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One of the many recorded adaptive responses in respiratory chain complex I deficient cells is the over-expression of the small metal binding proteins, metallothioneins (MTs). The antioxidant properties of MTs putatively protect the deficient cells against oxidative damage, thus limiting further damage and impairment of enzymes involved in energy production. Moreover, the role of metallothioneins in supplying metal cofactors to enzymes and transcription factors in order to promote energy metabolism was previously proposed, which could accompany their role as antioxidants. This view is supported by the observations that MT knockout mice tend to become moderately obese, implying a lower energy metabolic rate. Hence, the involvement of metallothioneins in mitochondrial function and disease cannot be ignored. However, this association is still very vague due to the diversity of their functions and the complexity of the mitochondrion. The use of systems biology technology and more specifically metabolomics technology was thus employed to clarify this association by investigating the metabolic differences between wild type and MT knockout mice in unchallenged conditions as well as when mitochondrial function (energy metabolism) was challenged with exercise and/or a high-fat diet. The metabolic differences between these mice were also studied when complex I of the respiratory chain was inhibited with rotenone. The metabolome content of different tissues and bio-fluids were examined in an untargeted fashion using three standardized analytical platforms and the data mined using modern metabolomics and related statistical methods. Clear metabolic differences were found between the wild type and MT knockout mice during unchallenged conditions. These metabolic differences were persisted and were often amplified when mitochondrial metabolism was specifically challenged through exercise, high-fat intake or complex I inhibition. The data pointed to an overall reduced metabolic rate in the MT knockout mice and possible insulin resistance after the interventions which imply (and confirm) the involvement of MTs in promoting energy metabolism in the wild type mice. Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012 One of the many recorded adaptive responses in respiratory chain complex I deficient cells is the over-expression of the small metal binding proteins, metallothioneins (MTs). The antioxidant properties of MTs putatively protect the deficient cells against oxidative damage, thus limiting further damage and impairment of enzymes involved in energy production. Moreover, the role of metallothioneins in supplying metal cofactors to enzymes and transcription factors in order to promote energy metabolism was previously proposed, which could accompany their role as antioxidants. This view is supported by the observations that MT knockout mice tend to become moderately obese, implying a lower energy metabolic rate. Hence, the involvement of metallothioneins in mitochondrial function and disease cannot be ignored. However, this association is still very vague due to the diversity of their functions and the complexity of the mitochondrion. The use of systems biology technology and more specifically metabolomics technology was thus employed to clarify this association by investigating the metabolic differences between wild type and MT knockout mice in unchallenged conditions as well as when mitochondrial function (energy metabolism) was challenged with exercise and/or a high-fat diet. The metabolic differences between these mice were also studied when complex I of the respiratory chain was inhibited with rotenone. The metabolome content of different tissues and bio-fluids were examined in an untargeted fashion using three standardized analytical platforms and the data mined using modern metabolomics and related statistical methods. Clear metabolic differences were found between the wild type and MT knockout mice during unchallenged conditions. These metabolic differences were persisted and were often amplified when mitochondrial metabolism was specifically challenged through exercise, high-fat intake or complex I inhibition. The data pointed to an overall reduced metabolic rate in the MT knockout mice and possible insulin resistance after the interventions which imply (and confirm) the involvement of MTs in promoting energy metabolism in the wild type mice. Doctoral

The induction of metallothionein (MT) expression in mitochondrial disorders has been well studied on the transcription level by means of RNA measurements in an attempt to understand and confirm the function of this protein in the deficient cells and organs (Olivier, 2004:42; Pretorius, 2006:44; Reinecke, 2004:89). However, MT expression induction still needs to be verified on protein (translation) level in order to confirm previous findings and to gain better perspective on the significance of MT expression induction. Therefore, it is necessary to use a technique that is capable of quantifying MT accurately in biological material. Due to the lack of sensitivity and selectivity of many commonly used techniques (Dabrio et al., 2002:125), it is necessary to develop a mass spectrometric based quantification technique to detect and quantify MT-2A selectively and accurately. For quantification of human MT-2A in biological material using a mass spectrometry-based method, a MT (MT-2A) standard similar to the native form but with a slightly different mass was required. Due to the lack of pure human MT standards and high cost of pure rabbit MT standards, it was decided to create a recombinant human MT-2A with different mass due to additional N-terminal amino acids. In addition, native human MT-2A is also required to develop and optimize an MS quantification technique in a future study. Therefore, pure (98 %) rabbit MT standard, which is highly similar to human MT-2A, was purchased to serve as a positive control for MS detection in this study and which can also be used to develop and optimize an MS quantification technique in a future study. An expression vector for human MT-2A was constructed with the use of recombinant DNA techniques. The correct construct was identified and characterized with PCR and verified by sequencing. This newly created expression vector was transformed into four E.coli BL21(DE3) strains to express a modified human recombinant MT-2A (MT-2AA) using induction with IPTG. This protein comprised of a full length human MT-2A sequence, but excluding the N-terminal Met and including an N-terminal His-tag. MT-2AA expression in the selected strains was extensively optimized and monitored with SDS-PAGE. Ecoli BL21 (DE3) CodonPlus-RIL cells proved to be the strain that expressed MT-2'A at the highest relative levels. Expressed MT-2'A was isolated and purified using a three step purification procedure which included heat treatment, metal chelating chromatography and RP-HPLC. Relative pure (70 %) MT-2'A was successfully obtained as confirmed with SDS-PAGE and mass spectrometry. Removal of the His-tag from MT-2'A with thrombin protease cleavage was, however, unsuccessful. In addition, it was observed that this protein was, compared to native commercially obtained MT-2A, unstable and after extensive purification still had a lower than required purity. It was concluded from this studies' results that, although it was successfully produced, this recombinant MT-2A protein would not be suitable as an internal standard for MS analysis of human MT-2A. On the other hand, rabbit MT-2E (as alternative) holds great promise as internal standard since it is stable and pure. Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2008.

Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2008. The induction of metallothionein (MT) expression in mitochondrial disorders has been well studied on the transcription level by means of RNA measurements in an attempt to understand and confirm the function of this protein in the deficient cells and organs (Olivier, 2004:42; Pretorius, 2006:44; Reinecke, 2004:89). However, MT expression induction still needs to be verified on protein (translation) level in order to confirm previous findings and to gain better perspective on the significance of MT expression induction. Therefore, it is necessary to use a technique that is capable of quantifying MT accurately in biological material. Due to the lack of sensitivity and selectivity of many commonly used techniques (Dabrio et al., 2002:125), it is necessary to develop a mass spectrometric based quantification technique to detect and quantify MT-2A selectively and accurately. For quantification of human MT-2A in biological material using a mass spectrometry-based method, a MT (MT-2A) standard similar to the native form but with a slightly different mass was required. Due to the lack of pure human MT standards and high cost of pure rabbit MT standards, it was decided to create a recombinant human MT-2A with different mass due to additional N-terminal amino acids. In addition, native human MT-2A is also required to develop and optimize an MS quantification technique in a future study. Therefore, pure (98 %) rabbit MT standard, which is highly similar to human MT-2A, was purchased to serve as a positive control for MS detection in this study and which can also be used to develop and optimize an MS quantification technique in a future study. An expression vector for human MT-2A was constructed with the use of recombinant DNA techniques. The correct construct was identified and characterized with PCR and verified by sequencing. This newly created expression vector was transformed into four E.coli BL21(DE3) strains to express a modified human recombinant MT-2A (MT-2AA) using induction with IPTG. This protein comprised of a full length human MT-2A sequence, but excluding the N-terminal Met and including an N-terminal His-tag. MT-2AA expression in the selected strains was extensively optimized and monitored with SDS-PAGE. Ecoli BL21 (DE3) CodonPlus-RIL cells proved to be the strain that expressed MT-2'A at the highest relative levels. Expressed MT-2'A was isolated and purified using a three step purification procedure which included heat treatment, metal chelating chromatography and RP-HPLC. Relative pure (70 %) MT-2'A was successfully obtained as confirmed with SDS-PAGE and mass spectrometry. Removal of the His-tag from MT-2'A with thrombin protease cleavage was, however, unsuccessful. In addition, it was observed that this protein was, compared to native commercially obtained MT-2A, unstable and after extensive purification still had a lower than required purity. It was concluded from this studies' results that, although it was successfully produced, this recombinant MT-2A protein would not be suitable as an internal standard for MS analysis of human MT-2A. On the other hand, rabbit MT-2E (as alternative) holds great promise as internal standard since it is stable and pure. Masters

MSc (Biochemistry), North-West University, Potchefstroom Campus The dysfunction of mitochondrial complex I (CI) impedes the most efficient mechanism feeding electrons into the respiratory chain (RC) — a system that, together with ATP synthase (CV), is responsible for the majority of cellular energy production via oxidative phosphorylation (OXPHOS). Although CI deficiency is the most common defect in mitochondrial energy metabolism, it is among the most complex, multifactorial, poorly understood disorders that currently lack an effective treatment. One of the most promising tools used to gain insight into this form of mitochondrial disease (MD), is the whole-body Ndufs4 knockout (Ndufs4/) mouse model. Although the neurological phenotype of these animals has been widely studied, the effect of CI deficiency on Ndufs4/skeletal muscle metabolism remains elusive. The lack of research on this tissue is largely owed to the view that these animals do not display strong muscle involvement. However, neuromuscular involvement is a hallmark feature of MD and considering skeletal muscle’s high energetic demand, metabolic activity, and integration with the nervous system, this tissue needs to be investigated. The aim of this study was, therefore, to combine hypothesis-generating metabolic profiling and biochemical strategies to gain insight into the energy metabolism of both glycolytic (white quadriceps) and oxidative (soleus) skeletal muscles from Ndufs4/mice. Profiling methods that utilise various high-content analytical platforms were employed in order to generate a broad metabolic phenotype of CI deficient muscles. This multi-platform metabolomics approach comprised of targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS), untargeted gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and proton nuclear magnetic resonance (1H-NMR) spectroscopy. However, extensive multi-platform metabolomics of mouse tissues presents a challenge due to the limited-quantity samples obtained — especially in the case of NMR spectroscopy, which is inherently insensitive. Therefore, one of the chief objectives of the study was to develop a 1H-NMR method that enables the analysis of small-quantity biological samples. In the first part of the study, we present a novel miniaturised 1H-NMR method utilising 2 mm NMR tubes, which enables metabolic profiling on a tenth of the sample quantity required by a well-established standard operating protocol (SOP). We demonstrate the miniaturised method’s acceptability regarding precision (CV < 15%), relative accuracy (80–120 %), linearity (R2 > 0.95) and statistical equivalence (p < 0.05) to the SOP when analysing spiked synthetic urine as well as mouse muscle extracts. In addition, we exhibit the novel method’s advantages for large-scale metabolomics when; i) adequate sample quantities are available by analysing increasingly concentrated versions (up to 10×) of samples to expand metabolome coverage; or ii) sample quantities are limited by performing a pilot metabolomics study on minute Ndufs4/(n = 3) and wild-type (WT; n = 3) solei, which identified five metabolites (previously linked to MD) strongly discriminating the two genotypes. In the second part of the study, we investigate the effects of CI deficiency in Ndufs4/skeletal muscles. Through multi-platform metabolic profiling of Ndufs4/(n = 19), and WT (n = 20) white quadriceps and soleus muscles, we provide the first empirical evidence of adaptive responses to CI dysfunction involving non-classical pathways that fuel the ubiquinone (Q)-cycle. By restoring the electron flux to CIII via the Q-cycle, these adaptive mechanisms could maintain adequate oxidative ATP production, despite CI deficiency — providing a possible explanation for the lack of muscle involvement in the Ndufs4/phenotype. We report a respective 48 and 34 discriminatory metabolites between Ndufs4/, and WT white quadriceps and soleus muscles, among which the most prominent alterations indicate the involvement of the glycerol-3-phosphate shuttle, the electron transfer flavoprotein system, respiratory chain CII, and the proline cycle in fuelling the Q-cycle. Enzyme (CI-CIV and CS) activity assays confirmed severely reduced (80 %) CI activity in both Ndufs4/(n = 12) muscle types, compared to WTs (n = 10), along with moderate reductions in CS (12 %) and CIII (18 %) activities in Ndufs4/solei. When comparing muscle fibre types, glycolytic fibres seemed to be more vulnerable to CI deficiency, as greater disturbances in metabolic profiles were evident, along with much lower residual CI activity (4.58 ± 1.67 nmol/min/mg) compared to oxidative fibres (14.74 ± 6.67 nmol/min/mg). Taken together, this study contributes to the natural science field in two ways. Firstly, through our novel miniaturised 1H-NMR method, we provide a cost-effective alternative solution for the current restrictions of NMR spectroscopy in the analysis of limited-quantity biological samples. Traditionally, cryoprobe technology is required for such studies; however, we show that a typical NMR spectrometer with a standard probe head can be used to analyse small sample quantities with adequate analytical efficiency. Secondly, through discover-phase multi-platform metabolic profiling, we provide novel mechanistic insight into CI deficiency — thereby highlighting the value of metabolomics in MD research. We report skeletal muscle-specific changes in several metabolic pathways that result from whole-body mitochondrial dysfunction, which upon further investigation could provide novel targets for therapeutic intervention in CI deficiency and potentially lead to the development of new treatment strategies. Masters

