published_or_final_version / Medicine / Master / Master of Philosophy

[Truncated abstract] Type 1 Diabetes is emerging as a significant public health problem faced by nearly every country in the world. It has major economic and social implications with considerable burden of illness. Approximately 140,000 Australians have been diagnosed with T1DM with an annual increase in incidence rate of 3% per year, comparable to the overall global increase. The management of T1DM requires insulin therapy which places considerable burden on the patient and their carers. Coping with daily insulin injections, dietary changes, modification of physical activity and vigilant monitoring of blood glucose levels, will impact on patient?s quality of life. The optimum goal for the treatment of type 1 diabetes is to safely achieve near-normal glycaemia and failure to maintain this goal accelerates the progression of the devastating long term complications of diabetes. Unfortunately attempts to achieve near normal glycaemia are limited by the risk of excessive lowering of blood glucose levels and hypoglycaemia remains a major barrier to strict glucose control of diabetes. In general this thesis focuses on two fundamental issues related to the epidemiology of severe hypoglycaemia. Namely, methodological consideration when analysing prospective observational data and application of the most robust methodology. A prospective open cohort study of the Princess Margaret Hospital diabetes clinic established in 1992, with 99% case ascertainment was used. This hospital is the only paediatric referral centre for type 1 diabetes and every child diagnosed in the state of Western Australia is treated at this centre. ... The results of this study showed that severe hypoglycaemia remains a major problem and recent approaches to therapy may be allowing a degree of improved control without the expected increased risk of severe hypoglycaemia. The study in chapter 7 investigates genetic risk factors related to severe hypoglycaemia. A significant relationship where the presence of the iv deletion (D) allele of the angiotensin-converting enzyme (ACE) increases risk of severe hypoglycaemia has been reported. This study concludes that the presence of D allele of the ACE gene does not predict a significantly higher risk of severe hypoglycaemia. In an attempt to optimize glycemic control, patients may suffer multiple episodes of severe hypoglycaemia which can adversely affect quality of life as well as educational and intellectual disadvantage. The study in chapter 8 investigates the factors related to recurrent severe hypoglycaemia. A rigorous and informative time-to-event approach is used to account for within child correlation, staggered enrolment and timevarying covariates. This allows important risk factors to change over time. Preschool children have an increased risk of experiencing recurrent severe hypoglycaemia. The findings of this thesis highlights the importance of selecting appropriate analytical methodology to identify risk factors associated with severe hypoglycaemia and also to dismiss factors that had previously been thought to be important. This will help in formulating management plans in order to limit the impact of severe hypoglycaemia.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Thesis (M.P.H.M. (Primary Health Care Management))--Mahidol University, 2007. / LICL has E-Thesis 0023 ; please contact computer services.

Basing on the traditional TCM interpretation, experience of recent research studies and our experimental findings, a few component herbs in Formulae 1 & 2 would be tentatively selected for a new formula. They were Radix Rehmanniae, Radix Astragali, Rhizoma Atractylodis Macrocephalae, Rhizoma Alismatis, Cortex Moutan and Rhizoma Smilacis Chinensis. Whether the new formula could give better efficacy would need to be tested in new clinical trials and experimental models. (Abstract shortened by UMI.) / Diabetes mellitus has long been a clinical problem for hundreds of years. More than 194 million people in the world now suffer from the disorder. About 15% of all diabetic patients would develop unhealing foot ulcers which compile significant proportion of nontraumatic lower-extremity amputations. Basing on the clinical experience of Prof. Xi Jiu Yi in Shanghai, literature review and an innovative interpretation of traditional Chinese medicine, two formulae (F1 & F2) derived from a well known herbal formula: the "Pills of Six Drugs with Rehmannia" were created for clinical trials. With the early successful limb salvage rate of over 80% observed in a clinical series studied at the Prince of Wales Hospital, Hong Kong, multi-directional studies on the two formulae were carried out. The aim was to find out the clinical efficacy of Formulae 1 & 2, and their component herbs, and the biological mechanism of action. A series of in-vitro, ex-vivo and in-vivo experimental models were completed for the latter purposes. / Granulation formation is an important issue essential for ulcer healing. Therefore a CRL-7522 fibroblast cell line and primary fibrobass from eight diabetic foot ulcer patients (ex-vivo) were used to detect the granulation enhancing activities of the Formulae 1 & 2 and component herbs. The two formulae and some of their component herbs viz, Radix Astragali (HQ), Radix Rehmanniae (SD) and Rhizoma Atractylodis Macrocephalae (BZ) showed significant enhancement effects on the cell viability and apparently facilitated granulation formation. Hence the Formulae 1 & 2, and the three component herbs were selected for further studies. The other nine component herbs of the formulae were found to have no significant enhancing effects on cell viability. With an established diabetic rat model (n0 STZ and n5 STZ), a piece of full-thickness skin was removed from the foot of the rat to develop a diabetic rat foot ulcer model. The ulcer area was measured by a specially designed area measuring programme, namely the Image Analytical Programme. The ulcer areas and their percentage reductions over time were recorded and analysed using statistical multilevel models with adjustments for weight, blood glucose level and the presence of extra ulcers. Results revealed that the ulcer area was significantly reduced by the Formulae 1 & 2, and one of their component herbs, Radix Rehmanniae (SD). / Lau Tai-Wai. / "February 2005." / Adviser: Ping Chung Leung. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0197. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 292-310). / 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.

