Doctoral Degrees (Chemical Pathology)

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    Cellular and biochemical effects induced by antiretroviral drugs.
    (2013) Anwar, Khawar.; Pillay, Tahir S.; Chuturgoon, Anil Amichund.
    In the treatment of HIV/AIDS, protease inhibitors (PIs) and nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs) are the major components of highly active antiretroviral therapy (HAART). The side effects of these drugs include various metabolic disorders including insulin resistance, dyslipidaemia and lipodystrophy. The precise mechanistic basis of these remains largely unknown. In this study we aimed to understand the molecular basis of these metabolic effects by analysing the effects on lipoprotein lipase (LPL) activity, insulin signaling and the cellular metabolic profile. It was previously shown by this group that indinavir inhibits insulin signaling at a proximal level. The study was extended to a wider range of ARVs and in particular, the effects of sodium salicylate (NaSal) and berberine chloride (BBR) were analysed to determine if they could reverse the effects of the drugs on insulin signalling. In addition, Chinese hamster ovary cells transfected with the human insulin receptor (CHO-IR) were used for the first time to study the effects of NRTIs on the insulin signaling pathways. The high level of expression of insulin receptor facilitated sensitive detection of any alteration in the phosphorylation of signaling proteins as compared to 3T3-L1 adipocytes. Three PIs, indinavir, nelfinavir and ritonavir were used in this study. Indinavir and nelfinavir treatment significantly reduced the insulin-stimulated phosphorylation of the IRβ, IRS-1, Akt and MAPK in CHO-IR cells. However phosphorylation of GSK-3α\β was not affected by the PIs. Ritonavir also decreased (not statistically significant) the phosphorylation of IR-β and IRS-1 but its inhibitory effect on MAPK was the same as by the other PIs. NRTI’s did not inhibit insulin-stimulated tyrosine phosphorylation of IRβ and IRS-1 but reduced phosphorylation at MAPK and Akt. In order to understand the role of NFκ-B pathway in blocking insulin-stimulated tyrosine phosphorylation, IKK-16, a selective inhibitor of IkB kinase (IKK) was used but no significant involvement of this pathway was found in blocking tyrosine phosphorylation at IRS-1. Similarly, NaSal and BBR were also used to reverse the effects induced by PIs and NRTIs in CHO-IR cells but no significant change was observed on Akt and MAPK. NaSal and BBR reduced (but not significantly) the effects of PIs (indinavir and nelfinavir) on IR-β and IRS-1. These findings suggest that PIs induce insulin resistance by affecting multiple steps in the signaling pathway. At the proximal end of the insulin signalling pathway, protease inhibitors affect IR-β and IRS-1 while at the distal end they affect phosphorylation of Akt and MAPK. CHO-IR cells were also used to measure LPL activity using a colorimetric method employing pNPB as substrate. The most commonly used ARVs were tested. These included four PIs and six NRTIs .The results showed that NRTIs stavudine and emtricitabine significantly inhibited the LPL activity from the CHO-IR cells. PIs indinavir and nelfinavir were also found to decrease LPL activity extracellularly when added to the assay reaction in vitro. Similarly nelfinavir and atazanavir sulfate inhibited the activity of the LPL from the CHO-IR cells after 16 hour treatment. This suggested that these drugs may interfere with the enzyme activity intracellularly either at the level of its synthesis or its transportation from cytoplasm to the cell surface. These finding suggests that protease inhibitors may play a role in inhibiting lipoprotein lipase activity in vivo, and may thereby induce metabolic disorders in HIV-positive patients being treated with protease inhibitors. Metabolomic analysis was performed on the supernatant of cells treated with PIs and NRTIs, with and without insulin stimulation. Many significant alterations and trends in amino acids and organic acids levels in CHO-IR cells supernatants (treated with PIs) were recorded using 1H-NMR. For example, PIs decreased the synthesis of threonine, phenylalanine, lysine, arginine, isoleucine butyrate, glutamate, histidine and 2-oxo-isovalerate. Furthermore overproduction of lactate and ketones were observed in the nelfinavir treated cells. This may be a consequence of the secondary effects of insulin resistance induced by the PIs. Similarly NRTIs (stavudine and tenofovir) treatment also induced changes in the levels of many amino acids and organic acids .NRTIs decreased the synthesis of acetate, acetoacetate, histidine, methionine, phenylalanine and tryptophan while significant increase in the acetone was observed in stavudine-treated cells. Taken together, the results of this study suggest that PIs and NRTIs, inhibits LPL activity and synthesis, affects insulin signalling pathways at different levels and alters the synthesis of different cellular metabolites which may affect the signalling pathways of insulin.
