Diabetic nephropathy is a chronic kidney disease and one of the more severe complications from diabetes mellitus type 2. The glomerular and tubular dysfunctions usually lead to end stage renal disease and the treatments of these patients (dialysis, kidney transplants) are a huge economic burden for the society. Due to an epidemiologic increase of type 2 diabetes, conventional diagnostic markers like creatinine and albumin are not sufficient, since they are only able to identify already existing kidney damage. With targeted metabolomics, the analysis of small molecules produced from metabolism, this project aimed at finding novel and more sensitive metabolic biomarkers from several different classes of metabolites. The different assays were performed with flow injection analysis, high performance liquid chromatography, gas chromatography and mass spectrometry, and with principal component analysis and discriminant analysis, up-and down-regulated metabolites could be identified and their respective biochemical pathways, if possible, explained. In diabetics significantly elevated concentrations of very long chain fatty acids (impaired peroxisomal β-oxidation), urinary sugars and acylcarnitines in plasma could be recognized. Markers indicating kidney damage included significantly increased plasma concentrations of asymmetric dimethylarginine (inhibition of nitric oxide synthase resulting in decreased endothelial functionality) and histamine (indication of uremic pruritus). Oxidative stress was also found to be a potential prognostic marker as indicated by the raised methionine-sulfoxide to methionine ratio in nephrotic patients. To summarize, this project succeeded in identifying metabolic biomarkers both for diabetes type 2 and nephropathy, which in the future might become important tools in slowing down progression or diagnosing these diseases.

<p>Diabetic nephropathy is a chronic kidney disease and one of the more severe complications from diabetes mellitus type 2. The glomerular and tubular dysfunctions usually lead to end stage renal disease and the treatments of these patients (dialysis, kidney transplants) are a huge economic burden for the society. Due to an epidemiologic increase of type 2 diabetes, conventional diagnostic markers like creatinine and albumin are not sufficient, since they are only able to identify already existing kidney damage. With targeted metabolomics, the analysis of small molecules produced from metabolism, this project aimed at finding novel and more sensitive metabolic biomarkers from several different classes of metabolites. The different assays were performed with flow injection analysis, high performance liquid chromatography, gas chromatography and mass spectrometry, and with principal component analysis and discriminant analysis, up-and down-regulated metabolites could be identified and their respective biochemical pathways, if possible, explained. In diabetics significantly elevated concentrations of very long chain fatty acids (impaired peroxisomal β-oxidation), urinary sugars and acylcarnitines in plasma could be recognized. Markers indicating kidney damage included significantly increased plasma concentrations of asymmetric dimethylarginine (inhibition of nitric oxide synthase resulting in decreased endothelial functionality) and histamine (indication of uremic pruritus). Oxidative stress was also found to be a potential prognostic marker as indicated by the raised methionine-sulfoxide to methionine ratio in nephrotic patients. To summarize, this project succeeded in identifying metabolic biomarkers both for diabetes type 2 and nephropathy, which in the future might become important tools in slowing down progression or diagnosing these diseases.</p>

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This dissertation brings to the fore the “Characterization of the SULT1A1 polymorphism in a South Africa Tswana population group.” The primary experimental group studied came from South African homogeneous Tswana individuals who participated voluntarily in an ongoing large-scale epidemiological Prospective Urban and Rural Epidemiological (PURE) study the North-West University (Potchefstroom Campus) participates in, as one of the 16 low- middleand high-income countries across the world. The primary aspect investigated was the comprehensive profile of the single nucleotide polymorphism (SNP) and copy number variation (CNP) of the SULT1A1 gene. Using the PCRbased RFLP method, SULT1A1 genotypes, and allele frequency distributions in an experimental