Tuberculosis (TB) claims the lives of millions of individuals each year, and is consequently the world’s second-most deadly infectious disease after acquired immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010 alone. Developing countries carry the heaviest burden, with the occurrence of multidrug-resistant (MDR) TB becoming more frequent, making more efficient vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1 gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and the Type Vll secretion system are thought to be responsible for the transport of extracellular proteins across the hydrophobic, and highly impermeable, cell wall of Mycobacterium, and consequently are thought to play a role in the virulence of this organism. To date, our understanding of tuberculosis pathophysiology and virulence has been described primarily using proteomic and genomic approaches. Subsequently, using the relatively new research approach called metabolomics, and interpreting the data using systems biology, we aimed to identify new metabolite markers that better characterise virulence and the proteins involved, more specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS metabolomics research approach, we compared the varying metabolomes of M. smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and subsequently identified those metabolite markers differing between these strains. Multivariate and univariate statistical analyses of the analysed metabolome were used to identify those metabolites contributing most to the differences seen between the two sample groups. A general increase in various carbohydrates, amino acids and lipids, associated with cell wall structure and function, were detected in the ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites associated with the antioxidant system, virulence protein formation and energy production in these mycobacteria, were also seen to differ between the two groups. This metabolomics investigation is the first to identify the metabolite markers confirming the role of the ESX-1 gene cluster with virulence and the underlying metabolic pathways, as well as its associated role with increased metabolic activity, growth/replication rates, increased cell wall synthesis and an altered antioxidant mechanism, all of which are believed to contribute to this organism’s increased pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Tuberculosis (TB) claims the lives of millions of individuals each year, and is consequently the world’s second-most deadly infectious disease after acquired immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010 alone. Developing countries carry the heaviest burden, with the occurrence of multidrug-resistant (MDR) TB becoming more frequent, making more efficient vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1 gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and the Type Vll secretion system are thought to be responsible for the transport of extracellular proteins across the hydrophobic, and highly impermeable, cell wall of Mycobacterium, and consequently are thought to play a role in the virulence of this organism. To date, our understanding of tuberculosis pathophysiology and virulence has been described primarily using proteomic and genomic approaches. Subsequently, using the relatively new research approach called metabolomics, and interpreting the data using systems biology, we aimed to identify new metabolite markers that better characterise virulence and the proteins involved, more specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS metabolomics research approach, we compared the varying metabolomes of M. smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and subsequently identified those metabolite markers differing between these strains. Multivariate and univariate statistical analyses of the analysed metabolome were used to identify those metabolites contributing most to the differences seen between the two sample groups. A general increase in various carbohydrates, amino acids and lipids, associated with cell wall structure and function, were detected in the ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites associated with the antioxidant system, virulence protein formation and energy production in these mycobacteria, were also seen to differ between the two groups. This metabolomics investigation is the first to identify the metabolite markers confirming the role of the ESX-1 gene cluster with virulence and the underlying metabolic pathways, as well as its associated role with increased metabolic activity, growth/replication rates, increased cell wall synthesis and an altered antioxidant mechanism, all of which are believed to contribute to this organism’s increased pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Concern on a global scale gave rise to the founding of the Stockholm Convention on persistent organic pollutants (POPs) with a view to restrict the use and production of these toxic chemicals. As a signatory, South Africa is legally bound to abide to the Convention’s objectives, including participating in relevant research and monitoring. However, developing countries such as South Africa have limited information concerning POPs, partially because these countries do not have sufficient analytical capabilities, and thus method development and refinement are necessary. One group of POPs consisting of polychlorinated dibenzo-pdioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dl-PCBs), collectively referred to as dioxins, are of particular concern due to their high toxicity and persistence. Additionally, the analysis of dioxins is recognised as one of the most analytically challenging of its kind. This study investigated the effect of different preparatory protocols on the semi-quantification of dioxins using the H4IIE-luc-reporter gene assay. The protocols evaluated were either Soxhlet or pressurised liquid extraction (PLE) combined with a manual acid digestion, gel permeation chromatography (GPC) and Florisil fractionation clean-up procedure as well as the automated Total Rapid Prep™(TRP) system which makes use of a PLE combined with a multi-layer silica, alumina and carbon column clean-up procedure. To evaluate the protocols, an eight point matrix matched calibration curve, two soil samples and a certified reference material (CRM) were used. The extracts were semi-quantified by the H4IIE-luc bio-assay. During the course of the assay, the appropriateness of different standards was investigated, and a mixed standard containing all 17 toxic PCDD/Fs was chosen for quantification. During the data review process, higher bioassay equivalent (BEQ) values were obtained from PLE compared to Soxhlet extraction, while no statistically significant difference (Kruskal-Wallis ANOVA: p > 0.05) was found between the assay quantifications for the different preparatory techniques. However, the results of the H4IIE bio-assay were larger than the expected values. The identity of the chemicals that were in all likelihood responsible for the higher response was investigated through instrumental analysis using comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GCxGC-TOFMS). Instrumental results indicated a high level of PAHs in the extracts, which could lead to super induction of the aryl hydrocarbon receptor (AhR) and therefore, to a positive bias in the results. Instrumental screening proved that all selected preparatory protocols were inadequate at removing interfering compounds and not sufficiently selective for PCDD/Fs, although the TRP was more successful in removing interferences. The high matrix interference hindered peak identification. Additionally, as indicated by instrumental analysis, the weak recovery of PCDD/Fs could be ascribed to high evaporation temperatures. The effect of different reference standards in the H4IIE bio-assay used during semi-quantification needs further investigation; similarly, the optimisation of extraction, evaporation and clean-up protocols and the use of different GCxGC-TOFMS column combinations aimed at more efficient separation needs to be investigated. The assistance of the National Metrology Institute (funded through the Department of Trade and Industry) towards this research is hereby acknowledged. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Concern on a global scale gave rise to the founding of the Stockholm Convention on persistent organic pollutants (POPs) with a view to restrict the use and production of these toxic chemicals. As a signatory, South Africa is legally bound to abide to the Convention’s objectives, including participating in relevant research and monitoring. However, developing countries such as South Africa have limited information concerning POPs, partially because these countries do not have sufficient analytical capabilities, and thus method development and refinement are necessary. One group of POPs consisting of polychlorinated dibenzo-pdioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dl-PCBs), collectively referred to as dioxins, are of particular concern due to their high toxicity and persistence. Additionally, the analysis of dioxins is recognised as one of the most analytically challenging of its kind. This study investigated the effect of different preparatory protocols on the semi-quantification of dioxins using the H4IIE-luc-reporter gene assay. The protocols evaluated were either Soxhlet or pressurised liquid extraction (PLE) combined with a manual acid digestion, gel permeation chromatography (GPC) and Florisil fractionation clean-up procedure as well as the automated Total Rapid Prep™(TRP) system which makes use of a PLE combined with a multi-layer silica, alumina and carbon column clean-up procedure. To evaluate the protocols, an eight point matrix matched calibration curve, two soil samples and a certified reference material (CRM) were used. The extracts were semi-quantified by the H4IIE-luc bio-assay. During the course of the assay, the appropriateness of different standards was investigated, and a mixed standard containing all 17 toxic PCDD/Fs was chosen for quantification. During the data review process, higher bioassay equivalent (BEQ) values were obtained from PLE compared to Soxhlet extraction, while no statistically significant difference (Kruskal-Wallis ANOVA: p > 0.05) was found between the assay quantifications for the different preparatory techniques. However, the results of the H4IIE bio-assay were larger than the expected values. The identity of the chemicals that were in all likelihood responsible for the higher response was investigated through instrumental analysis using comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GCxGC-TOFMS). Instrumental results indicated a high level of PAHs in the extracts, which could lead to super induction of the aryl hydrocarbon receptor (AhR) and therefore, to a positive bias in the results. Instrumental screening proved that all selected preparatory protocols were inadequate at removing interfering compounds and not sufficiently selective for PCDD/Fs, although the TRP was more successful in removing interferences. The high matrix interference hindered peak identification. Additionally, as indicated by instrumental analysis, the weak recovery of PCDD/Fs could be ascribed to high evaporation temperatures. The effect of different reference standards in the H4IIE bio-assay used during semi-quantification needs further investigation; similarly, the optimisation of extraction, evaporation and clean-up protocols and the use of different GCxGC-TOFMS column combinations aimed at more efficient separation needs to be investigated. The assistance of the National Metrology Institute (funded through the Department of Trade and Industry) towards this research is hereby acknowledged. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.