. In contrast, expression levels of Raldh2 and CYP26 are significantly reduced in embryos of diabetic mothers, and in embryos of non-diabetic mice cultured in vitro in hyperglycemic conditions. Moreover, embryos of diabetic mice show significantly reduced response to RA-induced up-regulation of CYP26. These findings suggest that the rate of degradation of RA is slower in embryos of diabetic mice and thus the teratogenic effect of RA is enhanced. / Leung Bo Wah. / "July 2005." / Adviser: Alisa S. W. Shum. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3779. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 158-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Leung, Siu Lun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 153-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Chan Wai-Hon. / "September 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 137-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

In this 15 year follow up study in a Chinese population, we confirmed that maternal gestational diabetic status significantly increased women’s future cardiometabolic risk. Glycaemic levels below the current criteria for a positive screening test for gestational diabetes and for the diagnosis of gestational diabetes still significantly predict women’s future risk. In utero hyperinsulinaemia, which caused by an intrauterine hyperglycaemic environment, was found to predict children’s AGT and adolescents’ overweight and MetS. The results had important implication that the current diagnostic criteria for gestational diabetes may not be discriminative in predicting both the mothers and their children’s future cardiometabolic risk. Although recent research has re-visited and emphasised on the diagnostic criteria of gestational diabetes which best predicted adverse pregnancy outcome, future study should also scrutinise on the optimal glycaemic threshold, either in screening or diagnostic test, that relate to the mothers’ and children offspring’s long term cardiometabolic risk. / Tam, Wing Hung. / Thesis (M.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 119-146). / Abstract also in Chinese. / LIST OF TABLES --- p.xxii / LIST OF FIGURES --- p.xxv / LIST OF ABBREVIATIONS --- p.xxvi / Chapter Chapter 1 --- Gestational diabetes & future cardiometabolic risk - an overview / Chapter 1.1 --- Historical background --- p.2 / Chapter 1.2 --- Pregnancy physiology vs. gestational diabetes --- p.5 / Chapter 1.3 --- Diabetes mellitus - a global epidemic --- p.6 / Chapter 1.4 --- History of gestational diabetes & progression to Type 2 DM --- p.7 / Chapter 1.5 --- History of gestational diabetes & cardiometabolic risk --- p.8 / Chapter 1.6 --- Type 2 DM among children and adolescents --- p.9 / Chapter 1.7 --- Type 2 DM among offspring of mothers with gestational diabetes --- p.10 / Chapter 1.8 --- Cardiometabolic risk in children exposed to maternal gestational diabetes --- p.12 / Chapter 1.9 --- Long term follow up on mothers & children cohort --- p.12 / Chapter Chapter 2 --- Research methodology / Chapter 2.1 --- Subjects --- p.16 / Chapter 2.2 --- Obstetric and neonatal information --- p.18 / Chapter 2.2.1 --- Maternal glycaemic indices at pregnancy --- p.18 / Chapter 2.2.2 --- Umbilical cord blood C-peptide & insulin levels --- p.18 / Chapter 2.2.3 --- Definition of antenatal variables --- p.19 / Chapter 2.3 --- Follow up assessment of the mothers --- p.19 / Chapter 2.4 --- Follow up assessment of the children and adolescents --- p.22 / Chapter 2.5 --- Definition of abnormal glucose tolerance and metabolic syndrome --- p.24 / Chapter 2.5.1 --- Definition of abnormal glucose tolerance --- p.24 / Chapter 2.5.2 --- Definition of metabolic syndrome in adult --- p.24 / Chapter 2.5.3 --- Definition of metabolic syndrome in adolescent --- p.25 / Chapter 2.6 --- Determination of insulin resistance and pancreatic beta cell function --- p.26 / Chapter 2.6.1 --- Definition of insulin resistance --- p.26 / Chapter 2.6.2 --- Definition of pancreatic beta cell function --- p.26 / Chapter 2.6.3 --- Measurement of insulin resistance and pancreatic β-cell function --- p.27 / Chapter 2.7 --- Statistical analysis --- p.31 / Chapter 2.7.1 --- Statistical programme --- p.31 / Chapter 2.7.2 --- Comparison between group differences --- p.31 / Chapter 2.7.3 --- General Linear Model --- p.32 / Chapter 2.7.4 --- Multivariate logistic regression --- p.33 / Chapter 2.7.5 --- Receiver operating characteristic analysis --- p.37 / Chapter 2.8 --- Ethics approval --- p.41 / Chapter 2.9 --- Funding --- p.42 / Chapter Chapter 3 --- History of gestational diabetes and women’s future cardiometabolic risk / Chapter 3.1 --- Maternal clinical parameters at the index pregnancy --- p.44 / Chapter 3.2 --- Maternal cardiometabolic status at 8 years post-delivery --- p.45 / Chapter 3.3 --- Maternal cardiometabolic status at 15 years post-delivery --- p.49 / Chapter 3.4 --- Prediction of cardiometabolic risk by maternal gestational diabetic status --- p.50 / Chapter 3.4.1 --- Abnormal glucose tolerance and metabolic syndrome at 8 years by maternal gestational diabetic status --- p.52 / Chapter 3.4.2 --- Abnormal glucose tolerance, DM, hypertension and metabolic syndrome at 15 years by maternal gestational diabetic status --- p.52 / Chapter 3.5 --- The role of insulin resistance in predicting women’s DM and metabolic syndrome --- p.55 / Chapter 3.6 --- Discussion --- p.57 / Chapter 3.7 --- Conclusion --- p.62 / Chapter Chapter 4 --- Glycaemic variables measured at mid-gestation of the index pregnancy predict women’s future cardiometabolic risk / Chapter 4.1 --- Glycaemic levels in pregnancy and perinatal outcome --- p.64 / Chapter 4.2 --- Glycaemic levels in pregnancy and women’s future cardiometabolic risk --- p.65 / Chapter 4.2.1 --- Prediction of women’s cardiometabolic risk at 8 and 15-year --- p.66 / Chapter 4.2.2 --- Optimal cut-off levels in predicting women’s future cardio- metabolic risk --- p.69 / Chapter 4.3 --- Discussion --- p.75 / Chapter 4.4 --- Conclusion --- p.78 / Chapter Chapter 5 --- Maternal gestational diabetes and offspring’s cardiometabolic risk / Chapter 5.1 --- Offspring’s cardiometabolic risk at 8 years age --- p.80 / Chapter 5.1.1 --- Baseline characteristics at pregnancy and delivery --- p.80 / Chapter 5.1.2 --- Children’s clinical and biochemical parameters at 8 years age --- p.82 / Chapter 5.2 --- Offspring’s cardiometabolic risk at 15 years age --- p.84 / Chapter 5.2.1 --- Adolescents’ clinical and biochemical parameters at 15 years age --- p.84 / Chapter 5.2.2 --- Clinical parameters of adolescents with abnormal glucose tolerance --- p.84 / Chapter 5.3 --- Discussion --- p.88 / Chapter 5.4 --- Conclusion --- p.90 / Chapter Chapter 6 --- In utero hyperinsulinaemia and offspring’s cardiometabolic risk / Chapter 6.1 --- Umbilical cord blood insulin and C-peptide --- p.92 / Chapter 6.1.1 --- Umbilical cord insulin and C-peptide concentrations in the original cohort --- p.92 / Chapter 6.1.2 --- Determination of in utero hyperinsulinaemia by umbilical cord insulin and C-peptide levels --- p.95 / Chapter 6.2 --- The effect of in utero hyperinsulinaemia on children’s abnormal glucose tolerance at 8 years of age --- p.98 / Chapter 6.2.1 --- Receiver operating characteristic analysis --- p.98 / Chapter 6.2.2 --- Logistic regression analysis --- p.98 / Chapter 6.3 --- The effect of in utero hyperinsulinaemia on adolescents’ cardio- metabolic risk at 15years of age --- p.102 / Chapter 6.3.1 --- Logistic regression analysis --- p.102 / Chapter 6.4 --- Discussion --- p.105 / Chapter 6.5 --- Conclusion --- p.108 / Chapter Chapter 7 --- Summary and conclusion / Chapter 7.1 --- Summary of the thesis --- p.110 / Chapter 7.1.1 --- Women’s long term cardiometabolic risk after a pregnancy with gestational diabetes --- p.110 / Chapter 7.1.2 --- The long term cardiometabolic risk of children born to mothers who had gestational diabetes --- p.111 / Chapter 7.1.3 --- New findings from the studies and their implications --- p.111 / Chapter 7.2 --- Strength and weakness in the study --- p.113 / Chapter 7.2.1 --- Unique cohort from universal screening --- p.113 / Chapter 7.2.2 --- Study design --- p.113 / Chapter 7.2.3 --- Response rate and loss to follow up --- p.114 / Chapter 7.2.4 --- Treatment effect of gestational diabetes --- p.115 / Chapter 7.3 --- Issues of future research --- p.115 / Chapter 7.3.1 --- Follow up study on the HAPO cohort --- p.115 / Chapter 7.3.2 --- Opportunity for international collaboration --- p.117 / Chapter 7.4 --- Conclusion --- p.118 / REFERENCES --- p.119