group of 1 867 individuals were determined. According to the literature this is by far the largest and most homogeneous group from which such information has been acquired to date. The SULT1A1*1, SULT1A1*1/*2 and SULT1A1*2 genotypes were found to be present at a percentage of 43.76, 47.12 and 9.11 respectively. In comparison to similar studies in other population groups, results from this study indicate that there are ethnic differences in the SULT1A1 genotypes incidence. Asian group differs from Caucasian and Tswana groups because of its exceptionally high prevalence of individuals with the SULT1A1*1 genotype and a very low incidence of the SULT1A1*2 genotype. The SULT1A1*1 genotype profiles of Caucasian and Tswana groups were comparable, but notable differences were observed for the SULT1A1*2 genotype. Using a quantitative multiplex PCR method for the CNV study, the numbers of copies of the SULT1A1 gene in the Tswana population were determined, and the results showed 1 to ~5 copies: only 0.65% of the subjects had a single copy, whereas 59.69% of the subjects had 3 or more copies. This result shows a significant discrepancy between the Caucasian-American samples, which showed that only 26% from that group had more than three copies. However, there is a significant relationship with the African-American population, which presented 63% with 3 or more copies. This finding confirms results from a much smaller African-American study, and suggests a possible genetic link between the African Tswana and the heritage of the African-Americans. These findings were submitted for publication to the South African Journal of Science, as that journal specializes in publication of new knowledge that has a regional focus on Africa. Simultaneous phenotypic consequences of the SNP and CNP of the SULT1A1 gene, as well as the thermo-stable and thermo-labile forms of the sulfotransferases were determined. For this, the formation of [35S]-4-nitrophenyl sulphate from 4-nitrophenol and [35S]-3’-phosphoadenosine- 5’-phosphosulfate ([35S]-PAPS) in platelet homogenates were measured, with the data normalized to a common platelet count. This investigation required fresh blood for enzyme activity. These samples came from 98 Caucasian subjects who voluntarily participated in this part of the study. The experimental data presented a unique challenge to develop a statistical model to accommodate the complexity of the distribution of the data in the phenotype and genotype components, which could be achieved by the development of a mixed model. The model indicated that product formation increased through increasing copy number, but did not differ for SULT1A1*1 and SULT1A1*1/*2. However, the rate of increase in product for the thermo-stable forms of the SULTs was greater than that of thermo-labile forms. In contrast, copy number effect for SULT1A1*2 differed considerably from that of the other two genotypes. Since genotype is also a significant factor, it was concluded from Tukey post-hoc tests that the population group means for product formation differ significantly (for all levels). These results are presently being prepared for publication in an accredited international journal. Finally, perturbations in 23 biochemical parameters measured in the PURE study were analyzed as a function of the SULT1A1 SNP and CNP were evaluated. No group separation in this regard could be found. It could be shown however, that sulfonation of the iodothyronines, which are endogenous substrates for the SULTs, was influenced by the SULT1A1 genotype. The relative concentrations in plasma of the sulphonated iodothyronines may be expressed as T2S > T3S >> T4S, which coincides with the substrate preference of the SULT1A1 enzymes. This observation may, however, only be qualitatively interpreted as (1) the targeted metabolomics mass spectrometric method used for the quantitative analysis of these substances needs further development, and (2) the influence of deiodonation was not taken into account in these studies. In conclusion, three perspectives are given at the end of the thesis which might be considered for further investigations. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