MSc (Biochemistry), North-West University, Potchefstroom Campus Mitochondrial disease is one of the most prevalent inherited paediatric disorders among children, with a prevalence of 1 in 5000 children. Leigh syndrome is the result of a Complex I (CI) deficiency and disrupts the redox balance needed for the function of various dehydrogenase enzymes. Due to the extensive heterogeneity of the disease, it remains a challenge to diagnose and treat mitochondrial diseases. One of the most overlooked areas for possible treatment is the solute carriers of the inner mitochondrial membrane (IMM). These solute carriers function together to form shuttle systems that transport electrons over the impermeable IMM, which can possibly aid in recovery of the disrupted redox balance. Targeted transcriptomic analysis was done on liver, heart and brain tissue collected from a CI deficient (Ndufs4 knockout) mice model to analyse the gene expression of the selected solute carriers part of the malate-aspartate shuttle and the citrate-pyruvate shuttle. The proteins of the glycerol-3-phosphate shuttle was also included due to its ability to also carry electrons over the IMM. This was done by making use of the Ion GeneStudioâ„¢ S5 Semiconductor Sequencer accompanied by the Ion Chefâ„¢ Instrument. Targeted metabolomics was also done on the selected tissue on the metabolites transported by the solute carriers, also to deduce if there are any changes in their abundance with diseases like Leigh syndrome. This was done via GC-TOF-MS and LC-MS/MS analysis. Transcriptomic analysis revealed for the liver tissue, Gpd1 (glycerol-3-phosphate dehydrogenase 1), Mdh1 (malate dehydrogenase 1), Me1 (malic enzyme 1) and Slc25a1 (solute carrier family 25 member 1, citrate carrier) were all down regulated in the knockout group. For the heart tissue, Slc25a22 (solute carrier family 25 member 22, glutamate carrier) and Me1 (malic enzyme 1) were also down regulated in the knockout group and for the brain tissue, Me3 (malic enzyme 3), Slc25a3 (solute carrier 25 member 3, phosphate carrier) and Slc25a22 (solute carrier family 25 member 22, glutamate carrier) were all up regulated in the knockout group. Changes in the expression of these genes all seem to be an effect of the redox imbalance due to complex I deficiency or revolve around an attempt to maintain energy production. Metabolomic analysis indicated an increase in abundance in metabolites associated with the down-regulated enzymes and shuttles. These changes all seem to be an effect of the complex I knockout and not an attempt to compensate for the disruption of the cells function. Considering this study involved an unconventional method for gene expression analysis, the overall success in the outcome of this study can be attributed to the combination of transcriptomics and metabolomics data to give a better understanding of the events at molecular level with abnormalities such as complex I deficiency. Masters

MSc (Biochemistry), North-West University, Potchefstroom Campus Mutations in mitochondrial DNA (mtDNA) result in primary mitochondrial disease (MD), which presents with a wide range of clinical phenotypes. There is currently no cure for MD, this can be attributed to the major phenotypical discrepancies that exist between MD patients. A single mutation can lead to different clinical phenotypes while different mutations can lead to the same phenotype, which makes diagnosis difficult. Heteroplasmy, a state where more than one mtDNA genotype coexist within the same cell, is thought to be one of the confounding factors influencing the differences in clinical phenotypes. Although the effect of heteroplasmy on MD severity has been widely studied in vitro, not much is known about the effect of heteroplasmy in vivo. This is due to a lack of animal models that harbour functional heteroplasmic mutations. This study employed the LB138 (uaDf5) C. elegans strain to investigate the characteristics that influence the suitability of this strain as a disease model for harbouring a range of mtDNA deletion heteroplasmy levels and ii) elucidate the cellular responses to increasing heteroplasmy levels in this model. To achieve the aim of this study, a combination of investigative approaches including genetic, biochemical, and metabolomic investigations were carried out in succession. First, genetic characterization was carried out to determine changes in mtDNA copy number and uaDf5 mtDNA heteroplasmy levels during larval development. While significant changes in mtDNA copy number were observed in the larval stages, alterations in heteroplasmy levels were not significant. Furthermore, comparing uaDf5 mtDNA heteroplasmy levels between parent and progeny, as well as among progeny from the same parent, showed considerable variation in heteroplasmy levels in individual nematodes. This suggests that it is difficult to predict the heteroplasmy levels of a large number of worms from the same sample (i.e., plate). Second, biochemical investigation indicated that the uaDf5 C. elegans strain presents with a mitochondrial disease phenotype, as evident by the significant reduction in basal respiration and complex (CIII) activity. Third, one of the main objectives of this study was to standardize a metabolite extraction method for metabolomics investigation on C. elegans. A method utilizing iv glass beads, to ensure efficient disruption of the outer protective cuticle of C. elegans, was successfully standardized. Using the standardized method, metabolomics investigations were performed on thirty C. elegans samples, harbouring different levels of uaDf5 mtDNA deletion across an established but narrow range. Metabolomics revealed no significant correlations between metabolite concentrations and the different levels of uaDf5 mtDNA. In conclusion, the uaDf5 C. elegans strain was deemed to be not suitable for investigating the effect of an mtDNA deletion along the heteroplasmy range, thus future studies should consider using model organisms that harbour predictable heteroplasmy levels. Masters

