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In recent years there has been an explosion in the rates of obesity, defined as a body mass index greater than 30kg/ m2, and associated cardiovascular disease. Alterations in peripheral glucocorticoid metabolism have been suggested to play a role in the development of obesity. Obesity occurs in both sexes, but the risk of associated metabolic disturbance and vascular dysfunction is greater in men. Although there is no accepted definition of obesity in rodents, the term is used to describe animals with a significant increase in fat pad mass often achieved by feeding a high fat diet. Although animal models of obesity have been useful in delineating potential mechanisms linking obesity with its metabolic and vascular sequelae, most studies have been in male animals and, thus, have not addressed sex differences. Additionally, emerging evidence shows that obesity during pregnancy is associated with increased cardio-metabolic and vascular disease in offspring, although the processes underlying such ‘programming’ effects are unclear. This thesis addresses the hypothesis that exposure to postnatal, or maternal obesity will alter both metabolism and vascular function in mice. Male and female mice maintained on a high fat and sugar diet from 5 weeks of age had increased adipose tissue deposition in adulthood. However there were striking sex differences in glucose homeostasis, mRNA levels and glucocorticoid metabolism, with males being more severely affected. Treatment of male mice with 17β-estradiol ameliorated a number of the effects of the high fat diet, including weight gain and altered glucose homeostasis; additionally estradiol altered glucocorticoid metabolism in the adipose so that it resembled that of females. Suprisingly, given the changes in metabolism, obesity in adult mice produced only small changes in vascular function and did not alter vascular remodelling following injury. The effects of maternal obesity were studied using male offspring aged 3 and 6 months. The offspring of obese mothers had similar body weight, adiposity, plasma lipid and plasma hormone concentrations to controls. In contrast, exposure to obesity in utero was associated with receptor specific changes in agonist-mediated contraction and decreased endothelium-dependent relaxation in male offspring. Despite these changes in vascular function, no alterations in blood pressure or vascular remodelling following injury were present. These results demonstrate that the more profound changes in glucose-insulin homeostasis associated with obesity in male humans can be recapitulated in rodent models and imply that estradiol plays a role in protecting the metabolism of female mice, potentially by alteration of glucocorticoid metabolism. Despite altered metabolism in postnatal obesity vascular function remained normal suggesting metabolic and vascular dysfunction are not intrinsically linked. Conversely, maternal obesity did not cause any overt changes in offspring metabolism but caused vascular dysfunction implying these parameters can be programmed independently.

published_or_final_version / Chemistry / Master / Master of Science

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Peripheral steroids and oxycholesterols are important lipid compounds controlling various functions in the human body. Steroid analysis of biological samples is routinely employed in the clinical environment as an essential source of information on endocrine and metabolic disorders. It has been reported that stress related neurosteroids have been implicated in the development and prognoses of neurodegenerative disorders such as Alzheimer’s disease (AD). These compounds have been identified as possible biomarkers in the diagnosis of AD and other neurodegenerative disorders. Therefore, methods for the simultaneous analysis of steroids from the four major classes (estrogens, androgens, progestogens and corticosteroids) are vital in providing useful and more comprehensive data. Homeostasis of cholesterol in the brain is maintained primarily by metabolism to oxysterols, including oxycholesterols. These oxycholesterols act as a transport form of cholesterol as it readily navigates the blood-brain barrier. Oxycholesterols are generally more bioactive than cholesterol and is of interest in pathophysiology. Moreover, if their production in cells and tissues and/or their introduction with dietary animal fat are excessive, oxycholesterols could indeed contribute to the pathogenesis of various disease processes. The first study in this thesis focuses on a novel supercritical fluid chromatography–tandem mass spectrometry method for targeted analysis of eighteen peripheral steroids. The method is simple and fast. It has sufficient sensitivity for quantification of 18 different steroids in small volume human plasma. Therefore, this novel method can be applied for screening many steroids within 5 minutes providing the possibility to use for routine healthcare practice. The second study involves the quantification of three adrenal steroids in plasma from domesticated White Leghorn (WL) chickens and Red Junglefowl (RJF) birds. The domestication effects on stress induced steroid secretion and adrenal gene expression in chickens are evaluated. The third study focuses on determination of more than ten oxycholesterols in biological samples with a gas chromatography–mass spectrometry method and a supercritical fluid–tandem mass spectrometry method.

