Hyperglycaemia caused by insulin deficiency is believed to play a major role in the de-velopment of neuropathy in diabetic patients. The clinical and pathological features of diabetic neuropathy vary considerably, although sensory and autonomic dysfunctions are the most common characteristics. Normalisation of the blood glucose level by ef-fective insulin treatment decreases the incidence of diabetic neuropathy in patients. However, intensive insulin therapy may result in more frequent hypoglycaemic epi-sodes than are provoked by less ambitious diabetes control. Neuropathy might also be induced by severe hypoglycaemia in diabetes or insulinoma. Accordingly, it seems that the diversity in clinical symptoms of diabetic neuropathy may be due to the combined effects of hyperglycaemia and hypoglycaemia. Based on that assumption, the general aim of this project was to study the relationship between severe hypoglycaemia and pe-ripheral neuropathy in diabetic rats. To understand how the development of neuropathy is related to glycaemic control, we needed to be aware of the glucose dynamics in the animal model that we used. The aim was to ascertain whether the diabetic rats were similar to type 1 diabetic patients with regard to such dynamics. To achieve that goal, we used a MiniMed continuous glucose monitoring system (CGMS®) to measure sub-cutaneous glucose in freely moving rats over a period of 72 hours. The glucose monitor worked well, and it showed that the insulin-treated diabetic BB/Wor rats with a hyper-glycaemic insulin regimen have a glycaemic status similar to that of type 1 diabetic patients with poor glycaemic control. The diabetic rats with a hypoglycaemic regimen generally had low blood glucose levels. Prolonged hypoglycaemia led to axonal de- and regeneration of large myelinated fibres in vagus nerve destined to the laryngeal muscle. Axonal de- and regeneration was also observed in the gastrocnemius and sural nerves, although the frequency of degeneration was much lower in the sural nerve. Small myelinated and unmyelinated nerve fibres were normal in these nerves. These results suggest that hypoglycaemia preferentially damages muscle-related nerve fibres. In contrast, in the diabetic rats exposed to pro-longed hyperglycaemia, only the sural nerve exhibited decreased myelinated fibre diameter in the absence of obvious axonal degeneration. In situ glucose measurements by microdialysis showed that the glucose concentrations in blood and subcutaneous tissue were similar in healthy, diabetic hyperglycaemic, and diabetic hypoglycaemic rats. In the healthy and hyperglycaemic animals, the lowest glucose level was found in the peripheral nerve. Moreover, in controls, the glucose level was lower in muscle than in blood. In hypoglycaemic rats, there were no signifi-cant differences in glucose concentrations between different tissues.

Master of Science / Department of Clinical Sciences / Thomas Schermerhorn / The use of continuous blood glucose monitors (CBGMs) has recently come into favor in human medicine for the control and monitoring of the diabetic patient. It allows for a higher degree of accuracy of the true glucose curve throughout a 72-hour period. With this information, physicians are better equipped to treat and manage diabetic patients. Recently, this modality has been verified for use in veterinary patients including cats and dogs. This is an excellent source of information, especially in the management of difficult to regulate veterinary patients. This device has potential for use in various applications, particularly for the monitoring of patients with various diseases under general anesthesia. In order to ensure accurate results do occur when an animal is under general anesthesia, the continuous blood glucose monitor was evaluated on apparently healthy patients under anesthesia for routine procedures such as ovariohysterectomies and orchiectomies. In this manner, the monitor was tested on anesthetized patients that had the potential to experience hypothermia, hypotension, and other anesthesia-associated complications that can be typical of patients that could benefit from the CGMS.

Diabetes is a group of metabolic diseases characterized by hyperglycaemia resulting from defects in insulin secretion, insulin action, or both. Several models have been developed for describing the glucose-insulin system. Silber and Jauslin developed a semi-mechanistic integrated glucose insulin (IGI) model which simultaneously describe glucose and insulin profiles in either healthy subjects or type 2 diabetis mellitus (T2DM) patients. The model was developed for describing the basal system, i.e. when no drugs are present in the body. In this thesis the IGI model was extended to also include the effects of anti-diabetic drugs on glucose homeostasis. The model was extended to describe postprandial glucose and insulin excursions in T2DM patients treated with either biphasic insulin aspart or the GLP-1 receptor agonist liraglutide. These extensions make the model a useful tool in drug development as it can be used for elucidating the effects of new products as well as for clinical trial simulation. In this thesis several modelling tasks were also performed to get a more mechanistic description of the glucose-insulin system. A model was developed which describes the release of the incretin hormones glucosedependent insulinotropic polypeptide and glucagon-like peptide-1 following the ingestion of various glucose doses. The effects of these hormones on the beta cell function were incorporated in a model describing both the C-peptide and insulin concentrations in healthy subjects and T2DM patients during either an oral glucose tolerance test or an isoglycaemic intravenous glucose infusion. By including measurements of both C-peptide and insulin concentrations in the model it could also be used to characterize the hepatic extraction of insulin.