PhD (Science with Biochemistry), North-West University, Mahikeng Campus The high prevalence of obesity and related metabolic disorders due to increased consumption of fructose-rich diet is a serious global threat to the public’s health among children and adolescents. Obesity is associated with several metabolic disorders including, type-2 diabetes, insulin resistance, inflammation, and oxidative stress. Furthermore, obesity is characterised by impaired lipid metabolism in key tissues such as the liver and adipose tissue. Curcumin, a hydrophobic polyphenol extracted from Curcuma longa (turmeric), possesses anti-obesity, anti-diabetic, anti-inflammatory, and antioxidant effects. Although curcumin has been reported to possess a range of biological activities, molecular targets of curcumin are not fully elucidated. The present study was also motivated by evidence suggesting that curcumin may regulate lipid metabolism, which plays a central role in the development of obesity and its complications. Therefore, the first objective of the present study reported the effect of neonatal intake of curcumin on key molecular biomarkers associated with lipid metabolism in the liver, namely, LKB-1, AMPK, CPT-1, and ACC-1. Likewise, the second objective reported the effect of neonatal intake of curcumin on key molecular biomarkers associated with glucose metabolism in the liver, namely, AKT-1, GLUT-2, GP, and PGM (AKT-1 was also considered as the biomarker for insulin resistance in this study). The third objective reported the effect of neonatal intake of curcumin on inflammatory biomarkers in the liver, viz., TNF-α and IL-6. Furthermore, the fourth objective reported the effect of neonatal intake of curcumin on oxidative stress by determining the antioxidant status of curcumin in the liver using FRAP and TEAC assays, as well as the antioxidant scavenging activity of curcumin using DPPH assay and the antioxidant enzyme activity of the SOD. Finally, the fifth objective reported the effect of neonatal intake of curcumin on levels of liver metabolites as well as the concentration of the lipid-bound fatty acids detected as fatty acid methyl esters in adipose tissues. In this study, forty male Sprague Dawley rats were divided into four groups and administered with either a 0.5% dimethyl sulfoxide (vehicle control), 500 mg.kg-1 body mass of curcumin, fructose (20%, w/v) or a combination of curcumin and fructose. The study was conducted in two phases, the first phase was the pre-weaning which began from postnatal day 6 to day 21, and the second phase was the post-weaning which began from postnatal day 21 to day 63. At the end of the treatments on postnatal day 63, rats were euthanized. Then the liver and visceral fat tissues were collected for further molecular analysis. Gene expression was analysed using real-time quantitative polymerase chain reaction while protein expression was analysed using western blot. The antioxidant status was determined using the FRAP and TEAC assays while the antioxidant scavenging activity was determined using the DPPH radical scavenging and SOD activity assays. The concentration levels of hepatic metabolites and the adipose tissue lipid-bound fatty acids were assessed using gas-chromatography-mass spectrometry. Results showed upregulation in the expression of genes and proteins associated with lipid metabolism when the high fructose diet was administered. These include AMPK and its direct activator LKB-1, as well as subsequent target molecules ACC-1 and CPT-1 in the liver tissues. Treatment with curcumin showed to reverse the adverse effects of high fructose diet by upregulating the expression of genes and proteins analysed in the lipid metabolism. This study found that high fructose diet feeding altered hepatic insulin signalling target molecule AKT-1 by downregulating its gene and protein expression. In comparison, treatment with curcumin upregulated the expression of AKT-1. On the one hand, the upregulation of GP and PGM gene and protein expression in the high fructose diet group suggest that hepatic glucose production was promoted. On the other hand, treatment with curcumin suppressed hepatic glucose production by downregulating the expression of GP and PGM. In this study, it was also noted that the administration of curcumin downregulated the gene and protein expression of TNFα and IL-6 whereas the high fructose diet abnormally upregulated their expression. Furthermore, treatment with curcumin showed to enhance hepatic antioxidant status. This was observed by the increase in FRAP and TEAC values, as well as in DPPH level and SOD activity. Finally, it was noted that hepatic carbohydrate metabolite and hepatic organic acids were reduced in the high fructose diet group compared to the control group, while treatment with curcumin showed no significant difference. In the adipose tissue fatty acids levels analysis, results showed an increase in concentration level in the high fructose diet group of all SFA analysed, whereas the opposite had been observed in some of the MUFA and PUFA. Treatment with curcumin showed a decrease in the elevated concentration level of SFA respectively. Moreover, treatment with curcumin increased the concentration level of omega-3 PUFA (EPA and clupanodonic acid), though with no significant difference. The study concluded that curcumin elicits beneficial effects to ameliorate obesity and its related metabolic disorders by regulating molecular biomarkers associated with lipid metabolism through AMPK activation and its subsequent target molecules such as ACC-1 and CPT-1 in the liver tissues. In glucose metabolism, we concluded that curcumin suppressed hepatic glucose production by regulating GP and PGM which are key hepatic glucose production biomarkers. The regulation of an insulin signalling biomarker (AKT-1) by curcumin could also be an alternative therapeutic target to alleviate obesity and its related metabolic disorders. We further confirmed that curcumin possesses anti-inflammatory potential by reducing key inflammatory biomarkers such as TNF-α and IL-6. Finally, using the FRAP and TEAC assays, we further confirmed that curcumin possesses antioxidant capacity in the liver. Similarly, DPPH and SOD activity assays confirmed the antioxidant scavenging activity of curcumin. Therefore, the present study demonstrated that neonatal intake of curcumin could prevent and protect against high fructose diet-induced obesity and its related metabolic disorders such as type-2 diabetes, inflammation, and oxidative stress. Doctoral

MSc (Biochemistry), North-West University, Potchefstroom Campus In recent years, zebrafish (D. rerio) have emerged as valuable research models, being widely used as models for aquaculture research, but also to model human metabolic diseases. The ethical guidelines stipulate that anaesthetics need to be applied before sampling of zebrafish. The effect of the anaesthetics on the metabolic profiles of zebrafish and the implications that this may have for zebrafish as research models, have mostly been overlooked. The primary hypothesis of this study was that anaesthetics will have an effect on zebrafish metabolism. The possibility for new hypotheses to be generated were not eliminated, since untargeted metabolomics are more hypothesis-generating than hypothesis-testing techniques. In this study, multi-platform metabolomics were used to investigate the metabolic alterations in zebrafish caused by three commonly used anaesthetics namely tricaine methanesulfonate (MS-222), eugenol and 2- phenoxyethanol (2-PE). The metabolomic analyses indicated that anaesthesia caused minimal metabolic alterations, with the concentration of only a small number of metabolites altered. It is hypothesized that these concentration changes are caused by ATP depletion and a stress response, which both lead to the upregulation of protein and lipid catabolism. Glycolysis and tricarboxylic acid cycle (TCA) intermediates remained relatively unaffected, but there are some indications that gluconeogenesis may be slightly upregulated. These effects are, however, limited by the short induction times of the anaesthetics. It was also determined that eugenol exhibited significantly less metabolic perturbations than the other anaesthetics with only three significantly altered metabolites, making it the preferred anaesthetic for metabolomic studies in zebrafish. General pathways impacted during anaesthesia include but are not limited to ẞ-oxidation, gluconeogenesis, urea cycle and amino acid transamination. In conclusion, anaesthetics used during sampling are not expected to have a significant effect on the suitability of zebrafish as research models. Further investigations are needed to confirm the hypotheses generated in this study. Masters

Abalone is currently considered South Africa's most successfully produced aquacul‐ ture export product, with a 76% share of the total value generated by the aquaculture sector. A major risk factor for this sector is slow growth rates experienced during farming. Abalone feeds are often supplemented with amino acids in an attempt to enhance abalone growth. This is a first investigation of the effect of added proline to standard abalone feed, on the metabolite profile of slow‐growing abalone. A targeted liquid chromatography tandem mass spectrometry metabolomics research approach was followed to recognise the metabolic response of abalone showing slower growth performance. The addition of proline to the standard abalone diet was found to serve as a substrate for amino acid catabolism in slower growing abalone, by means of pro‐ line breakdown to assist with energy production via the tricarboxylic acid cycle. Other amino acids and urea cycle intermediates, that is, arginine, asparagine, ornith‐ ine and creatine further support energy production via the action of protein catabo‐ lism in slow‐growing abalone. Additionally, the importance of understanding how abalone respond metabolically to modified feed highlights the use of metabolomics to answer abalone aquaculture farming questions

Mitochondrial diseases (MD), such as Leigh syndrome (LS), present with severe neurological and muscular phenotypes in patients, but have no known cure and limited treatment options. Based on their neuroprotective effects against other neurodegenerative diseases in vivo and their positive impact as an antioxidant against complex I deficiency in vitro, we investigated the potential protective effect of metallothioneins (MTs) in an Ndufs4 knockout mouse model (with a very similar phenotype to LS) crossed with an Mt1 overexpressing mouse model (TgMt1). Despite subtle reductions in the expression of neuroinflammatory markers GFAP and IBA1 in the vestibular nucleus and hippocampus, we found no improvement in survival, growth, locomotor activity, balance, or motor coordination in the Mt1 overexpressing Ndufs4-/- mice. Furthermore, at a cellular level, no differences were detected in the metabolomics profile or gene expression of selected one-carbon metabolism and oxidative stress genes, performed in the brain and quadriceps, nor in the ROS levels of macrophages derived from these mice. Considering these outcomes, we conclude that MT1, in general, does not protect against the impaired motor activity or improve survival in these complex I-deficient mice. The unexpected absence of increased oxidative stress and metabolic redox imbalance in this MD model may explain these observations. However, tissue-specific observations such as the mildly reduced inflammation in the hippocampus and vestibular nucleus, as well as differential MT1 expression in these tissues, may yet reveal a tissue- or cell-specific role for MTs in these mice.