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

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2010.

published_or_final_version / abstract / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

Diabetes Mellitus (DM) é uma síndrome metabólica complexa, causada pela secreção diminuída ou ausente de insulina pelas células beta pancreáticas, levando a hiperglicemia. A hiperglicemia promove a glicação de proteínas e, conseqüentemente, o aparecimento de produtos finais da glicação avançada (AGEs). Atualmente, os pacientes diabéticos são monitorados pela determinação dos níveis de glicemia e hemoglobina glicada (HbA1c). As complicações geradas pela hiperglicemia podem ser divididas em micro e macrovasculares, representadas por retinopatias, nefropatias, neuropatias e doenças cardiovasculares. A albumina (HSA) é a proteína sérica mais abundante no organismo humano e está sujeita à glicação. A protoporfirina XI (PpIX) é a molécula precursora da síntese do heme, componente estrutural da hemoglobina. Ensaios in vitro e em animais indicaram que a hiperglicemia promove uma diminuição de sua concentração em eritrócitos. A espectroscopia de fluorescência é uma técnica bastante utilizada na área biomédica. A autofluorescência corresponde à fluorescência intrínseca presente em algumas moléculas, estando esta associada à estrutura das mesmas. O objetivo deste trabalho foi utilizar a técnica de espectroscopia de fluorescência para mensurar os níveis de autofluorescência da PpIX eritrocitária e AGE-HSA em pacientes diabéticos e indivíduos saudáveis e compará-los com os níveis de glicemia e HbA1c. Este estudo foi realizado com 151 indivíduos (58 controles e 93 diabéticos). Os dados epidemiológicos de pacientes e controles foram obtidos nos prontuários médicos. Para os indivíduos controle, os valores de glicemia foram adquiridos dos prontuários médicos e os níveis de Hb1Ac obtidos pela utilização de kits comerciais. A determinação da autofluorescência da PpIX foi realizada com excitação de 405 nm e emissão de 632 nm. Para a determinação do AGE-HSA foi realizada excitação de 370 nm e emissão de 455 nm. Aproximadamente 50% dos diabéticos apresentaram lesões micro ou macrovasculares decorrentes da hiperglicemia. Não foram observadas diferenças significativas nos valores de intensidade de emissão de PpIX entre os grupos estudados (P=0,89). Na análise do AGE-HSA observou-se diferenças significativas dos valores de intensidade de emissão entre os dois grupos, sendo este valor 1,45 vezes maior para o grupo de indivíduos diabéticos (P<0,0001). Os pacientes com complicações diabéticas apresentavam intensidade de emissão de fluorescência 1,19 vezes maior que os indivíduos sem complicações decorrentes da doença (P= 0,01), mesmo não havendo diferenças significativas nos valores de HbA1c entre os dois grupos. Concluímos que a espectroscopia de fluorescência foi uma técnica eficaz na identificação da autofluorescência da PpIX e do AGE-HSA. A PpIX não foi um biomarcador eficiente para o acompanhamento do DM. A determinação dos níveis de autofluorescência do AGE-HSA foi eficiente para a discriminação entre os grupos e para o monitoramento da progressão da doença, podendo ser mais eficiente que a dosagem de HbA1c. A espectroscopia de fluorescência é uma técnica simples, rápida e de baixo custo para o acompanhamento de indivíduos diabéticos. / Diabetes Mellitus (DM) comprises a complex metabolic syndrome, caused by reduced or absent secretion of insulin by pancreatic beta cells, leading to hyperglycemia. Hyperglycemia promotes glycation of proteins and, consequently, the appearance of advanced glycation end products (AGEs). Currently, diabetic patients are monitored by determining levels of glucose and glycated hemoglobin (HbA1c). The complications caused by hyperglycemia may be divided into micro and macrovascular complications, represented by retinopathy, nephropathy, neuropathy and cardiovascular disease. Albumin (HSA) is the most abundant serum protein in the human body and is subject to glycation. The Protoporphyrin IX (PpIX) is the precursor molecule of heme synthesis, structural component of hemoglobin. The in vitro and animals studies have indicated that hyperglycemia promotes a decrease in its concentration in erythrocytes. The fluorescence spectroscopy is a technique widely used in biomedical field. The autofluorescence corresponds to the intrinsic fluorescence present in some molecules, this being associated with the same structure. The aim of this study was to use fluorescence spectroscopy to measure levels of erythrocyte PpIX autofluorescence and AGE-HSA in diabetic and healthy subjects and compare them with levels of blood glucose and HbA1c. This study was conducted with 151 subjects (58 controls and 93 diabetics). Epidemiological data of patients and controls were obtained from medical records. For control subjects, blood glucose levels were obtained from medical records and levels of Hb1Ac obtained by using commercial kits. The determination of the PpIX autofluorescence was performed with excitation at 405 nm and emission at 632 nm. Determination of AGE-HSA was performed with excitation at 370 nm and emission at 455 nm. Approximately 50% of diabetic had micro and macrovascular lesions resulting from hyperglycemia. There were no significant differences in the PpIX emission intensity values between groups (P = 0.89). In the analysis of AGE-HSA was observed significant differences in the values of emission intensity between the two groups, and this value was 1.45-fold greater for the group of diabetic (P <0.0001). Patients with diabetic complications had fluorescence emission intensity of 1.19-fold higher than individuals without disease complications (P = 0.01), even with no significant differences in HbA1c values between the two groups. We conclude that fluorescence spectroscopy was an effective technique in the identification of the PpIX autofluorescence and AGE-HSA. The PpIX was not an effective biomarker for the monitoring of diabetes. The determination of AGE-HSA autofluorecência was efficient for the discrimination between groups and monitoring disease progression, may be more effective than HbA1c dosage. The fluorescence spectroscopy is a simple, fast and low cost for the monitoring of diabetic patients.