This dissertation brings to the fore the “Characterization of the SULT1A1 polymorphism in a South Africa Tswana population group.” The primary experimental group studied came from South African homogeneous Tswana individuals who participated voluntarily in an ongoing large-scale epidemiological Prospective Urban and Rural Epidemiological (PURE) study the North-West University (Potchefstroom Campus) participates in, as one of the 16 low- middleand high-income countries across the world. The primary aspect investigated was the comprehensive profile of the single nucleotide polymorphism (SNP) and copy number variation (CNP) of the SULT1A1 gene. Using the PCRbased RFLP method, SULT1A1 genotypes, and allele frequency distributions in an experimental group of 1 867 individuals were determined. According to the literature this is by far the largest and most homogeneous group from which such information has been acquired to date. The SULT1A1*1, SULT1A1*1/*2 and SULT1A1*2 genotypes were found to be present at a percentage of 43.76, 47.12 and 9.11 respectively. In comparison to similar studies in other population groups, results from this study indicate that there are ethnic differences in the SULT1A1 genotypes incidence. Asian group differs from Caucasian and Tswana groups because of its exceptionally high prevalence of individuals with the SULT1A1*1 genotype and a very low incidence of the SULT1A1*2 genotype. The SULT1A1*1 genotype profiles of Caucasian and Tswana groups were comparable, but notable differences were observed for the SULT1A1*2 genotype. Using a quantitative multiplex PCR method for the CNV study, the numbers of copies of the SULT1A1 gene in the Tswana population were determined, and the results showed 1 to ~5 copies: only 0.65% of the subjects had a single copy, whereas 59.69% of the subjects had 3 or more copies. This result shows a significant discrepancy between the Caucasian-American samples, which showed that only 26% from that group had more than three copies. However, there is a significant relationship with the African-American population, which presented 63% with 3 or more copies. This finding confirms results from a much smaller African-American study, and suggests a possible genetic link between the African Tswana and the heritage of the African-Americans. These findings were submitted for publication to the South African Journal of Science, as that journal specializes in publication of new knowledge that has a regional focus on Africa. Simultaneous phenotypic consequences of the SNP and CNP of the SULT1A1 gene, as well as the thermo-stable and thermo-labile forms of the sulfotransferases were determined. For this, the formation of [35S]-4-nitrophenyl sulphate from 4-nitrophenol and [35S]-3’-phosphoadenosine- 5’-phosphosulfate ([35S]-PAPS) in platelet homogenates were measured, with the data normalized to a common platelet count. This investigation required fresh blood for enzyme activity. These samples came from 98 Caucasian subjects who voluntarily participated in this part of the study. The experimental data presented a unique challenge to develop a statistical model to accommodate the complexity of the distribution of the data in the phenotype and genotype components, which could be achieved by the development of a mixed model. The model indicated that product formation increased through increasing copy number, but did not differ for SULT1A1*1 and SULT1A1*1/*2. However, the rate of increase in product for the thermo-stable forms of the SULTs was greater than that of thermo-labile forms. In contrast, copy number effect for SULT1A1*2 differed considerably from that of the other two genotypes. Since genotype is also a significant factor, it was concluded from Tukey post-hoc tests that the population group means for product formation differ significantly (for all levels). These results are presently being prepared for publication in an accredited international journal. Finally, perturbations in 23 biochemical parameters measured in the PURE study were analyzed as a function of the SULT1A1 SNP and CNP were evaluated. No group separation in this regard could be found. It could be shown however, that sulfonation of the iodothyronines, which are endogenous substrates for the SULTs, was influenced by the SULT1A1 genotype. The relative concentrations in plasma of the sulphonated iodothyronines may be expressed as T2S > T3S >> T4S, which coincides with the substrate preference of the SULT1A1 enzymes. This observation may, however, only be qualitatively interpreted as (1) the targeted metabolomics mass spectrometric method used for the quantitative analysis of these substances needs further development, and (2) the influence of deiodonation was not taken into account in these studies. In conclusion, three perspectives are given at the end of the thesis which might be considered for further investigations. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

