published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 91-134) Also available in print.

The incidence of malformed newborns is higher in human pregnancies complicated by diabetes mellitus, as compared to non-diabetic pregnancies. Neural tube and cardiac defects predominate among the fetal malformations induced by hyperglycemia. Non-specific maternal immune stimulation is protective in mice against birth malformations caused by chemical or physical teratogens, or by maternal diabetes mellitus. Insulin dependent diabetes was induced in ICR females to study the late gestation fetal heart by morphometric analysis. Diabetic females treated with Freund's compete adjuvant (FCA) or interferon-gamma (IFNγ) were also generated to elucidate potential positive effects of maternal immune stimulation during the diabetic pregnancy by morphometric analysis and pathologic scoring. Insulin-dependent CD1 females were generated to analyze late gestation fetal myocardial apoptosis by flow cytometric analysis and by real time-polymerase chain reaction (RT-PCR) analysis of a panel of 5 genes involved in apoptosis/proliferation (Bcl-2, P53, Caspase3, Caspase9 and PkC-e). The morphometric analysis of fetal hearts revealed visibly obvious dilation of ventricular chambers and outflow channel of the left ventricle, and reduction of total myocardial ventricular area in late gestation fetuses, as predominant changes seen in the offspring of diabetic dams. Pathologic scoring revealed that maternal immune stimulation, particularly with FCA, in part alleviated fetal heart changes of cavitary dilation and myocardial reduction. Increased rate of apoptosis/necrosis in the fetal myocardium in late gestation during the diabetic pregnancy was evidenced by flow cytometric analysis. Particularly there was a significant increase in percentage of early apoptotic cells in the fetal myocardium detected by cell markers annexin V and propidium iodide. There was also a significant increase in percentage of late apoptotic/necrotic fetal myocardial cells in the diabetic group compared to the control group. These results suggest that maternal treatment with FCA may in part protect the heart from high hyperglycemia by reducing the number of myocardial cells undergoing apoptosis and necrosis. The RT-PCR analysis revealed subtle changes in gene expression for all the genes except Bcl-2. A paradoxical and dramatic up-regulation of this anti-apoptotic gene was observed in late gestation fetal myocardium from the insulin-dependent hyperglycemic groups. Possibly, this could be a mechanism to protect the fetal myocardial cell from the chronic exposure to a severe hyperglycemic insult and consequent apoptosis. In conclusion, maternal insulin-dependent diabetes caused morphological changes in the late gestation fetal heart. Such changes were in part related to dysregulation of myocardial apoptosis. Maternal immune stimulation with FCA improved fetal heart morphology, by a mechanism that may in part relate to normalizing fetal myocardial apoptosis. / Ph. D.

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Orientador: Vera Therezinha de Medeiros Borges / Coorientador: Iracema de Mattos Paranhos Calderon / Banca: Roberto Antônio de Araújo Costa / Banca: Meliza Goi Roscani / Resumo: Introdução: Diabetes Melito Gestacional (DMG) constitui risco para desenvolver diabetes melito tipo 2 e os seus possíveis desdobramentos futuros. É considerado fator de risco independente para doença cardiovascular, a principal causa de morte entre a população diabética. Objetivos: Identificar e comparar as alterações estruturais e da função diastólica cardíaca em gestantes portadoras de diabetes gestacional e gestantes sem patologias, e verificar se existem correlações entre características clínicas e variáveis bioquímicas maternas com o índice de massa ventricular (iMVE) e a razão entre pico de velocidade do fluxo diastólico transmitral no início da diástole (E') e pico de velocidade do fluxo diastólico transmitral durante a contração atrial (A'). Sujeitos e Métodos: Foi realizado estudo prospectivo e transversal em gestantes com DMG (n=21) e gestantes sem patologias (n=23). Todas as gestantes realizaram ecocardiograma entre a 34ª. e a 37ª. semanas de gestação, cujos resultados foram analisados estatisticamente para comparação entre os grupos estudados, adotando-se o limite mínimo de significância de 95% (p<0,05). Resultados: Entre as características clínicas e variáveis bioquímicas estudadas, os valores de idade, paridade, índice de massa corpórea pré-gestacional e gestacional, glicemia de jejum e hemoglobina glicada foram significativamente maiores no grupo DMG. Das variáveis estruturais, a espessura da parede posterior, do septo interventricular, a massa do VE e o iMVE foram significativamente maiores ... / Abstract: Background: Gestational Diabetes Mellitus (GDM) is a risk condition for developing type 2 diabetes and its possible future developments. It is considered an independent risk factor for cardiovascular disease, the leading cause of death among diabetic patients. Objective: To identify and compare the structural and diastolic function in pregnant women with gestational diabetes mellitus and healthy pregnant women and verify possible correlation between clinical and biochemical variables with maternal ventricular mass index (LVMI) and the ratio of peak of transmitral diastolic flow velocity in early diastole (E') and peak velocity of transmitral diastolic flow during atrial contraction (A'). Subject and Methods: This was a prospective cross-sectional study in women with GDM (n = 21) and healthy pregnant women (n = 23). All the women underwent echocardiography between the 34th. and the 37th. weeks of gestation, and the results were statistically analyzed for comparison between groups, adopting the threshold of significance of 95% (p <0.05). Results: Clinical features and biochemical variables, values for age, parity, body mass index gestational and before pregnancy, fasting glucose and hemoglobin A1c were significantly higher in GDM group. Posterior wall thickness, interventricular septum, LV mass and LV mass index were significantly higher in GDM group. The E' and E'/A' were significantly lower in the GDM group, which also showed a positive correlation between LVMI, fasting glucose and pregnancy body mass index. Conclusion: Pregnant women with GDM have different diastolic profile, near to dysfunctional pattern, but within the range of normal values for the parameters studied. Even so, as the dysfunctional pattern tends to evolve with advancing age, it is recommended to these women more rigorous monitoring of glycemic control and cardiovascular system / Mestre

