This thesis describes biochemical and molecular methods diagnostic for a spectrum of steroid metabolic diseases. Deficiency of any enzyme in the steroid hormone biosynthetic pathways leads to disorders including congenital adrenal hyperplasia, while some cause disorders of sex development (DSD). A gas chromatography mass spectrometry-based analytical technique called urinary steroid profiling (USP) has been shown to be a useful diagnostic test for these diseases and for steroid-secreting adrenocortical tumours. To test the hypothesis that this approach would be effective in our local population, we interpreted 482 USP results using reference intervals set up from 371 local healthy subjects. Characteristic steroid metabolite excretion patterns were found in 39 patients, including 21 patients with 21-hydroxylase deficiency (21OHD) where there were grossly increased 17-hydroxyprogesterone metabolites, 12 patients with 5α-reductase 2 deficiency (5ARD) with extremely low 5α- to 5β-reduced steroid metabolite ratios, and five patients with adrenocortical carcinoma with markedly raised tetrahydro-11-deoxycortisol and 3,16,20-pregnenetriols levels. / The genetic basis of 21OHD in various populations is mainly due to conversion between the CYP21A2 and the CYP21A1P genes but this has not yet been explored in our population. By using DNA sequencing and multiplex ligation-dependent probe amplification, 74 mutations were found in 35 patients with 21OHD. Gross deletion/conversion of the CYP21A2 gene accounted for 27%. c.290-13A/C>G was the most common point mutation (27%), followed by p.Ile172Asn (17.6%). One novel mutation c.1367delA was also detected. Their prevalence in our patients differs from those in other populations. / The most common cause of 46,XY DSD in Western populations is androgen insensitivity syndrome (AIS) but this has not been verified locally. A prospective study was conducted where 64 patients were recruited for comprehensive hormonal profiling and targeted molecular analysis. In this study, a genetic diagnosis was established in 22 patients, with 5ARD being the most common disease, followed by AIS. Traditionally the diagnosis of 5ARD relies on measuring dihydrotestosterone. However, with our experience in diagnosing this condition based on USP and mutational analysis of the SRD5A2 gene, two new diagnostic algorithms for 46,XY DSD were proposed where dihydrotestosterone is not required. / In vitro study is the preferred method for characterising the function of novel genetic variants. However, clinical laboratories rarely have the facilities and resources for it. In silico prediction programmes appear to be practical alternatives but their performance on testing non-synonymous variants in genes related to steroid metabolism has not been verified. Three web-based in silico prediction programmes, namely Sorting Intolerant From Tolerant, PolyPhen-2 and Pathogenic-Or-Not-Pipeline, were tested by analysing 797 published non-synonymous genetic variants in 12 genes related to steroid metabolism. The results of in vitro functional study and/or clinical phenotype were used as gold standards. The performance of these three programmes were: sensitivity (76.6%, 84.1%, 70.0%), specificity (56.6%, 56.3%, 89.4%) and accuracy (70.1%, 75.2%, 76.8%), respectively. / In conclusion, USP is a valuable biochemical phenotyping technique that helps to select patients for subsequent genetic confirmation. Since the mutation spectrum of 21OHD and the aetiological basis of 46,XY DSD in our population differ from the others, laboratory diagnostic algorithms and molecular analytical strategies must be adjusted accordingly. / Chan, On Kei Angel. / Thesis (M.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 250-269). / Appendixes includes Chinese.

Non-alcoholic fatty liver disease (NAFLD) results from an unequal uptake/storage and export/oxidation of lipids within the liver and is often a secondary disease to type II diabetes (22). NAFLD causes this imbalance of lipids by altering glucose and lipid metabolism, which corresponds to a decrease in mitochondrial function leading to failure of the liver. One established treatment for type II diabetes and NAFLD is the drug metformin, which has similar properties to the newly discovered CTRP 3 protein which is part of a group of bioactive molecules secreted by adipose tissue, collectively termed adipokines (2-4). Both have similar effects on hepatic glucose and lipid metabolism and both specifically suppress hepatic gluconeogenesis (11, 17, 27, 29). The revolutionary Seahorse extracellular flux analyzer was used to measure the metabolism of H4IIE hepatocytes without use of radiolabeling (1). By detecting the Oxygen Consumption Rate (OCR) of hepatocytes the level of metabolic function within mitochondria can be measured. Once an effective protocol was established using this new technology, hepatocytes treated with metformin had a significantly lower OCR compared to control treated hepatocytes treated. However, H4IIE hepatocytes treated with metformin and palmitate had a significant increase in OCR and eventually equilibrated with the lower OCR of hepatocytes solely treated with metformin. With similar effect, hepatocytes treated with CTRP3 and palmitate caused a drastic increase in OCR while hepatocytes treated with only CTRP3 had a decrease in OCR. This suggests that CTRP3 increases fatty acid oxidation which decreases lipid concentrations within hepatocytes which could mean future protection of liver against NAFLD. In conclusion, our Seahorse XF analyzer models compare metformin and CTRP3’s similarities and suggest the possible liver protective functions of CTRP3. Our results will aid in future research of CTRP3 to further determine its possible uses as a treatment for liver-associated diseases.

