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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

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

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