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Since its discovery in 1988 the powerful vasoconstrictor endothelin-1 (ET-1) has been widely implicated in the pathophysiology of chronic kidney disease (CKD) as well as the cardiovascular disease with which it is associated. ET receptor antagonists have favourable effects in experimental models of these conditions and orally acting antagonists are now licensed for the treatment of pulmonary arterial hypertension. However, there is a paucity of human data regarding the role of ET-1 in CKD. In this thesis, I have therefore explored the utility of ET-1 as a biomarker in CKD, and, using selective ET receptor antagonists, the beneficial renal and cardiovascular effects of ET receptor antagonism in CKD. I have shown that as glomerular filtration rate (GFR) declines plasma ET-1 increases linearly whereas urinary ET-1 shows an exponential increase. Furthermore, urinary ET-1 may be a useful marker of disease activity in patients with lupus nephritis. Its levels are high in those with biopsy-proven active renal inflammation and these fall with treatment. I have shown that in subjects with stable non-diabetic proteinuric CKD, acute selective ETA receptor antagonism reduces blood pressure and arterial stiffness and that these systemic benefits are associated with an increase in renal blood flow and reduction in proteinuria. Importantly, these effects are seen on top of those achieved with maximal therapy with angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers. Following a study confirming unchanged pharmacokinetics in CKD, I have used an oral selective ETA receptor antagonist to show that the reductions in BP, arterial stiffness and proteinuria seen in my acute studies are maintained longer term. This results of this study also suggest that the mechanism for the reduction in proteinuria is haemodynamic and relates to a reduction in GFR and filtration fraction. In summary, these studies suggest that ET-1 may act as a potential biomarker of renal inflammation, and confirm its role in the pathophysiology of the systemic and renal vasoconstriction seen in CKD. They also suggest that selective ETA receptor antagonism may provide a novel therapeutic approach in proteinuric CKD on top of standard therapies. Larger and longer term studies are now warranted to confirm this potential.

Atherosclerosis is a chronic inflammatory disease leading to plaque buildup in the major arteries. The plaques consist of cholesterol, calcium, inflammatory cells, extracellular matrix and fibrous material. Under inflammatory conditions IFN-• stimulation of human monocytes and macrophages generates reduced pteridine, 7,8-dihydroneopterin (78NP) which has been shown to be an effective cytoprotective agent to some cell types against oxidative damage by reactive oxygen species (ROS). 7,8-dihydroneopterin is oxidized to fluorescent neopterin in the presence of hypochlorite (HOCl). Although a considerable amount of work has been published on the composition of neopterin in atherosclerotic plaques, very little is known about the variation of 78NP and other oxidative biomarkers across the length of the carotid and femoral and their contribution to plaque progression, which was researched in this work. Atherosclerotic plaques excised from patients with carotid and femoral plaques were sliced into 3-5 mm sections, and each section was analyzed for concentrations of neopterin, 7,8- dihydroneopterin, •-tocopherol, TBARS, DOPA, cholesterol, dityrosine, protein carbonyls •- aminoadipic semialdehyde (AAS) and •-glutamic semialdehyde (GGS), free and esterified 7- ketocholesterol (7-KC). Cultured live plaque as a source of 7,8-dihydroneopterin and neopterin was also investigated in this study. It was shown that carotid plaques significantly vary from femoral plaques, in the levels and range of most oxidative biomarkers. Carotid plaques showed a high variation in the biomarker concentrations between plaques but also between sections of an individual plaque. Femoral plaques on the other hand showed lower amounts of biomarkers with very little variation in biomarker concentrations. High variation with pterin concentrations and other biomarkers suggests dynamic and active changes in inflammation within the plaque. Collectively, it was observed that every plaque was unique with respect to its composition and correlations between the biomarkers. Though shown to be a well-known antioxidant and a radical scavenger, there is no published literature on 7,8-dihydroneopterin’s mode of entry into and out of the cell. To understand how it enters the cells could explain the difference in its protective ability of different cell types Abstract xxviii against oxidative stress-mediated cell death. Knowledge of transport of 7,8-dihydroneopterin will provide insights about its protection of monocyte/macrophage cell death which could potentially reduce atherosclerotic plaque growth and progression. As 7,8-dihydroneopterin is produced from guanosine, a nucleoside that is transported using specialized nucleoside transporters (equilibrative nucleoside transporters (ENT's) and concentrative nucleoside transporters (CNT's), their role was examined and characterized for 7,8-dihydroneopterin