Adipocytes express angiotensin II (AngII) receptors; however the direct effects of AngII at the adipocyte remain unclear. Knockout mouse models of renin-angiotensin system components exhibit reduced body weight, reduced adiposity, improved glucose tolerance, and improved blood pressure when fed high fat diets, which may be due to reduced action of AngII through the AT1aR in adipocytes. Additionally, hypercholesterolemic AT1aR deficient mice are protected from AngII-induced increases in atherosclerosis and abdominal aortic aneurysm (AAA) formation. We hypothesized that deficiency of AT1aR in adipocytes would reduce the development of obesity, obesity-induced disorders, and vascular diseases. To test this hypothesis, we created a mouse model of adipocyte AT1aR deficiency using the Cre/LoxP system. Adipocyte-AT1aR deficiency confers no protection from the development of obesity or obesity- associated parameters. However, low fat fed adipocyte-AT1aR deficient mice exhibit remarkable adipocyte hypertrophy and reductions in adipocyte differentiation. These results demonstrate that AngII is a stimulus for adipocyte differentiation and adipocyte hypertrophy alone is insufficient to initiate obesity- associated disorders. In hypercholesterolemic mice, adipocyte AT1aR deficiency conferred no protection from diet or AngII-induced vascular diseases. Overall these studies suggest the primary role of adipocyte AT1aRs is to promote adipocyte differentiation and the development of small adipocytes.

Diseases, such as cardiovascular disease (CVD), are linked to chronic low levels of inflammation. This inflamed state is the product of risk factors including exposure to environmental pollutants, such as polychlorinated biphenyls (PCBs), which are correlated with increased risk for CVD and diabetes. In response to this health risk, our research addresses the mechanisms by which coplanar PCBs elicit an inflammatory response and the mitigation of PCB-induced inflammation through dietary intervention using docosahexaenoic acid (DHA), an omega-3 lipid. Investigators from the University of Kentucky Engineering Department are developing remediation technologies that detoxify PCBs through dechlorination. We studied the cellular toxicity of coplanar PCB 77 remediation products in primary vascular endothelial cells. The dechlorination products elicited different toxicological responses, which were less than the parent compound and contributed to the overall inflammatory response. The presence of PCB 77 at any concentration was sufficient to promote an inflammatory response, which was attenuated with complete dechlorination. PCB 77 is a good model for coplanar PCB-induced toxicity, but in environmental and human samples, coplanar PCB 126 is detected more frequently. Using different doses of PCB 126, we determined that acute exposure to 5 μmol PCB 126/kg mouse was sufficient to produce an inflammatory response without inducing a toxic wasting phenotype. PCB-induced inflammation was attenuated in vitro by DHA-derived neuroprostanes. Applying this information, we fed mice a DHA-enriched diet and exposed them to PCB 126. Liver and adipose lipid profiles confirm an increase in omega-3 fatty acid composition and DHA metabolites, and changes in gene expression indicate a heightened anti-oxidant response in the presence of PCB-induced inflammation. These data provide an overview of the in vivo response to a PCB-induced inflammation after DHA dietary feeding. We have demonstrated that PCB-induced endothelial dysfunction is propagated through lipid domains called caveolae. Caveolae are also signaling domains for toll-like receptor 4 (TLR4), and receptor for lipopolysaccharide (LPS). Similar to PCBs, TLR4 signaling is inhibited by DHA. We compared the caveolae-associated signaling response after exposure to coplanar PCB 126 or LPS. The domain localization of caveolae was altered by both PCB 126 and LPS. Our study determined that PCB 126-induced inflammation was not inhibited by a TLR4-specific inhibitor, but caveolae-based signaling was critical to both PCB 126- and LPS-induced inflammation. Environmental pollutants, such as coplanar PCBs, are risk factors in the development of chronic diseases. Here we investigate possible signaling pathways associated with environmental toxicity and apply potential dietary interventions with omega-3 lipids.

Type 2 diabetes mellitus is a metabolic disease that affects one in ten people in the United States. It is caused by a combination of genetics and lifestyle factors. Disease progression begins with insulin resistance in peripheral tissues followed by pancreatic beta-cell failure. The mechanisms behind disease progression are not completely understood. PH domain leucine rich repeat protein phosphatase 1 (PHLPP1) is a known regulator of Akt and other members of the AGC kinase family. Akt has been established to play a role in numerous metabolic signaling pathways, including insulin action. It is hypothesized that as a regulator of Akt, PHLPP1 would have an important function in glucose homeostasis. Glucose tolerance tests performed on 8-week old Phlpp1-/- mice revealed no significant difference in glucose tolerance compared to wild type, however these mice did exhibit increased fasting blood glucose levels. Glucose tolerance tests were repeated at 20 weeks on the same mice and, interestingly, they displayed impaired glucose tolerance compared to wild type. Insulin tolerance tests showed that 8-week old mice have increased insulin sensitivity, however, the 20-week old mice were insulin-resistant compared to control animals. The 20-week old knockout mice also had significantly higher fasting blood glucose levels compared to 8-week old mice. To determine if the increased fasting blood glucose levels are due to increased hepatic glucose output, pyruvate tolerance tests were performed on both the 8 & 20 week old mice. Old mice displayed significantly increased hepatic glucose production compared to wild type. EchoMRI done on 24-week old mice showed significantly increased fat mass and decreased lean mass in the Phlpp1-/- mice compared to wild type littermates. Western blot analysis of liver samples from 32 week old Phlpp1-/- mice indicates loss of Akt signaling accompanied by a decrease in IRS2 protein levels, a common indicator of insulin resistance. These data suggest that Phlpp1-/- mice mimic the development of type 2 diabetes in humans, and provide a unique animal model to study the progression of type 2 diabetes and diabetes-associated complications.