Oesophageal adenocarcinoma (OAC) is a leading cause of cancer death in the UK and current systemic therapies are ineffective for the majority of patients. The central aim of this work was to explore strategies to identify novel therapeutic targets. Research has failed, thus far, to identify a dominant oncogene in OAC, although the tumour suppressor p53 is frequently mutated. Inhibiting the mitotic kinase, polo-like kinase 1 (PLK-1), was proposed as a synthetic lethal strategy. PLK-1 was demonstrated to be over-expressed in both verified OAC cell lines and human OAC tissue compared to non-transformed cells and epithelium. Mutation of p53 was associated with over-expression of PLK-1 in both OAC and ovarian cancer tissue. Using a carefully validated viability assay, both an established and novel PLK-1 inhibitor were demonstrated to induce a G2/M arrest and reduce OAC cell proliferation. Relative selectivity was demonstrated for OAC compared to non-transformed cells. This therapeutic window could be enhanced with the induction of cancer cell cytotoxicity by pulsed administration of a short half-life inhibitor. Immunotherapeutics offer potential tumour-selectivity but no OAC-specific proteins have been defined. A comparative proteomic approach was employed to identify OAC-specific proteins as potential therapeutic targets. A tissue resource was established and methods to lyse fresh frozen biopsies optimised. An isobaric quantitative proteomic workflow was applied to OAC and matched normal biopsies and quantitative accuracy confirmed for 6 candidate proteins by immunohistochemistry. Proteome coverage and quantitative dynamic range were compared between isobaric and label-free systematic sequencing proteomic strategies applied to further patients’ tissues. The challenges of combining incomplete datasets were approached with a Bayesian framework to estimate the probability that a protein was missed during an experiment compared to not being present in the sample. This method was applied to generate a complete set of protein identifications and relative tissue expression. To gain insight into the dysregulated cellular processes in human OAC tissue, a network analysis was applied to the quantitative proteomic data. Enriched functional clusters were identified suggesting deranged glucose metabolism, potentially due to the Warburg effect. These findings were duplicated and candidate tumour-specific proteins identified in a further set of biopsies using the optimised quantitative proteomic method. The combined quantitative oesophageal proteomic dataset represents the largest in OAC to date. This thesis demonstrates a hypothesis-driven, synthetic lethal approach can yield cancer-selective therapeutic effects. Novel candidate therapeutic targets are also revealed through the development of quantitative proteomic methods and the application of network analysis.

<p>Understanding the mechanisms that control T cell function and differentiation is crucial to develop new strategies to modulate immune function and prevent autoimmune and inflammatory disease. The balance between effector (Teff; Th1, Th2 and Th17) and regulatory (Treg) T cells is critical to provide an appropriate, but not excessive, immune response and therapies to induce Treg or inhibit Teff are likely promising treatment strategies. It has recently become clear that T cell metabolism is important in both T cell activation and differentiation. T cells undergo a metabolic reprogramming upon activation and not all differentiated T cell subsets utilize the same metabolic fuels or programs.</p><p>These metabolic differences are not trivial, as T cell metabolism is tightly</p><p>regulated and dysregulation can lead to cell death or reduced immunity. An</p><p>understanding of the metabolic differences between Teff and Treg may lead to a new direction for treating inflammatory diseases by modulating the Teff:Treg balance through metabolic inhibition. Previous studies have shown that Teff express higher levels of the glucose transporter Glut1 than Treg, however the role of Glut1, and importantly, the cell-intrinsic role of glucose metabolism in T cell differentiation and inflammation was not previously examined. The work presented here examines the role of Glut1 in T cell differentiation. We show that effector CD4 T cells were dependent on Glut1 for proliferation and function both in vitro and in vivo. In contrast, Treg were Glut1-independent and capable of suppressing colitis in the absence of Glut1 expression.</p><p>Additionally, previous studies have shown broad metabolic differences between Teff and Treg, however the specific metabolic profiles of Teff and Treg are poorly understood. Here, Teff and Treg metabolism is examined to test if dependence on distinct metabolic pathways will allow selective targeting of different T cell populations. We show that pyruvate dehydrogenase kinase 1 (PDHK1) is differentially expressed in the T cell subsets and inhibition of PDHK1 selectively suppresses Th17 and promotes Treg differentiation and function. Because Teff and Treg have distinct metabolic profiles, we hypothesized that the Treg-­specific transcription factor FoxP3 may drive the Treg oxidative metabolic program. We therefore examined the role of FoxP3 in T cell metabolism and determined that FoxP3 promotes glucose and lipid oxidation and suppresses glycolytic metabolism. Importantly, we show that promoting glycolysis with transgenic expression of Glut1 inhibits Treg suppressive capacity. Together, these data suggest that FoxP3 drives an oxidative metabolic program that is critical to Treg function. Overall, this work examines the metabolic phenotypes and regulation of Teff and Treg and potential metabolic targets that could be used to treat autoimmune and inflammatory disease.</p> / Dissertation