transport. It was found that 7,8-dihydroneopterin and neopterin are transported via nucleoside transporters in U937 cells, THP-1 cells and human monocytes. ENT 2 was the major transporter in U937 cells while ENT 1 transported bulk of 7,8-dihydroneopterin in THP-1 cells. Both ENT's and CNT's are involved in 7,8-dihydroneopterin uptake in human monocytes. In all the cell lines tested, 7,8-dihydroneopterin protection against AAPH mediated oxidative cell death was inhibited by nucleoside transport inhibitors, suggesting that nucleoside transporters are indispensible for 7,8-dihydroneopterin mediated intracellular protection against oxidative stress. Accurate measurement of neopterin, as a biomarker of inflammation in plaques and cells is critical aspect to assess disease progression. The current C18 HPLC method used in our laboratory for neopterin measurement lacks sensitivity due to interference of acetonitrile (ACN) over time. Acidic tri-iodide conversion of 7,8-dihydroneopterin to neopterin was also variable at times giving inconsistent measurement of neopterin so the manganese oxide (MnO2) method was looked at as an alternative. Electrochemical detector (ECD) was another option studied as it did not require any precolumn oxidation of 7,8-dihydroneopterin to neopterin. A new method using strong cation exchange (SCX) column was developed for a precise, sensitive neopterin assay which got rid of the ACN interference completely. The MnO2 method of 7,8-dihydroneopterin oxidation did not work with biological samples such as serum or plaque homogenates. Electrochemical detection was also found to be very unreliable and inconsistent.

Selected Ion Flow Tube-Mass spectrometry (SIFT-MS) is an analytical measurement technology for the real-time quantification of volatile organic compounds in gaseous samples. This technology has current and potential applications in a wide variety of industries, although the focus of this research is in medical science. In this field, SIFT-MS has potential as a diagnostic device, capable of determining the presence of a particular disease or condition. In addition, SIFT-MS can be used to monitor the progression of a disease state, or predict deviations from expected behaviour. Lastly, SIFT-MS can be used for the identification of biomarkers of a particular disease state. All these possibilities are available non-invasively and in real-time, by analysing breath samples. SIFT-MS produces an extensive amount of data, requiring specific mathematical methods to identify biomarker masses that differ significantly between populations or time-points. Two classification methods are presented for the analysis of SIFT-MS mass scan data. The first method is a cross-sectional classification model, intended to differentiate between the diseased and non-diseased state. This model was validated in a simple test case. The second method is a longitudinal classification model, intended to identify key biomarkers that change over time, or in response to treatment. Both of these classification models were validated in 2 clinical trials, investigating renal function in humans and rats. The first clinical trial monitored changes in breath ammonia, TMA and acetone concentrations over the course of dialysis treatment. Correlations with the current gold standard plasma creatinine, and blood urea nitrogen were reported. Finally, biomarkers of renal function were identified that change predictably over the course of treatment. The second trial induced acute renal failure in rats, and monitored the change in renal function observed during recovery. For comparison and validation of the result, a 2-compartment model was developed for estimating renal function via a bolus injection of a radio-labelled inulin tracer, and was compared with the current gold standard plasma creatinine measurement, modified using the Cockcroft-Gault equation for rats. These two methods were compared with SIFT-MS monitoring of breath analytes, to examine the potential for non-invasive biomarkers of kidney function. Results show good promise for the non-invasive, real-time monitoring of breath analytes for diagnosis and monitoring of kidney function, and, potentially, other disease states.

Background: Although Bevacizumab (BVZ) and Sunitinib (SNT) prolong survival in cancer patients, an unanticipated side-effect is cardiotoxicity. Early indices of left ventricular (LV) systolic dysfunction would be useful to address the cardiac safety of anti-cancer drugs. Objective: Whether cardiac biomarkers, tissue velocity imaging (TVI), and/or strain rate (SR) can detect early cardiac dysfunction. Methods: A total of 95 C57Bl/6 mice received one of the following drug regimens: i) 0.9% saline; ii) BVZ; or iii) SNT and followed for 14 days. Serial blood pressure, high sensitivity troponin I (hsTnI), and echocardiography were performed. Results: BVZ- and SNT-treated mice demonstrated an increase in mean arterial blood pressure, hsTnI, cardiac apoptosis, and loss of cell integrity. TVI and SR values confirmed early LV systolic dysfunction at day 8, compared to conventional LVEF at day 13. Conclusions: Novel imaging techniques can detect early LV systolic dysfunction in a model of drug-mediated cardiomyopathy.