MSc (Biochemistry), North-West University, Potchefstroom Campus Mitochondrial diseases (MDs) are the most common inborn errors of metabolism, with an estimated prevalence of approximately 1 in 5 000 live births, and are mainly caused by deficiencies of complex I (CI) of the oxidative phosphorylation (OXPHOS) system. Clinical presentations of CI deficiency are highly heterogeneous, with the most commonly reported, being Leigh syndrome (LS) – a devastating progressive, multi-systemic, neurodegenerative disorder. The Ndufs4 gene, which encodes for an 18 kDa subunit of CI, is a mutational hotspot in LS patients. To date, the efficacy of the limited available therapeutic interventions remains inconclusive, and can, in large, be attributed to our poor understanding of the pathological mechanisms behind these highly complex diseases. Fortunately, with a whole-body Ndufs4 knockout (KO) mouse model available, researchers have a great opportunity to gain a better understanding of this commonly reported MD. What remains lacking, however, is the incorporation of multi-platform metabolomics using urine. This biofluid shows promise in revealing global metabolic perturbations in MDs, and thus possesses the potential to elucidate disease mechanisms. The aim of this study, therefore, was to investigate the metabolic consequences of Ndufs4 deficiency by analysing the urine of the whole-body Ndufs4 KO mouse model. This was accomplished by implementing two main objectives: firstly, by validating the mouse model via genetic and phenotypic evaluation and the measurement of CI activity in the liver; and secondly, by comparing the urinary metabolome of Ndufs4 KO and wild-type mice, acquired via both untargeted and targeted analyses, in order to obtain a comprehensive view of the metabolic consequences. In this study, the mouse model was successfully validated on the genetic and phenotypic level, with Ndufs4 KO mice displaying well-reported phenotypic characteristics, including growth retardation, transient alopecia and hunched back posture. Biochemically, the mouse model was further confirmed with Ndufs4 KO mice exhibiting 15% residual CI activity in the liver. Urinary metabolomic analyses revealed multiple metabolic perturbations in the Ndufs4 KO mice. Most notably, were the markers classically observed in MDs and commonly believed to be the result of an altered redox status, namely elevated levels of pyruvate, lactate and alanine as well as some tricarboxylic acid cycle intermediates (2-ketoglutarate, fumarate and malate). A downregulation in protein/amino acid catabolism was observed, as indicated by decreased levels of numerous amino acids (e.g. glutamine, glutamate, leucine, isoleucine, valine and phenylalanine), 3-methylhistine (index of skeletal muscle breakdown) and metabolites associated with the urea cycle (arginine, citrulline and N-acetylglutamate). Similarly, lipid/fatty acid catabolism also appeared to be downregulated, as shown by lowered levels of glycerol as well as numerous carnitine- and glycine fatty acid conjugates (octanoyl- and decanoylcarntine; butyryl-, valeryl- and hexanoylglycine). Metabolites present in pathways associated with biosynthetic processes and/or ROS scavenging (including the pentose phosphate pathway, one-carbon metabolism and de novo pyrimidine synthesis) were also decreased. Taken together, the implementation of urinary metabolomics proved to be successful in revealing global metabolic perturbations in Ndufs4 KO mice. Masters

North-West University, Potchefstroom Campus MSc (Biochemistry), North-West University,Potchefstroom Campus Mitochondria, the organelles found throughout the cytoplasm of most eukaryotic cells, have essential functions which have been implicated in the etiology of numerous metabolic and degenerative diseases. The mitochondrial oxidative phosphorylation (OXPHOS) system produces up to 90% of cellular energy. It comprises the respiratory chain (RC) of four enzyme complexes and the ATP synthase complex. Genetic mutations that affect the OXPHOS system cause a clinically heterogenous group of disorders which fall under the umbrella term, primary mitochondrial disease (MD). Collectively, MDs are the most common among the inborn errors of metabolism in humans. These diseases generally present with severe, detrimental clinical phenotypes and primarily affect tissues with a high energy demand. An isolated OXPHOS complex I (CI) deficiency is the most commonly observed childhood-onset MD. It is often caused by a mutation in the nuclear coded NADH dehydrogenase (ubiquinone) iron-sulfur protein 4 (Ndufs4) gene. The resulting phenotype, known as Leigh syndrome, is characterised by progressive neurodegeneration in specific brain regions that drives disease progression and premature death. Currently, the mechanisms governing the brain’s regional susceptibility to a CI deficiency are unclear and therapeutic strategies are lacking. Using the Ndufs4 knockout (KO) mouse, an accurate model of Leigh syndrome, this study aimed to determine whether brain regional differences in RC enzyme activities or metabolic profiles could be correlated with neurodegeneration. A combination of spectrophotometric enzyme activity assays and multi-platform metabolomics techniques were applied to investigate four selected brain regions: three neurodegeneration-prone regions (brainstem, cerebellum and olfactory bulbs) and a neurodegeneration-resilient region (anterior cortex). These were obtained from male Ndufs4 KO and wild-type mice. The enzyme assays (biochemical investigation) confirmed that CI activity was significantly reduced (60% to 80%) in the KO brain regions. Additionally, the findings suggested that lower residual CI activity, as well as higher OXPHOS requirements, or differential OXPHOS organisation, could underlie region-specific neurodegeneration. In accordance, a global disturbance in cellular metabolism distinguished the metabolic profiles (metabolomics investigation) of the KO brain regions. These global disturbances seemed to reflect a compensatory response in classic and non-classic metabolic pathways to alleviate the consequences of a CI deficiency. However, these adaptative responses seemed sub-optimal since they are susceptible to the detrimental effects of a CI deficiency and entail maladaptive features. Furthermore, the global metabolic perturbations had a gradient of severity across the brain regions which correlated with neurodegeneration and lower residual CI activity. It therefore seemed that the neurodegeneration-prone brain regions had greater requirements of the sub-optimal compensatory pathways which ultimately reached a detrimental threshold. This then triggered neurodegenerative processes. The impairment of various redox-sensitive reactions also suggested that a lower cellular NAD+/NADH ratio in the neurodegeneration-prone brain regions might augment neurodegenerative processes. In addition, a few discriminatory metabolites unique to the anterior cortex suggested that inherent regional differences in metabolism might play a role in regional neurodegeneration. Conclusively, the results enabled a better understanding of the regional neurodegeneration in Ndufs4 KO mice. The potential metabolic targets for treatment and for monitoring disease progression or therapeutic interventions revealed in this study, warrant further investigation. Masters