Thesis (Ph.D.)--University of Wisconsin--Madison, 2006 / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--University of Wisconsin--Madison, 2006 / Includes bibliographical references. Also available on the Internet.

Os objetivos da tese são, investigar (i) as estruturas terciária e quaternária de diversas proteínas em solução e (ii) as mudanças conformacionais de proteínas induzidas por ligantes e/ou temperatura, variando-se a concentração dos mesmos, etc. A principal técnica utilizada foi a de espalhamento de raios X a baixos ângulos (no inglês SAXS de mall-angle X-ray scattering), associada a outras metodologias como cristalografia e modelagem de proteínas. As contribuições dos estudos realizados de várias proteínas de interesse científico foram as seguintes: (i) propôs-se pela primeira vez um modelo de um receptor nuclear (RXR) composto por dois domínios, um de ligação ao ligante e outro ao DNA; (ii) elucidou-se o estado configuracional da fosfo-enol-piruvato carboxoquinase em solução; (iii) foi determinado o efeito de agregação e mudança conformacional induzido por ligantesna isoenzima fosfofrutoquinase e (iv) determinou-se a estrutura em solução da interleucina humana 22 e propôs-se modelos de interação com seus receptores. Além disso, (i) foi desenvolvido um método para determinar o estado oligomérico de uma proteína em solução, em quaisquer condições de tampão e concentração, utilizando medidas de SAXS em escala relativa, e (ii) mostrou-se que a densidade das proteínas é uma função do seu peso molecular, contrariando conceitos clássicos, tendo as proteínas pequenas (< 20kDa) uma densidade mais elevada que as maiores. / The purpose of this thesis is to investigate (I) the tertiary and quaternary structure of several proteins in solution and (II) conformational changed that such proteins undergo when they bind to ligands or temperature, pH, etc. conditions are varied. The main experimental technique utilized in this study was Small Angle X-Ray Scattering (SAXS) associated to other techniques and approaches like protein crystallography and homology modeling. The main contributions related to this work are: (i) for the first time a model for a nuclear receptor (RXR) containing two domains was proposed, one DNA binding domain and one ligand binding domain; (ii) the oligomeric state of the phosphor-enol-pyruvate caboxykynase in solution was detennined; (iii) the conformation changed induced by ligants to the isoenzyme phosphofructokinase was determined and (iv) the solution structure of human interleukin revealed and a model with its receptor proposed. Additionally, (i) a method for determining the oligomeric state of a protein in solution under any buffer conditions and concentration using SAXS in relative scale is proposed and (ii) it was also shown that proteins density is a molecular weight dependent function, where small proteins with less than 20kDa have in average a larger density than bigger ones.

No description available.

Hypoxia is a significant low oxygen state that has complex and diverse impacts on organisms. In aerobes, various adaptive responses to hypoxia are observed that vary depending on the level of oxygen depletion and previous adaptation, hence the continued attention to hypoxia as an important abiotic stressor. Adaptive responses to hypoxia are primarily governed by the hypoxia-inducible factors (HIFs), which activate downstream genetic pathways responsible for oxygen transport and metabolic plasticity. In aquatic habitats, oxygen availability can vary greatly over time and space. Therefore, aquatic organisms’ adaptation to hypoxia is likely pervasive, especially in genotypes originating from waterbodies prone to hypoxia. Here we report the transcriptional response to severe hypoxia in the freshwater crustacean Daphnia magna. We observe improved survival in media containing elevated calcium ion (Ca2+) concentrations. Additionally, we observe changes in lactate and pyruvate concentrations within tissues. To elucidate the transcriptome basis of these effects, we examine transcripts with known gene ontologies indicating roles in Ca2+ homeostasis and signaling, and in pyruvate metabolism, including gluconeogenesis (GNG). We observe the up-regulation of numerous transcripts encoding GNG pathway enzymes, including the rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK-C) and fructose-1,6-bisphosphatase (FBP). In contrast, no transcripts involved in Ca2+ homeostasis or signaling showed any significant differential expression. Some GNG transcripts are more up-regulated in clones from permanent waterbodies not prone to hypoxia, inconsistent with the hypothesis about its protective effects. One exception is the FBP transcript, which has been identified to be up-regulated in some hypoxia-tolerant aquatic organisms.