Colorectal cancer (CRC) is one of the most common cancer types and leading causes of cancer death worldwide. Since CRC is a heterogenic disease, there is a demand for increased knowledge of the underlying genetic and epigenetic mechanisms. The aim of this thesis was to investigate heredity and potential tumour markers in relation to prognosis. In paper I, survival of patients with CRC and a positive family history of CRC in first-degree relatives was analysed. Patients with colon cancer and positive family history of CRC had improved survival compared to patients with negative family history. This improvement in survival could not be explained by known clinico-pathological factors. In paper II, we investigated the prognostic value of Tryptophanyl t-RNA synthetase (TrpRS) in tissues from patients operated for CRC. Low protein expression of TrpRS in primary tumour tissues correlated with increased risk of recurrence and poorer survival. In paper III, the prognostic value of microsatellite instability (MSI) and the correlation to heredity for CRC in first-degree relatives was investigated. Patients with proximal colon cancer and MSI had improved cancer specific survival. There were no correlation between MSI and heredity. In paper IV, we evaluated the potential use of proximity ligation assay (SP-PLA) in patients with CRC, by simultaneous analysis of 35 proteins in only 5 μl plasma. SP-PLA is a suitable method for protein detection and might give valuable guidance in pursuing new prognostic and predictive tumour markers. However, none of the markers selected for present SP-PLA analyses gave better prognostic information than CEA. In conclusion, heredity is related to better survival independent of MSI in patients with CRC and MSI is associated with better prognosis in proximal colon cancer. Detection and increased knowledge of molecular mechanism in CRC is important, however it needs to be further investigated and validated in clinical use.

Mining has negative impacts on the environment, and is one of the main contributors to environmental pollution. This holds a potential hazard for ecosystems surrounding mining areas and also for public health in the surrounding communities. There is therefore a need for ecotoxicological research in order to assess these possible risks and find ways to minimize the harmful effects. One way in which to assess soil vitality are soil enzymes which are produced by plants and microorganisms and will therefore be more abundant in healthy soils. Earthworms have been proven to be useful bioindicators for metal contamination of soil, as they are able to accumulate metals from their environment into their body tissue. The aim of this study was to use earthworm bioassays, neutral red retention time analysis (NRRt), enzymatic analysis and RAPD-PCR to determine the effect of mining activity on the environment. This was done by assessing the ecotoxicity of tailings collected from three different mines, viz. gold-, opencast chrome- and coal mines. The metals chosen for analyses included four (Cr, Co, Ni and Pb) of the seven (Cd, Cr, Ni, Pb, Zn, Cu, Co) environmentally important trace elements as described for South African soils. Arsenic was also chosen since it is associated with gold mine tailings. Tailings and soil were collected from three sites at each of the mines which included unrehabilitated (TDF-U) and rehabilitated (TDF-R) substrates from tailings disposal facilities (TDFs) and reference soils (RS) in close proximity to TDFs. The physical and chemical properties of these different substrates were determined in terms of pH, particle size as well as metal contents. In addition to this, they were analysed for microbial community function by means of enzymatic activity, which has been proven to be useful in evaluating contamination of soil. The enzymes analysed in this study included dehydrogenase, ßglucosidase, acid phosphatase, alkaline phosphatase and urease. Earthworms (Eisenia andrei) were exposed to different material for 28 days during which their biomass, reproduction, mortality and lysosomal membrane stability were monitored weekly. Hereafter, they were removed from the material while the cocoons were left behind for a further 56 days. The hatched and unhatched cocoons as well as the juvenile worms were then counted to determine reproduction patterns in the materials. Metal concentrations in the substrates and earthworm body tissues were compared to selected benchmarks. Results indicated that when comparing the different materials from each mine, enzymatic activity proved to be a very sensitive parameter. Enzymatic activity showed significant differences (p < 0.05) between RS, TDF-R and TDF-U materials. Biomass was not a sensitive parameter (p > 0.05) for the worms exposed to the gold and chrome mine tailings, but it was a sensitive parameter (p < 0.05) for the coal mine exposed earthworms, showing early differences between the worms from the different sites (RS, TDF-R and TDF-U). The NRR-t assay was very sensitive (p < 0.05), indicating clear differences between the worms from each investigated site. In terms of reproduction, the production of cocoons showed clear differences (p < 0.05) between the different sites and could therefore be considered a sensitive parameter. Hatching success however, was not a sensitive parameter. The reason being that there were so little cocoons produced that it is not possible to determine the correct percentage of juvenile worms hatching from, for example, only one or two cocoons. Mortality was also not a sensitive endpoint as it was only observed in the coal mine material. RAPD - PCR results indicated genetic differences between earthworms exposed to the control- and the tailings materials, indicating either DNA alterations due to possible genotoxic effects, or genetic variation between individuals of the same species. Since mine waste materials often contain complex mixtures of metals that might be toxic on their own or in combination with other factors, it is difficult to attribute any observed genotoxic effect to any of the specific metals. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2012.