Autism spectrum disorder (ASD) is characterized by phenotypic heterogeneity and a complex genetic architecture which includes distinctive epigenetic patterns. We report differential DNA methylation patterns associated with ASD in South African children. An exploratory whole‐epigenome methylation screen using the Illumina 450 K MethylationArray identified differentially methylated CpG sites between ASD and controls that mapped to 898 genes (P ≤ 0.05) which were enriched for nine canonical pathways converging on mitochondrial metabolism and protein ubiquitination. Targeted Next Generation Bisulfite Sequencing of 27 genes confirmed differential methylation between ASD and control in our cohort. DNA pyrosequencing of two of these genes, the mitochondrial enzyme Propionyl‐CoA Carboxylase subunit Beta (PCCB) and Protocadherin Alpha 12 (PCDHA12), revealed a wide range of methylation levels (9–49% and 0–54%, respectively) in both ASD and controls. Three CpG loci were differentially methylated in PCCB (P ≤ 0.05), while PCDHA12, previously linked to ASD, had two significantly different CpG sites (P ≤ 0.001) between ASD and control. Differentially methylated CpGs were hypomethylated in ASD. Metabolomic analysis of urinary organic acids revealed that three metabolites, 3‐hydroxy‐3‐methylglutaric acid (P = 0.008), 3‐methyglutaconic acid (P = 0.018), and ethylmalonic acid (P = 0.043) were significantly elevated in individuals with ASD. These metabolites are directly linked to mitochondrial respiratory chain disorders, with a putative link to PCCB, consistent with impaired mitochondrial function. Our data support an association between DNA methylation and mitochondrial dysfunction in the etiology of ASD. Autism Res 2020, 13: 1079‐1093. © 2020 The Authors. Autism Research published by International Society for Autism Research published by Wiley Periodicals, Inc. Lay Summary Epigenetic changes are chemical modifications of DNA which can change gene function. DNA methylation, a type of epigenetic modification, is linked to autism. We examined DNA methylation in South African children with autism and identified mitochondrial genes associated with autism. Mitochondria are power‐suppliers in cells and mitochondrial genes are essential to metabolism and energy production, which are important for brain cells during development. Our findings suggest that some individuals with ASD also have mitochondrial dysfunction.

Background and Aims: Black boys (6–8 years of age) were shown to have higher pulse wave velocity with potential early vascular compromise. We aimed to compare predefined urinary metabolites in black and white boys to explore associations of pulse wave velocity with these metabolites. Methods and Results: We included 40 white and 40 black apparently healthy boys between the ages of 6 and 8 years. Femoral pulse wave velocity was measured along with various metabolites using liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) methods. Pulse wave velocity and diastolic blood pressure were higher in the black compared to the white boys (both p ≤ 0.002). Isovalerylcarnitine was lower and 1-metylhistidine tended to be lower (p = 0.002 and p = 0.073, respectively), whereas L-proline levels tended to be higher (p = 0.079) in the black compared to the white boys. In single, partial, and multiple regression analyses, pulse wave velocity correlated inversely with β-alanine (β = –0.414; p = 0.008) and 1-methylhistidine (β = –0.347; p = 0.032) and positively with L-proline (β = 0.420; p = 0.008), threonic acid (β = 0.977; p = 0.033), and malonic acid (β = 0.348; p = 0.030) in black boys only. Conclusion: Our study is the first to discover the associations of pulse wave velocity with β-alanine, 1-methylhistidine, and L-proline in children from South Africa, which may suggest potential early compromise in cardiac protective metabolic pathways in black boys as young as 6 years of age.

MSc (Biochemistry), North-West University, Potchefstroom Campus Nanotechnology has increasingly received attention the last few decades and the term refers to the categories of applied science and technology, where the combining key subject is the study of matter in scales of 1 to 100 nm and the designing of devices within that size range. Gold nanoparticles have especially drawn massive scientific attention; the reasons being that these particles exhibit high chemical stability and unique optical properties. Furthermore, gold nanoparticles can be easily synthesised and modified, while providing great potential for a drug delivery vehicle. There is however a gap in current research with regards to the safety and effect of these particles, especially in the field of metabolomics. This study thus aimed to provide a more comprehensive view of the effect of gold nanoparticles on the metabolome, by implementing an animal model. Two groups of Sprague-Dawley rats were monitored: one control group and one treatment group. The control group received a 0.9% saline solution and the treatment group received a solution of gold nanoparticles dispersed in citrate (90μg/500μl). Urine was collected at different time points over the course of 48 hours. The study utilised three different, but effective, platforms popular within the field of metabolomics, namely 1-dimensional Nuclear Magnetic Resonance (1H-NMR) spectroscopy, Liquid Chromatography Mass Spectrometry and Gas-chromatography Time-Of-Flight Mass Spectrometry (GC-TOF/MS). Urine samples were analysed via these platforms using both untargeted and targeted metabolomics approaches, investigating an array of metabolites (including amino acids, acylcarnitines and organic acids). The data was subjected to bio-statistical analysis to identify the relevant changes in metabolite levels and produce a full list of significant compounds affected by the intervention of gold nanoparticles. The significant metabolites brought forth evidence of possible perturbation within the pathways of energy metabolism as well as carbon- and amino acid metabolism. The results were found to produce a similar profile to that of many heavy metals, in the sense that binding with sulphur-containing molecules occurred readily and consequently inhibiting the function of several proteins and enzymes. The most prominent findings were linked to the enzyme dehydrogenase family and the thiol-rich compounds of the amino acid pathways, which are often associated with a phenotype similar to heavy metal poisoning. Therefore, it is reasoned as is in the case of other heavy metals, that gold (even in nanoform) possesses a high affinity for sulphur-containing compounds and will promptly replace these bonds, by displacing the original ion. National Research Foundation (NRF) Masters