Intracellular sterol redistribution is an important step in the lipid homeostasis of organisms, a process directly linked to the organizational arrangement in the plasma membrane (PM) of cells. Previous studies in the budding yeast Saccharomyces cerevisiae have demonstrated that the ARV1 (ACAT-related enzyme-2 required for viability 1) protein is a major regulator of sterol transport from the endoplasmic reticulum to the plasma membrane, contributing to the structural organization of the PM, rendering it resistant to anti-fungal compounds as well as maintaining ER integrity. This study assessed the significance of ARV1 in the plant pathogenic fungus Cryphonectria parasitica (Cparv1) and investigated its role in the pathogenesis and virulence of the fungus. C. parasitica is the causative agent of Chestnut blight, which has wreaked havoc on the American chestnut species. Genomic analysis revealed that the Cparv1 gene is very closely linked to another gene that putatively encodes a cyanamide hydratase (Cpcah). An initial gene deletion event resulted in the elimination of both genes and a highly deformed phenotype in C. parasitica that was fully recoverable by complementation. PCR-based expression analysis determined that the lack of Cparv1 was responsible for the debilitated phenotype of the double mutant, with no transcript detectable from Cpcah. Subsequent complementation of the Cparv1 gene was also observed to restore the wildtype phenotype. Mass spectrometry-based (MS) results indicated a decrease in sterol content of the DCparv1 mutant strain compared to wildtype EP155 thus confirming a role for Cparv1 in sterol homeostasis. It has been shown that infection of C. parasitica with virulence-attenuating hypoviruses altered intracellular lipid content and protein secretion. Ultrastructure studies conducted on the Cparv1 strain showed disrupted organelle integrity and the presence of cytoplasmic double membrane stretches. Decreased sterol content in C. parasitica infected with CHV1-EP713 was observed similar to DCparv1 suggesting a connection between the hypovirus-infected phenotype and Cparv1. Furthermore, a non-targeted metabolomic study on all three strains identified 324 metabolites. Through the subsequent pathway analysis, we have investigated the pleiotropic effects in the C. parasitica strains and established a mechanistic linkage between this the activity of the ARV-1 protein and the hypovirus-infected phenotype.

In this thesis, an analytical platform was designed and applied to various in vitro bacterial and eukaryotic cell cultures. An extraction and an analytical protocol were developed for comprehensive and simultaneous analysis of both lipid and polar metabolites for intra- and extracellular metabolomics using HILIC-LC-TOF-MS. This analytical platform was applied to four diverse research questions such as the effect of oxygen environment on growth, the interplay between gene expression and metabolism, metabolic changes that occur with age, and PAH toxicity. Specifically: (i) the effect of oxygen on the growth, physiology and metabolism of the Gram positive Streptococcus intermedius were investigated by comprehensive intra- and extracellular metabolomes and transcriptome. (ii) Metabolic insights into the role of the multipartite genome of the Gram negative bacteria Sinorhizobium meliloti and its metabolic preferences in a nutritionally complex environment. (iii) Age-associated metabolic dysregulation in murine bone marrow-derived macrophages during bacterial lipopolysaccharide-induced inflammation. (iv) Comprehensive intracellular metabolomic profiles of Sinorhizobium meliloti to sub-lethal exposure of individual or mixtures of polycyclic aromatic hydrocarbon revealed additive and dose-dependent effects. This thesis has demonstrated the versatility of the designed analytical platform and its use for diverse research in cell biology. / Thesis / Doctor of Philosophy (PhD)