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    Control of prostaglandin biosynthesis by the intrauterine tissues in primary dysfunctional human labour.
    (1987) Reddi, Kogie.; Norman, R. J.
    No abstract available.
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    Non-insulin-dependent diabetes in young Indians : a clinical and biochemical study.
    (1982) Jialal, Ishwarlal.
    One of the earliest recorded references to polyuria is found in the Papyrus Ebers (1500 BC) and much later the occurrence of "honey urine" was noted by an ancient Hindu physician, Sushrutha, in old Indian Sanskrit (400 BC). However, the first good clinical description of the disease is ascribed to Celsus, although the name "diabetes" was introduced by Aretaeus of Cappadocia. The body of knowledge which has accumulated since these early recordings to the present state of the art reflects a most impressive sojourn, punctuated by many milestones, each adding impetus to future attempts in a relentless endeavour to unravel the aetiopathogenesis of this common malady. However, this "sweet evil" (diabetes) remains an enigma in many ways. There is little doubt today that there are 2 major types of diabetes: juvenile onset diabetes, presently known as insulin-dependent diabetes mellitus (IDDM) and maturity onset diabetes, referred to as non-insulin dependent diabetes mellitus (NIDDM). In NIDDM aggregation of HLA types, evidence of cell mediated immunity and the presence of circulating islet cell antibodies, which are characteristically associated with IDDM, are not found. There is also a vast difference in concordance of diabetes in the co-twins between the two types of diabetes suggesting that a different mixture of genetic and environmental factors is operative in the pathogenesis of these two types of diabetes. In I960, Fajans and Conn drew attention to the existence of a form of diabetes with an onset before the age of 35 years. Their patients showed a substantial improvement in glucose tolerance when treated with an oral hypoglycaemic agent, tolbutamide. Subsequent to this report numerous studies from various parts of the world confirmed this entity of non-insulin dependent diabetes in the young (NIDDY) in White Caucasians. There are, however, several different syndromes presenting as mild carbohydrate intolerance in the first two to three decades of life. The classical form of NIDDY is a mild non-insulin requiring form of diabetes in which the disorder is inherited as a dominant trait; there is little progression of glucose intolerance, if any, with time, and the diabetes is rarely accompanied by vascular complications. This subtype of diabetes is referred to as MODY (maturity onset diabetes in the young) and thus constitutes a subset under the broad umbrella of NIDDY. However, recently compelling evidence for heterogeneity within MODY has been presented. This evidence is based on the prevalence of certain HLA antigens, insulin responses to oral glucose, occurrence of vascular complications, progression of hyperglycaemia to the stage of insulin requirement and failure to demonstrate autosomal dominant inheritance in some families studied. In the South African Indian population which has a high prevalence of diabetes, Campbell was the first to draw attention to NIDDY in Indians more than two decades ago. Since this initial report, nobody has really studied NIDDY in any depth in South Africa and certainly not in the Indian population. NIDDY in the local Indian population is of particular interest for the obvious reason that diagnostic and management problems arise daily in a population with a high prevalence of non-insulin dependent diabetes. It is vital that the clinical features, endocrine and associated biochemical aberrations be known in detail if this condition is to be managed appropriately and adequately. A study of these aspects therefore became the primary task of this thesis. To pre-empt any challenge that patients were not really diabetic, the strict criteria of the W.H.O. for the diagnosis of diabetes were chosen. It should therefore be borne in mind throughout this study that a group of rather severe diabetics were selected by design. The patients studied represent the rather extreme end of the spectrum. But, in the event, this selection proved advantageous in that it covered an unstudied part of the spectrum and some light could be shed on the natural history of the disorder. In the long term the purpose was to prepare the ground for what must become the thrust of future studies, namely the biochemical pathogenesis of NIDDM. If it is true that some forms of NIDDY are inherited dominantly, existing techniques should make it possible to identify a gene(s) locus and if this is done the biochemical basis of this disorder must be identifiable. In the present study direct examination of these aspects were not undertaken, but an attempt was certainly made to pinpoint those biochemical abnormalities which are perhaps primary or central to the whole disorder.