MSc (Biochemistry), North-West University, Potchefstroom Campus, 2018 Mycobacterium tuberculosis is estimated to infect approximately one-third of the world’s population, which can lead to an active, symptomatic disease called tuberculosis (TB), or to asymptomatic states, often referred to as latent TB infection. In 2015 alone, 10.4 million new TB cases were reported, resulting in an estimated 1.8 million deaths. Since the discovery of M. tuberculosis in 1882 by Robert Koch, a vast amount of genomics, proteomics and transcriptomics data have been generated, leading to our current understanding of M. tuberculosis and TB. Most of the data generated from studies used M. tuberculosis cultures; however, it is well-known that this organism’s metabolism and growth in culture differs greatly from growth in the human host, where many different growth mechanisms and energy substrates are preferentially used. Furthermore, very little research to date has focused on the adaptations of M. tuberculosis to the host’s defence mechanisms or growth environment, or for that matter, the host’s adaptations or altered metabolic state in response to the infectious pathogen. This is important since the pathophysiology of M. tuberculosis is directly linked to its metabolism and complex physiology, and to that of the host. Additionally, this pathogen can utilise numerous growth substrates, either by scavenging this from the host or via de novo biosynthesis, in order to ensure its own survival. Metabolomics has served well to expand the current knowledge of the disease and has contributed towards the improved diagnosis and treatment thereof, due to its unique capacity for identifying new disease biomarkers. Metabolomics is defined as the unbiased identification and quantification of the entire metabolome in a specific biological system, with the use of highly advanced analytical instruments, together with various statistical, computational and mathematical analyses. Metabolomics has enabled the identification of new metabolite markers in sputum, blood and urine from TB patients, describing novel M. tuberculosis metabolic pathways and host adaptations. Apart from their possible diagnostic applications, many of these new TB metabolite markers have contributed to the existing knowledge of the biology of the causative pathogen, including various underlying disease mechanisms related to M. tuberculosis drug resistance and virulence, as well as the mechanisms of TB drug action and related side-effects in the host. To date, however, very little data has been published on urine from TB patients, which can be considered an ideal sample matrix to identify markers associated with this host–pathogen interaction. Considering this, in this investigation, a combined semi-targeted liquid and gas chromatography mass spectrometry metabolomics approach was used to compare the urinary fatty acylcarnitines, amino acids and selected organic acids of active TB patients with that of healthy individuals, in order to better characterise the TB-induced alterations to the host metabolome. The generally elevated concentrations of the fatty acylcarnitines and amino acids are most likely due to TB-cachexia. However, the significantly elevated concentrations of arginosuccinate, asparate (and associated asparagine), ornithine (and associated proline and hydroxyproline) and glutamate (and associated glutamine) in particular, indicate a urea cycle abnormality, due to inhibition of N-acetylglutamate synthase by the accumulating propionyl-CoA, isovaleryl-CoA and methylmalonyl-CoA in TB patients. Furthermore, elevated propionylcarnitine, methylmalonate and methylcitrate in the TB patient urine are associated with a vitamin B12 deficiency, which deserves further investigation. Lastly, various metabolites indicative of lactic acidosis, ketoacidosis, oxidative stress and liver damage were identified in the urine of the TB patients Masters

PhD (Biochemistry), North-West University, Potchefstroom Campus Mitochondrial disease (MD) is a subgroup of inborn errors of metabolism, which can be caused by a mutation in either the nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). One of the most common mtDNA disease causing mutations is the m.3243A>G point mutation, which affects the incorporation of the amino acid leucine into mitochondrial proteins and thus the oxidative phosphorylation system (OXPHOS) system. This mutation was initially linked to mitochondrial myopathy, encephalopathy lactic acidosis and stroke like episodes (MELAS), but various other phenotypes and symptoms was later linked to this mutation, including progressive external ophthalmoplegia (PEO), maternally inherited diabetes-deafness (MIDD) and myopathy. However, the reason why these patients presents with such a broad spectrum of symptoms, even though they harbor the same mutation, remains unknown. Therefore, the aim of this study was to investigate the urine metabolome of a cohort of m.3243A>G diagnosed patients, presenting with different phenotypes (MELAS, MIDD and myopathy), using a multi-platform metabolomics approach. This multi-platform metabolomics approach consisted of untargeted as well as targeted analytical methods. The untargeted analyses consisted of gas chromatography−mass spectrometry (GCMS), nuclear magnetic resonances (NMR) spectroscopy, and liquid chromatography mass spectrometry with ion mobility (LC-IM-MS), in negative as well as positive ionization mode, while the targeted analyses consisted of liquid chromatography tandem-mass spectrometry (LC-MS/MS). Using this multi-platform metabolomics approach enabled us to analyze a larger portion of the metabolome compared to using a single analytical technique. In the first part of the study, we investigated 9 patients presenting specifically with MELAS and 29 healthy controls. We were able to identify 36 metabolites that were altered in the patient group when compared to healthy controls. When investigating these 36 metabolites further, we were able to link them to redox imbalance as a result of a defective OXPHOS system and stalled fatty acid oxidation. Our investigation also resulted in the first association between MELAS and an intricate web of affected pathways consisting of the one-carbon metabolism, methylation cycle and the transsulfuration pathway. In order to validate the 36 markers identified, a new cohort consisting of two MELAS patients and seven controls were used. We demonstrate complete separation of the MELAS patients and controls using principle component analysis, thus indicating that the 36 markers are not unique to the initial cohort used and thus have potential for diagnosis or treatment monitoring. In the second part of the study, we expanded on the findings by investigating two additional m.3243A>G associated phenotypes, MIDD (n = 30) and myopathy (n = 18). These two phenotypes, together with the MELAs cohort were compared to healthy controls, and to one another, in order to find not only metabolic similarities between the different phenotypes, but also phenotypic specific perturbations. Our novel findings indicate, especially in the MELAS patients, increased de novo fatty acid synthesis (FAS) in the mitochondria. We hypothesize that this increased FAS is probably due to lipoic acid synthesis, an essential cofactor for pyruvate dehydrogenase, 2-ketoglutarate dehydrogenase as well as the glycine cleavage system. Furthermore, we show specific metabolic perturbations in each of the three phenotypes. Investigating the metabolic similarities, we found three metabolites that were perturbed in all three phenotypes, 2-hydroxyglutaric acid, glycolic acid and 4-pentenoic acid. We conclude that these metabolites should be further investigated for diagnostic potential. The strength of our study was the utilization of different analytical platforms to generate the robust metabolomics data reported here. We show that urine may be a useful source for disease-specific metabolomics data. Our study contributed to the mitochondrial disease research field by providing significant insight into metabolic alterations caused by the m.3243A>G mutation. Firstly, results obtained in both parts of this study showcased the valuable information that could be obtained when implementing metabolomics as investigation tool. Secondly our results highlighted the potential for mitochondrial disease biosignatures for disease mechanistic understanding. Finally, we pointed out several important metabolic pathways affected in these patients that could be investigated in future studies. Ultimately, understanding the m.3243A>G mutation could lead to better diagnostic and treatment options, which both doctors and patients would benefit from immensely. Doctoral