Orientador: Iracema de Mattos Paranhos Calderon / Coorientador: Adenilda Cristina Honorio-França / Banca: Lígia Maria Suppo de Souza Rugolo / Banca: Patrícia Palmeiras / Banca: Solange Barros Carbonare / Banca: Silvana Andrea Molina Lima / Resumo: Não disponível / Abstract: Not available / Doutor

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

The study objective was to determine whether early 2 nd trimester amniotic fluid (AF) erythropoietin (EPO) was associated with and predictive of (a) development of maternal gestational diabetes (GDM) and (b) the infant outcome parameters of (i) gestational age at birth (GAAB) assessed exclusively among spontaneous vaginal deliveries or (ii) birth weight (measured in grams and percentiles). Enzyme-linked-immunosorbent assay was used to determine the EPO concentration of 170 biobanked AF samples. Student's t-test revealed no difference between GDM and non-GDM subjects. AF EPO was not predictive of GAAB despite being significantly greater among preterm infants compared to post-term infants. In contrast, AF EPO was significantly higher among the smallest infants using both birth weight classification schemes. However, following inclusion of known covariates AF EPO was predictive of gram birth weight only. Early 2nd trimester AF EPO may emerge as a useful biomarker of fetal nutritional status and/or growth.

Pregestational and gestational diabetes (GDM) places the mother and her offspring at an increased risk for later development of insulin resistance and type 2 diabetes. Oxidative stress may mediate long-term disturbances in glucose homeostasis associated with type 2 diabetes and the metabolic syndrome. This thesis describes a cross-sectional study examining serum concentrations of free radical generated oxysterols as markers of oxidative stress in a cohort of teenage daughters from pregnancies with and without GDM. Daughters of GDM-pregnancies had a tendency of higher levels of serum oxysterols (7beta-hydroxycholesterol); however, this difference was not statistically significant after adjustment for total cholesterol. Serum oxysterols were significantly correlated with obesity measures such as waist circumference and BMI, which likely accounted for the tendency for higher measures of oxysterol concentrations in the GDM daughters. Oxysterols represent potentially important biomarkers for oxidative stress in adolescent girls as their levels track with the metabolic syndrome risk factors. / Le diabète pré-gestationnel et le diabète de gestation (DG) augmentent le risque dedéveloppement d'une future résistance à l'insuline et de diabète de type 2 autant pourla mère que pour l'enfant. Le stress oxydatif est un facteur potentiel impliqué dans ledéséquilibre du glucose sanguin associé au diabète de type 2 et au syndromemétabolique. La présente thèse est une étude sectionnelle croisée, ayant pour but demesurer des marqueurs du stress oxidatif, notamment la concentration des oxystérolsgénérés par les radicaux libres dans le sérum d'adolescentes, nées de mères ayantprésenté ou non un diabète de gestation. Nos résultats montrent des concentrationsd'oxystérols (7P-hydroxycholesterol) plus élevées dans le sérum de filles issues degestations diabétiques à comparer aux filles de mères n'ayant pas eu de DG.Cependant, la différence entre les deux groupes n'était pas statistiquementsignificative après un ajustement au cholestérol total. La concentration d'oxystérolsétait significativement corrélée aux marqueurs d'obésité, notamment la circonférencede la taille et l'index de masse corporelle, possiblement à l'origine de la tendance desoxystérols à être plus élevés dans le cas des adolescentes issues de gestationsdiabétiques.

Thesis (Ed. D.)--Illinois State University, 2004. / Title from title page screen, viewed November 17, 2005. Dissertation Committee: Dent Rhodes (chair), Kenneth Jerich, Cheri Toledo, Denise Charron-Prochownik. Includes bibliographical references (leaves 96-105) and abstract. Also available in print.