Mining has negative impacts on the environment, and is one of the main contributors to environmental pollution. This holds a potential hazard for ecosystems surrounding mining areas and also for public health in the surrounding communities. There is therefore a need for ecotoxicological research in order to assess these possible risks and find ways to minimize the harmful effects. One way in which to assess soil vitality are soil enzymes which are produced by plants and microorganisms and will therefore be more abundant in healthy soils. Earthworms have been proven to be useful bioindicators for metal contamination of soil, as they are able to accumulate metals from their environment into their body tissue. The aim of this study was to use earthworm bioassays, neutral red retention time analysis (NRRt), enzymatic analysis and RAPD-PCR to determine the effect of mining activity on the environment. This was done by assessing the ecotoxicity of tailings collected from three different mines, viz. gold-, opencast chrome- and coal mines. The metals chosen for analyses included four (Cr, Co, Ni and Pb) of the seven (Cd, Cr, Ni, Pb, Zn, Cu, Co) environmentally important trace elements as described for South African soils. Arsenic was also chosen since it is associated with gold mine tailings. Tailings and soil were collected from three sites at each of the mines which included unrehabilitated (TDF-U) and rehabilitated (TDF-R) substrates from tailings disposal facilities (TDFs) and reference soils (RS) in close proximity to TDFs. The physical and chemical properties of these different substrates were determined in terms of pH, particle size as well as metal contents. In addition to this, they were analysed for microbial community function by means of enzymatic activity, which has been proven to be useful in evaluating contamination of soil. The enzymes analysed in this study included dehydrogenase, ßglucosidase, acid phosphatase, alkaline phosphatase and urease. Earthworms (Eisenia andrei) were exposed to different material for 28 days during which their biomass, reproduction, mortality and lysosomal membrane stability were monitored weekly. Hereafter, they were removed from the material while the cocoons were left behind for a further 56 days. The hatched and unhatched cocoons as well as the juvenile worms were then counted to determine reproduction patterns in the materials. Metal concentrations in the substrates and earthworm body tissues were compared to selected benchmarks. Results indicated that when comparing the different materials from each mine, enzymatic activity proved to be a very sensitive parameter. Enzymatic activity showed significant differences (p < 0.05) between RS, TDF-R and TDF-U materials. Biomass was not a sensitive parameter (p > 0.05) for the worms exposed to the gold and chrome mine tailings, but it was a sensitive parameter (p < 0.05) for the coal mine exposed earthworms, showing early differences between the worms from the different sites (RS, TDF-R and TDF-U). The NRR-t assay was very sensitive (p < 0.05), indicating clear differences between the worms from each investigated site. In terms of reproduction, the production of cocoons showed clear differences (p < 0.05) between the different sites and could therefore be considered a sensitive parameter. Hatching success however, was not a sensitive parameter. The reason being that there were so little cocoons produced that it is not possible to determine the correct percentage of juvenile worms hatching from, for example, only one or two cocoons. Mortality was also not a sensitive endpoint as it was only observed in the coal mine material. RAPD - PCR results indicated genetic differences between earthworms exposed to the control- and the tailings materials, indicating either DNA alterations due to possible genotoxic effects, or genetic variation between individuals of the same species. Since mine waste materials often contain complex mixtures of metals that might be toxic on their own or in combination with other factors, it is difficult to attribute any observed genotoxic effect to any of the specific metals. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2012.

Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for multiple sclerosis (MS) with now more than 600 documented cases in the medical literature. Long-term remission can be achieved with this therapy, but when is it justified to claim that a patient is cured from MS? In attempt to answer this question, the outcome of the Swedish patients is described, mechanisms behind the therapeutic effect are discussed and new tools for demonstration of absence of disease have been developed. In Swedish patients treated with HSCT for aggressive MS, disease free survival was 68 % at five years, and no patient progressed after three years of stable disease. Presence of gadolinium enhancing lesions prior to HSCT was associated with a favorable outcome (disease free survival 79 % vs 46 %, p=0.028). There was no mortality and no patient required intensive care. The immune system of twelve of these patients was investigated further. In most respects HSCT-treated patients were similar to healthy controls, demonstrating normalization. In the presence of a potential antigen, leukocytes from HSCT-treated patients ceased producing pro-inflammatory IL-17 and increased production of the inhibitory cytokine TGF-β1 suggesting restoration of tolerance. Cytokine levels and biomarkers of tissue damage were investigated in cerebrospinal fluid from a cohort of MS patients. The levels were related to clinical and imaging findings. A cytokine signature of patients with relapsing-remitting MS could be identified, characterized by increased levels of CCL22, CXCL10, sCD40L, CXCL1 and CCL5 as well as down-regulation of CCL2. Further, we could demonstrate that active inflammation in relapsing-remitting MS is a tissue damaging process, with increased levels of myelin basic protein and neurofilament light. Importantly, relapsing-remitting MS patients in remission displayed no tissue damage. In secondary progressive MS, moderate tissue damage was present without signs of active inflammation. From a clinical vantage point, it seems that we confidently can claim cure of relapsing-remitting MS patients after five years absence of disease activity. The new tools for evaluation of disease can strengthen this assertion and may enable earlier prediction of outcome.

Metabolism is an integrated network of biochemical pathways involving a series of enzymecatalysed anabolic or catabolic reactions in cells. Metabolites are chemical compounds that are involved in or are products of metabolic pathways, and the metabolome is defined as the total complement of all the low molecular weight metabolites present in a cell at any given time. Metabolomics is a relatively new research technology utilised for the global investigation, identification and quantification of the metabolome. Three aims were defined for the metabolomics study presented here: • The use of metabolomics technology to generate new biological information; • Application of the metabolomics technology to gain information on the three natural perturbations, namely the menstrual cycle, pregnancy and aging; and • Reflection on metabolomic studies as a hypothesis-generating approach. I obtained three sets of urine samples from women during their menstrual cycle, samples from sixteen pregnant and eleven non-pregnant women for the second natural perturbation, and data sets from previous investigations on infant and child groups, as well as thirty-two urine samples from adults for the study of the metabolomic profiles due to age. These urine samples were analysed to determine the organic acid metabolite profiles. The metabolites were identified by means of AMDIS and were manually quantified. Data matrixes were compiled, which underwent certain data reduction steps, prior to statistical analysis. Different statistical approaches were used to generate information on these three natural perturbations due to the clear differences between the three experimental groups used. The investigation of the menstrual cycle did not show a distinct difference between the three phases involved in the cycle, whereas the pregnancy perturbation showed a difference between pregnant groups and non-pregnant groups. The most pronounced difference in metabolite profiles were found when the different age groups were compared to one another. Finally a hypothesis on the effect of age on metabolism was defined and an experimental approach was proposed to evaluate this hypothesis. In conclusion three proposals were formulated from this investigation: 1. If it appears that an insufficient number of participants can be generated for a metabolomics study, such a study should be discarded in the interest of a more feasible investigation. 2. It is advisable that a number of appropriate analytical validation parameters should be incorporated in the early stages of a metabolomics study, specifically linked to the context of the perturbation chosen for the investigation. 3. The control and experimental groups should be homogenous that is to say as comparable as possible with regard to age, ethnicity, diet, and gender, lifestyle habits and other possible confounding influences, except for the specific perturbation being studied. In a perfect world this would be possible, specifically when hypothesis formulation, testing and finally the expansion of scientific knowledge is a desired outcome of the investigation. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.