PhD,School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand / Tissue specific regulation of the progesterone receptor is central to female health. The synthetic steroid, RU486 is a partial progesterone and oestrogen receptor antagonist, functioning to actively silence progesterone receptor gene associated transcription. For this reason, it has been used both as a contraceptive and quite controversially as an abortive agent. In this study, both cellular and gene specific effects of RU486 were investigated in a rat model of early pregnancy, this including the key phases of the plasma membrane transformation, the window of receptivity and early implantation. As all of these stages are hormonally regulated by progesterone and oestrogen, the focus here was to elucidate the mechanisms of action of a single dose of RU486, used as a postcoital contraceptive, at day 3.0 of pregnancy, to successfully prevent implantation of a viable blastocyst and subsequent pregnancy. In association with the cellular preparation of uterine epithelial cells for implantation, selected molecular targets and events were investigated at a protein and gene expression level, both prior to and after RU486 treatment, to assess the effects of either a deficit or excessive expression of these gene products on uterine preparation and eventual implantation. Factors here included the progesterone receptor, markers of apoptosis (Bax and Bcl2), mediators of angiogenesis (VEGF, bFGF and PDGF) and biomarkers of endometrial implantation (LIF, Calcitonin and Muc-1). Together, an ultrastructural and light microscopy analysis showed RU486 to morphologically alter the uterine endometrial cells and to disrupt the plasma membrane transformation of early pregnancy, predisposing these cells towards apoptosis. In association with this, progesterone receptor gene and protein expression was ubiquitously decreased throughout pregnancy. With regards to the implantation process of early pregnancy, the luminal epithelial cells undergo apoptosis to allow the hatching blastocyst to penetrate and implant within the uterine wall. This is partially mediated by the ratio of the expression of the apoptotic factors Bax and Bcl-2. Surprisingly here, RU486 caused an overall anti-apoptotic environment, despite previously observed high levels of apoptotic activity. This indicates that factors other than Bax and Bcl-2 influence the RU486-induced apoptosis. A crucial event of early pregnancy is the establishment of an adequate blood supply to sustain and nourish the implanting blastocyst. There was a decided reduction in the angiogenic response of early pregnancy, as a direct consequence of RU486 treatment; the normally high levels of VEGF and bFGF during early pregnancy, were markedly decreased at all three days of pregnancy. This was reflected in the lack of increased vascularisation as normally signalled by the indicator dye, Pontamine Sky blue. In contrast to the overall increase in VEGF and bFGF at the time of blastocyst implantation during early pregnancy, increased PDGF expression was localised to the implantation sites, strongly suggesting a role for this angiogenic factor in endothelial cell proliferation. v The endometrial biomarkers are indicative of implantation, their expression patterns varying around the phase of implantation. These markers are essential to implantation, as when LIF and Calcitonin are deregulated and Muc-1 persists on the apical surface of the endometrium, implantation fails. These events are precisely what occur following RU486 treatment. In summary, the overall effects of RU486 in the rat model of early pregnancy, when used as a postcoital contraceptive, indicate highly effective inhibition of progesterone and oestrogen effects on the endometrium, mediated by their receptors. More specifically, the structural and molecular events mirror those described in ovariectomised animal models, suggesting a hormonally under-stimulated endometrium. Clearly from the present study, the precise priming of the endometrium in preparation for blastocyst implantation is severely impaired by RU486 through a number of signalling pathways, thus predisposing the uterus to pregnancy failure.