Lee, Man Yuen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 191-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

Introdução: O Diabetes mellitus tipo 1 (DM1) cursa com produção ausente ou irrisória de insulina e é a forma responsável pelos casos mais graves de distúrbios glicêmicos. O DM1 exerce forte influência sobre o crescimento fetal. Enquanto a hiperglicemia estimula o crescimento fetal devido à hiperinsulinemia, nas pacientes com vasculopatias a placentação inadequada pode levar o feto à restrição. Objetivos: identificar alterações de crescimento fetal e avaliá-las quanto a sua associação com o controle glicêmico materno e de marcadores laboratoriais de função renal. Métodos: foram avaliadas, de forma prospectiva em coorte observacional, 60 gestantes com DM1 que iniciaram o pré-natal no primeiro trimestre. A associação entre a classificação de peso ao nascimento com as seguintes variáveis foi analisada: média glicêmica, frequência de hipo e hiperglicemia, frequência de hipo e hiperglicemia grave, hemoglobina glicada, frutosamina, ácido úrico, creatinina e proteinúria de 24 horas. A predição do crescimento fetal também foi estudada. Resultados: Desvios do crescimento fetal em pacientes com DM1 ocorreram em 41% dos casos (n=25). Observou-se que 10% das gestações resultaram em PIG (n=6) e 31%, em GIG (n=19). Níveis aumentados de média glicêmica (p =0,006), baixa frequência de hipoglicemias (p = 0,027) e alta frequência de hiperglicemias (p = 0,014) se associaram a GIG no terceiro trimestre. Em todos os trimestres, valores séricos mais elevados de ácido úrico, creatinina e proteinúria de 24hs, se associaram de maneira significativa, ao grupo PIG. Foi construído um modelo, com taxa de acerto de 80.3%, para a predição de crescimento fetal com os valores de terceiro trimestre da média glicêmica e da creatinina. Conclusões: Foram identificadas variáveis relacionadas ao controle glicêmico materno e à marcadores laboratoriais de função renal que se associaram a alterações no crescimento fetal. Usando algumas dessas variáveis foi possível construir um modelo para predição do crescimento fetal com boa acurácia / Introduction. Diabetes mellitus type 1 (DM1) is described as absent or negligible production of insulin and it is responsible for the most severe cases of glycemic disorders. DM1 has a strong influence on fetal growth. In pregnant women the hyperglycemia stimulates fetal growth, and the vasculopathy influences the placentation process, which may lead to growth restriction. Objective. To identify fetal growth disorders and their association with maternal glycemic control and laboratory markers of renal function. Methods. Sixty pregnant women with DM1 were prospectively followed from the first trimester in an observational cohort. The association between birthweight classification with the following parameter were investigated: glycemic mean, frequency of hypo and hyperglycemia, frequency of severe hyper and hypoglycemia, glycated hemoglobin, fructosamine, uric acid, creatinine and proteinuria of 24 hours. The prediction of fetal growth was also investigated. Results. Abnormal fetal growth was observed in 41% (n= 25). Large for gestational age (LGA) was observed in 31.7% (n=19) and small for gestational age (SGA) in 10% (n= 6). High values of glycemic mean (p = 0.006), low frequency of hypoglycemia (p = 0.027) and high frequency of hyperglycemia (p = 0.014) were significantly associated with LGA fetal growth in the third trimester. In all trimesters, the SGA fetal growth was significantly associated with higher serum values of uric acid, creatinine and proteinuria of 24 hours. The prediction model for fetal growth, using values of glycemic mean and creatinine, was significant in the third trimester with an accuracy of 80.3%. Conclusions. The maternal glycemic control and the laboratory markers of renal function associated with the fetal growth disorders in pregnancies with DM1 were identified. Using these parameters it was possible to predict with a good accuracy the fetal growth in DM1