PhD (Biochemistry), North-West University, Potchefstroom Campus The South African abalone, "perlemoen" (as it is called locally) industry is largely based on farming with Haliotis midae, which has been commercially cultured in man-made shore-based systems with great success for the last 20 years. Due to the basic dynamics of abalone aquaculture being well-known, the high market value and the demand for this delicacy, this sector is commercially, the largest of all aquaculture sectors in SA. However, knowledge of abalone metabolism and the biochemical processes associated with abalone growth and development are lacking. Since maximising growth and health of abalone is the primary goal for optimising production and revenue on abalone farms, research on abalone metabolism could lead to a better understanding of their metabolic responses to specific perturbations and subsequently, to better growth. Metabolomics, one of the newest additions to the "omics" research technologies, aims to investigate the metabolism holistically, and is considered a powerful tool for new biomarker identification and better elucidation of the observed phenotypical changes associated with a perturbation. Considering this, the effects of 1) functional and environmental hypoxia and 2) diet and abalone age as experienced within the standard farming environment, were investigated in Haliotis midae in this thesis. By analysing different tissue samples (adductor muscle, foot muscle, left gill, right gill, haemolymph and epipodial tissue), using a multiplatform (nuclear magnetic resonance spectroscopy, gas chromatography mass spectrometry and liquid chromatography mass spectrometry), standardised metabolomics approach, growth and metabolism of abalone could be elucidated. Univariate statistical methods were used to identify those features of significance, to which metabolite identifiers were assigned, based on well-defined identification guidelines, which were subsequently used in pathway analyses and for biological interpretation of perturbations in relation to growth of abalone. The results show that functional and environmental hypoxia result in a metabolic imbalance in H. midae, with the resulting energy deficit being compensated for by phosphoarginine reserves. This initial response is later supplemented by anaerobic glycolysis, whereby glucose is converted to pyruvate, and then to lactate or opines, in order to replenish the dwindling nicotinamide adenine dinucleotides required as substrates for further adenosine triphosphate production. Furthermore, the metabolomics results also suggest that stressors such as hypoxia, causes abalone to redirect their energy utilisation towards those metabolic pathways essential to the survival of the animal, at the expense of growth. In contrast, the metabolomics analysis done on the adductor muscle samples of abalone, with comparatively good growth rates, showed that faster growing individuals utilise energy pathways and reserves (via elevated insulin production) in such a way that they promote protein synthesis. Furthermore it is suggested that modified artificial abalone feed stimulates mitochondrial function, enabling juvenile abalone to catabolise proline for energy production, while in adult abalone, proline was utilised primarily towards improving energy production through ß-oxidation pathways. From this metabolomics investigation, it becomes evident that abalone have well-developed metabolic mechanisms ensuring survival during periods of oxygen depletion, however, this does inhibit growth, and in the absence of such stress, the metabolism of abalone would favour protein synthesis. At this stage the reasons as to why some individuals utilise amino acid reserves more rapidly for protein synthesis, under the same growth conditions are still debatable. Furthermore, this study proves that metabolomics is an extremely valuable tool for investigating the altered metabolic processes related to growth in abalone, and hence, could be considered a valuable tool for the abalone aquaculture industry, for identifying biomarkers for growth and health monitoring. Doctoral

MSc (Biochemistry), North-West University, Potchefstroom Campus, 2017 Gold nanoparticles (GNPs) and differentially functionalised or ligand exchanged GNPs (Lig-GNPs) present promising advantages in a variety of fields. Surface functionalisation of GNPs with ligands is believed to improve the biocompatibility of the GNPs. However, the effects of these particles on biological systems remain unclear due to contradictions and several limitations in the literature, such as unstandardised protocols. Standardised methods and compatibility of assays with GNPs are essential in accurately determining the overall effect of GNPs on biological systems. Metabolomics analysis may present answers to the possible effects observed in the literature as it presents the metabolites, which are the closest to the functional phenotype of a biological system. However, research in these two fields as a combination is greatly limited. This study reveals the metabolic variation that occurs in HepG2 cells when treated with PVP-GNPs, PSSNa-GNPs and Citrate-GNPs by using standardised, pre-evaluated protocols. The results show that even though differentially functionalised GNPs seem to improve the biocompatibility of the particles, GNPs do induce variation on metabolome level; however, the variation is not necessarily linked with cytotoxicity. This field presents opportunities to further elucidate the effects that GNPs may have on biological systems. Masters

MSc (Biochemistry), North-West University, Potchefstroom Campus, 2016 Mitochondria are the cell’s main energy producing site, found in the cytoplasm of nearly all eukaryotic cells. These organelles generate cellular energy in the form of adenosine triphosphate (ATP), mostly by means of the oxidative phosphorylation (OXPHOS) system, consisting of the respiratory chain (RC) and ATP synthase complex. When one of the four complexes (which form the RC) becomes impaired, it is called a respiratory chain deficiency (RCD). Diagnosing RCDs is a major challenge and requires a multi-disciplinary approach, which includes clinical, histochemical, molecular and biochemical assessment. The golden standard for diagnosing a RCD is enzyme analyses on a muscle sample obtained from a muscle biopsy, which is an invasive procedure. A urinary biosignature was proposed, that has the potential to be used as a screening tool for selecting patients that have a potential RCD. The proposed biosignature however consisted of 12 features that still required verification. The aim of this study was to improve this proposed biosignature, by verifying the 12 features and expanding the biosignature by analysing the identical sample cohort with alternative analytical platforms in order to discover additional features. This study was conducted in two separate phases, a verification phase and an expanding phase. Two levels of verification were performed for the LC-MS features of the proposed biosignature. Five of the 12 features could be verified and only one could be identified to a certain extent. For the expansion part of this study, two additional platforms were used, a gas-chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analysis. Following evaluation of the methods used and sample analysis, a number of data mining steps and statistical analyses were performed to compile a list of top ranked features for each platform. The LC-MS, GC-MS and NMR features were considered for the improved biosignature, by using a variety of statistical analyses the number and best combination of features were selected. A list of five features was compiled and the discriminative power was evaluated. Results indicated that the improved biosignature was unable to classify samples 100% accurately with some of the clinical control samples classified as RCDs, however the biosignature could still be helpful in limiting the inclusion of CRC patients in the biopsy process which gives it the potential to be used in the diagnostic workflow. Masters

PhD (Biochemistry), North-West University, Potchefstroom Campus, 2016 There has been growing concern in recent years about the alarmingly high prevalence and severity of hypertension and other cardiovascular diseases in individuals from newly (or recently) westernised countries such as South Africa. This is especially true for the Black ethnic group where higher average blood pressure values are seen, compared to their Caucasian counterparts. There is already an established connection between urbanisation and increased prevalence of lifestyle diseases in developed countries such as the USA. However, despite the global effort of clinicians and scientists investigating the aetiology of hypertension, regarding its involvement in cardio-metabolic disease no definitive biological mechanism has been elucidated, especially in the Black ethnic group of South Africa. This study thus aimed to investigate hypertension in Black and Caucasian South Africans in a holistic manner, utilising a metabolomics-based approach together with clinical data and targeted biochemical measured markers. Two metabolomics platforms were used to ensure wider metabolome coverage, namely gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Study participants were divided into gender and ethnic groups and each group was further divided into quintiles according to average 24-hour ambulatory systolic blood pressure values. Only data from quintile 1 (normotensives) and quintile 5 (extreme hypertensives) were used in statistical analyses to ensure optimal separation between blood pressure groups. In the hypertensive Black males perturbations in several systems involved in ethanol metabolism were evident, being driven by a shifted global NADH/NAD+ ratio. Alterations in the bile acid metabolism of the hypertensive Black females were seen, while a more classical pre-diabetic insulin resistant state was observed in the hypertensive Caucasian females. In the hypertensive Caucasian males, disruptions in fatty acid metabolism and liver damage was evident, along with perturbations in detoxification systems. Obesity and perturbations in gut flora metabolism were evident in most of the hypertensive groups. Results from this study serve to demonstrate the power of applied metabolomics in the field of cardiovascular research, as novel metabolic pathways not previously associated with the pathogenesis of hypertension were found to be perturbed in hypertensives compared to their normotensive counterparts. Doctoral