O fato de esteróides de plantas apresentarem tanto funções fisiológicas quanto ecológicas, talvez tenha sido fator limitante de seu uso em quimiossistemática. Até hoje, os trabalhos do nosso grupo têm se restringido a marcadores com apenas funções ecológicas. Neste trabalho, utilizamos esteróides como marcadores quimiossistemáticos, com o intuito de descobrir se eles poderiam ser analisados com a metodologia usual desenvolvida por nosso grupo. Devido à ampla distribuição dessas moléculas, limitamos o trabalho a angiospermas. Inicialmente, realizamos um levantamento bibliográfico no Chemical Abstracts (até dezembro de 1986) e, em uma segunda etapa, codificamos e armazenamos os dados em um microcomputador IBM-PC. A presença de alguns esteróis (campesterol, sitosterol, colesterol e estigmasterol) em praticamente todos organismos, levou-nos a eliminar deste trabalho esse grupo de substâncias, pois características imutáveis não têm valor quimiossistemático. Baseados em semelhanças estruturais, separamos os esteródes em oito tipos, a saber: esteróides, brassinolídeos, ecdisonas, sapogeninas, witanolídeos, pregnanos, cardenolídeos e bufodienolídeos. Esta classificação foi legitimada por análises estatísticas (testes não-paramétricos). Os índices de oxidação e especialização de esqueleto (metodologia do nosso grupo) não revelaram muitas diferenças dentro de cada tipo esteroidal. De um modo geral, estes tipos esteroidais apresentam-se predominantemente distribuídos em certos grupos de angiospermas. Este é, por exemplo, o caso das ecdisonas em Caryophyllales e Lamiales, dos witanolídeos em Solanales e, dos cardenolídeos e pregnanos em Gentianales. Por outro lado, enquanto superordens, como Magnoliiflorae, apresentam apenas esteróis (tipo menos oxidado e menos especializado), outras superordens, como Ranunculiflorae, Solaniflorae e Liliiflorae, apresentam uma fantástica diversidade de tipos. As monocotiledôneas se caracterizam pela presença de saponinas, e os outros tipos (exceto esteróis) aparecem com distribuição esparsa. Comparando nossos dados com os de pteridófitas gimnospermas (apenas esteróis e ecdisonas), podemos concluir que a diversidade dessa classe de micromoléculas em angiospermas coloca os esteróides na posição de uma característica evoluída de plantas. / Plant steroids perform not only ecological, but also physiological functions. Hence it was thought a priori that their use as chemosystematic markers should be of doubtful value. The present work aimed to evaluate the correctness of this concept. In view of the vast natural distribution of steroids we restricted our efforts to the angiosperms. Initially we undertook a literature survey in Chemical Abstracts (up to 1986) and then we codified and stored the data in a microcomputer IBM-PC. Some of the steroids (campesterol, colesterol, sitosterol and stigmasterol) occur practically in alI organisms. Since constant characters are of little systematic value, such compounds were omitted from our study. According to structural analogies we separated plant steroids in eight types: simple steroids, brassinolides, ecdysons, sapogenins, withanolides, pregnanes, cardenolides and bufodienolides. This classification was authenticated by statistical analyses (non parametric tests).The skeletal speciaIizations and the oxidation levels (according to methodology developed by our group) did not differ greatly wthin each steroidal group. However the general distribution of steroids in angiosperms is quite selective. This is the case e.g. for ecdysons in Caryophyllales and Lamiales. On the other hand, while superordens such as Magnoliiflorea are characterized only by steroids (the least specialized and oxygenated type), other superordens such as Ranunculiflorae, Solaniflorae and Liliiflorae are characterized by a very considerable number of types. The monocotyledons contain chiefly saponins, other types (except steroids) appearing more sparsly distributed. In contrast to angiosperms pteridophytes and gymnosperms posses only few steroidal types (simple steroids and ecdysons). Hence diversity of this micromolecular class is an indication of phylogenetic advance in the plant kingdom.