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: While antiretroviral therapy decreases HIV/AIDS morbidity and mortality, long-term treatment results in insulin resistance and cardiovascular diseases. A possible cause of such adverse effects may be an increase in oxidative stress resulting from protease inhibitor (PI)-induced mitochondrial dysfunction. We therefore hypothesized that PI treatment, specifically Lopinavir/Ritonavir, results in increases in myocardial reactive oxygen species (ROS), leading to downstream outcomes, i.e. elevated apoptosis. Moreover, we proposed that increased ROS levels in this instance might occur as a result of PI-mediated induction of the non-oxidative glucose pathways (NOGPs). In light of this, we also investigated the effect of PI treatment on the NOGPs by employing both in vitro and in vivo samples. For the in vitro work we employed a rat cardiomyoblast cell line, while tissues (heart, liver) were collected from two separate experimental models, i.e. a) Group A exposed to PIs via mini-osmotic pump for a period of eight weeks, and b) Group B administered PIs via a jelly-based method for 16 weeks. We found that PIs increased mitochondrial ROS levels in vitro but that this was not accompanied by a parallel rise in programmed cell death. Moreover, we found no induction of the NOGPs in response to PI exposure (for both in vitro and in vivo models here employed). However, we found that the AGE pathway was significantly down-regulated in the liver of Group A. Investigation into a proposed mechanism for this observation proved inconclusive and further studies are thus required to clarify the significance in terms of metabolic dysfunction found in the Group A model. Our study thus shows that PIs can increase ROS levels (in vitro) but that compensatory antioxidant mechanisms may prevent this in vivo. Subsequently, downstream effects were limited i.e. we did not observe NOGP induction and programmed cell death. An intriguing finding emerged, however, i.e. that PIs can elicit an impact on the AGE pathway. We propose future studies with modifications to the current rat and cell models in order to evaluate the downstream effects of PIs on the NOGPs and programmed cell death. / AFRIKAANSE OPSOMMING: Terwyl antiretrovirale terapie MIV/VIGS morbiditeit en mortaliteit verlaag, veroorsaak langtermyn behandeling insulienweerstandigheid en kardiovaskulêre siekte. 'n Moonltike oorsaak van sulke newe-effekte kan 'n toename in oksidatiewe stres veroorsaak deur die protease inhibeerder (PI)-geïnduseerde mitochondriale wanfunskionering. Ons hipotetiseer dat PI behandeling, spesifiek Lopinavir/Ritonavir, versoorsaak 'n toename in miokardiale reaktiewe suurstofspesies (ROS), wat aanleiding gee tot afstroom uitkomste, i.e. verhoogde apoptose. Verder, stel ons voor dat verhoogde ROS vlakke in hierdie geval onstaan as gevolg van PI-gemedieerde induksie van die nie-oksidatiewe glukose weë (NOGWe). In die lig hiervan het ons ook die effek van PI behandeling op die NOGWe ondersoek deur beide in vitro en in vivo monsters te gebruik. Vir die in vitro werk het ons van 'n rot kardio-mioblastsellyn gebruik gemaak, terwyl weefsels (hart, lewer) versamel is van twee afsonderlike eksperimentele modelle, i.e. a) Groep A blootgestel aan PIs via mini-osmotiese pomp vir 'n periode van agt weke, en b) Groep B PIs is toegedien via 'n jellie gebaseerde metode vir 16 weke. Ons het bevind dat die die PIs mitochondriale ROS vlakke in vitro verhoog maar dat dit nie vergesel is met 'n paralelle toename in apoptose. Verder is geen induksie van die NOGWe in reaksie op PI blootstelling waargeneem (vir beide in vitro en in vivo modelle). Hoewel ons het bevind dat die AGE weg in die lewer van Groep A beduidend afgereguleer is. Ondersoek na 'n moontlike megansime vir hierdie waarneming was onoortuigend en verdere ondersoek is nodig om die betekenis in terme van die metaboliese wanfunskionering in die Groep A model vas te stel. Ons studie toon dus aan dat PIs, ROS vlakke (in vitro) verhoog, maar dat kompensatoriese anti-oksidant meganismes in die hierdie in vivo model verhoed word. Gevolglik is die afstroom effekte beperk i.e. ons het geen NOGWe induksie en aptoptose waargeneem nie. 'n Interesante bevinding het wel uitgestaan, i.e. PIs kan 'n impak hê op die AGE weg. Ons stel dus voor dat toekomstige studies met modifikasies, tot die huidige rot- en sel-modelle gemaak word om die afstroomeffekte van PIs en apoptose te evalueer.