La documentation scientifique fait état de la présence, chez l’adulte, de cellules souches et progénitrices neurales (CSPN) endogènes dans les zones sous-ventriculaire et sous-granulaire du cerveau ainsi que dans le gyrus denté de l’hippocampe. De plus, un postulat selon lequel il serait également possible de retrouver ce type de cellules dans la moelle épinière et le néocortex des mammifères adultes a été énoncé. L’encéphalopathie de Wernicke, un trouble neurologique grave toutefois réversible qui entraîne un dysfonctionnement, voire une défaillance du cerveau, est causée principalement par une carence importante en thiamine (CT). Des observations récentes laissent envisager que les facteurs en cause dans la prolifération et la différenciation des CSPN pourraient également jouer un rôle important lors d’un épisode de CT. L’hypothèse, selon laquelle l’identification de nouveaux métabolites entrant dans le mécanisme ou la séquence de réactions se soldant en une CT pourraient en faciliter la compréhension, a été émise au moyen d'une démarche en cours permettant d’établir le profil des modifications métaboliques qui surviennent en de telles situations. Cette approche a été utilisée pour constater les changements métaboliques survenus au niveau du foyer cérébral dans un modèle de rats déficients en thiamine (rats DT), particulièrement au niveau du thalamus et du colliculus inférieur (CI). La greffe de CSPN a quant à elle été envisagée afin d’apporter de nouvelles informations sur la participation des CSPN lors d’un épisode de CT et de déterminer les bénéfices thérapeutiques potentiels offerts par cette intervention. Les sujets de l’étude étaient répartis en quatre groupes expérimentaux : un premier groupe constitué de rats dont la CT était induite par la pyrithiamine (rats DTiP), un deuxième groupe constitué de rats-contrôles nourris ensemble (« pair-fed control rats » ou rats PFC) ainsi que deux groupes de rats ayant subi une greffe de CSPN, soit un groupe de rats DTiP greffés et un dernier groupe constitué de rats-contrôles (rats PFC) greffés. Les échantillons de foyers cérébraux (thalamus et CI) des quatre groupes de rats ont été prélevés et soumis à des analyses métabolomiques non ciblées ainsi qu’à une analyse visuelle par microscopie à balayage électronique (SEM). Une variété de métabolites-clés a été observée chez les groupes de rats déficients en thiamine (rats DTiP) en plus de plusieurs métabolites dont la documentation ne faisait pas mention. On a notamment constaté la présence d’acides biliaires, d’acide cynurénique et d’acide 1,9— diméthylurique dans le thalamus, alors que la présence de taurine et de carnosine a été observée dans le colliculus inférieur. L’étude a de plus démontré une possible implication des CSPN endogènes dans les foyers cérébraux du thalamus et du colliculus inférieur en identifiant les métabolites-clés ciblant les CSPN. Enfin, les analyses par SEM ont montré une amélioration notable des tissus à la suite de la greffe de CSPN. Ces constatations suggèrent que l’utilisation de CSPN pourrait s’avérer une avenue thérapeutique intéressante pour soulager la dégénérescence symptomatique liée à une grave carence en thiamine chez l’humain. / Endogenous neural-stem progenitor cells (NSPC) have been documented to be found in the subventricular and subgranular zones, the dentate gyrus, and suggestions of the possibility of these cells being found in the spinal cord and neocortex in adult mammalian brain have been postulated. Thiamine deficiency (TD) is the major cause of Wernicke's Encephalopathy, a reversible neurological disorder that results in cerebral dysfunction and impairment. Recent evidence suggests factors involved in neural NSPC proliferation and differentiation are involved during TD. By means of a current approach for profiling metabolic changes occurring in focal areas of the TD rat brain, specifically the thalamus and the inferior colliculus (IC), it was hypothesized that new metabolites that might offer a better understanding into the sequel and/or mechanism of TD could be identified. It was also considered that the use of NSPC transplantation could offer new information into the involvement of NSPC and potential therapeutic benefit in TD. Non-targeted metabolomics analysis, fluorescences microscopy, and scanning election microscopy (SEM) analysis visualization was performed on samples of the focal areas (thalamus and IC) of pyrithiamine induced TD rats (PTD), pair-fed controls (PFC) rats, and NSPC transplanted TD and PFC rats. Various key metabolites were identified in rats with TD, including previous undocumented metabolites such as bile acids, kynurenic acid, and 1,9-dimethyluric acid in the thalamus and taurine and carnosine in the IC. The study also demonstrated a possible involvement of endogenous NSPC in focal areas of the thalamus and IC identifying key metabolites targeting NSPC and showed tissue amelioration (observed through SEM) following NSPC transplantation. The findings suggested that NSPC could offer a therapeutic alternative to alleviate some of symptomatic degeneration of TD.