Thesis (MSc)--Stellenbosch University, 2001 / ENGLISH ABSTRACT: The effect of various organic reducing agents, in the. form of monosaccharide reducing sugars, on the rate of atom transfer radical polymerization (ATRP) of n-butyl methacrylate and methyl methacrylate is reported in this study. The addition of the reducing sugars has a positive effect on the rate of ATRP. Up to 100% increase in the rate of polymerization was recorded, in some cases. These organic reducing agents have little effect on the molecular weight and molecular weight distribution of the polyin-butyl methacrylate) and polydispersity indexes remain well below 1.2. The molecular weight of the poly(methyl methacrylate), when glucose and galactose are added to the reaction mixture, compares well with the theoretical expected values. An explanation for these observations is the ability of the reducing sugars to reduce part of the Cu(II) species, that serves to deactivate the growing radicals, to Cu(I), thereby ensuring a shift in the equilibrium between active and dormant chains in the direction of the former and a resulting increase in the rate of polymerization. uvNIS spectroscopy and cyclic voltammetry were used to investigate the mechanism behind the polymerization rate enhancement. / AFRIKAANSE OPSOMMING: In hierdie studie word die effek van verskeie organiese reduseermiddels, in die vorm van monosakkaried reduserende suikers, op die tempo van polimerisasie van ATRP gerapporteer. Hierdie reduserende suikers het 'n positiewe effek op die polimerisasie tempo. In sommige gevalle word 'n toename van 100% in die polimerisasie tempo waargeneem. Die organiese reduseermiddels het 'n minimale effek op die molekulere massa en molekulere massa verspreiding (in meeste gevalle minder as 1.2) van die poly(n-butiel metakrielaat). In die geval van die poly(metiel metakrielaat), wanneer glukose en galaktose by die reaksie mengsel gevoeg word, stem die molekulere massas goed ooreen met die teoreties voorspelde molekulere massas. Die waargenome toename in die polimerisasie tempo kan toegeskryf word aan die vermoe van die reduserende suikers om die Cu(II), wat dien om die groeiende radikale te deaktiveer, gedeeltelik te reduseer na Cu(l). Hierdeur word verseker dat die ewewig tussen die aktiewe en dormante kettings in die rigting van die eersgenoemde verskuif word, wat dus aanleiding gee tot 'n toename in die polimerisasie tempo. Ultraviolet spektroskopie en sikliese voltammetrie is ook gebruik om lig te werp op die meganisme agter die toename in die tempo van polimerisasie.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

Maternal lifestyle and nutrient intake during pregnancy can have long-lasting effects on the health of offspring as well as the mother. This dissertation focuses on the impact of maternal exercise during pregnancy on offspring insulin sensitivity and glucose uptake and the maternal effects of exercise during pregnancy. The first aim of this dissertation was to investigate if exercise prior to and during pregnancy and nursing would improve glucose uptake and insulin sensitivity in mice and rats. In both mice and rats, it was concluded that maternal exercise could enhance whole-body insulin sensitivity and increase glucose uptake into skeletal muscle and adipose tissue in adult offspring compared with offspring from sedentary dams. Maternal exercise also positively influenced male but not female adult offspring body composition; male offspring from exercised dams had significantly decreased fat mass and increased lean mass compared with offspring from sedentary dams. The second aim of this dissertation was to test whether exercise during pregnancy would improve glucose disposal in mouse dams with diet-induced obesity. Maternal running was effective in reducing fat mass accumulation and glucose intolerance associated with high fat feeding during pregnancy. In high fat diet mice, exercise was also able to improve insulin sensitivity in adipose tissue compared to tissue from sedentary high fat diet mice. The findings in this dissertation provide new insight into the long-term effects exercise during pregnancy can have on offspring health. Women may be encouraged to start an exercise regimen before and during their pregnancy if they are aware of the life-long benefits it can have for their children. The findings from the second aim present new insight into how exercise can affect pregnancies complicated by maternal obesity and glucose intolerance, and the animal model can be used in the future studies to investigate the offspring effects of maternal exercise during a diabetic pregnancy.