Lungs donated after cardiac death (DCD) are an underutilized resource for a dwindling donor lung transplant pool. Our study investigates the potential of a novel preservation solution, Somah, to better preserve statically stored DCD lungs, for an extended time period, when compared to low-potassium dextran solution (LPD). We hypothesize that Somah is a metabolically superior organ preservation solution for hypothermic statically stored porcine DCD lungs, possibly improving lung transplant outcomes. Porcine DCD lungs (n = 3 per group) were flushed with and submerged in cold preservation solution. The lungs were stored up to 12 h, and samples were taken from lung tissue and the preservation medium throughout. Metabolomic and redox potential were analyzed using high performance liquid chromatography, mass spectrometry, and RedoxSYS (R), comparing substrate and pathway utilization in both preservation solutions. Glutathione reduction was seen in Somah but not in LPD during preservation. Carnitine, carnosine, and n-acetylcarnosine levels were elevated in the Somah medium compared with LPD throughout. Biopsies of Somah exposed lungs demonstrated similar trends after 2 h, up to 12 h. Adenosine gradually decreased in Somah medium over 12 h, but not in LPD. An inversely proportional increase in inosine was found in Somah. Higher oxidative stress levels were measured in LPD. Our study suggests suboptimal metabolic preservation in lungs stored in LPD. LPD had poor antioxidant potential, cytoprotection, and an insufficient redox potential. These findings may have immediate clinical implications for human organs; however, further investigation is needed to evaluate DCD lung preservation in Somah as a viable option for transplant.

Active pharmaceutical ingredients (APIs) and their metabolites are ubiquitous in the environment and their occurrence in the aquatic environment is of growing concern. However, despite the fact that these may cause harmful effects in organisms found within this niche, little is currently known about the effects of APIs in the aquatic environment. Chiral pharmaceuticals are of particular concern as the enantiomers may be metabolised differently, with the potential for the production of an array of harmful compounds. There are many racemic APIs for treating human and animal conditions, and even in these target organisms the pharmacodynamic effects of the enantiomers are not always known. Within recent years the importance of the interactions of these compounds within the aquatic environment has been realised and information regarding the fate and biodegradation of such environmental pollutants is of great importance. The advent of post-genomic technologies has proved advantageous in the study of the effects of these environmental pollutants. In this thesis, the effects of a range of chiral APIs, and other environmental pollutants, on environmentally relevant microorganisms were investigated at the metabolome level. The effects of chiral APIs were investigated in a number of prokaryotic and eukaryotic systems in order to provide a comprehensive study of the effects of the APIs in the aquatic environment. FT-IR spectroscopy was employed for metabolic fingerprinting of some environmentally relevant bacteria and GC-MS was subsequently employed for metabolite profiling of two pseudomonads that had shown differential chiral effects with Propranolol. In addition, FT-IR microspectroscopy was employed for the investigation of the phenotypic and localised effects of chiral APIs in a eukaryotic system. Furthermore, the effects of a range of environmental pollutants on a complex bacterial community were investigated with the use of FT-IR spectroscopy and multivariate analysis. Initial results indicated a large phenotypic response in relation to phenol, and this was further explored with a range of ageing experiments and metabolic fingerprinting. An FT-IR peak was found to be characteristic of the phenotypic changes in the actively degrading communities and this was likely to be a degradation product of phenol, and armed with this knowledge the activated sludge community was monitored during the active degradation of phenol with the use of GC-MS.The work presented in this thesis has shown for the first time that metabolomics allows subtle phenotypes in microorganisms to be revealed when they are exposed to chiral forms of APIs which are commonly found in the aquatic environment. Despite these APIs not being designed for any interaction with bacteria and aquatic life in general these are significant findings and may have implications as more and more APIs become detectable and concentrated in the environment due to continued use in man and indeed animals or aquaculture.

Climate change is causing droughts, which are affecting crop production globally, and disrupting plant metabolism. Due to the unpredictable natural droughts that occur, causing tea farmers significant losses in tea estates, a Short-time Withering Assessment of Probability for Drought Tolerance (SWAPDT) method for distinguishing between drought tolerant (DT) and drought susceptible (DS) Camellia sinensis cultivars was developed based on cultivars from the Tea Research Foundation for Central Africa in Malawi, and validated on 400 samples from the Tea Research Institute in Kenya. From the results, a sample size of 20 tea trees was deemed sufficient to accurately determine the drought susceptibility of a large tea field of approximately 5 - 20 hectares, containing 50 000 - 200 000 tea trees, were the difference between their mean values is approximately 6%. Tea production and subsequently its quality rely on evenly distributed rainfall. Tea consumers concern themselves with the quality of tea, in particular its flavour and aroma. To breed for these phenotypic traits is challenging due to these being qualitative traits inherited from parents, and influenced by environment. Two C. sinensis populations, 60 Commercial cultivars and 250 NonCommercial cultivars (TRFK St. 504 and TRFK St. 524) were employed in a part of this study to identify the Quantitative Trait Loci (QTL) responsible for yield, drought tolerance and quality centred on a genetic map constructed using the DArTseq platform. The map comprised 15 linkage groups analogous to chromosome haploid number of tea plant (2n = 2x = 30) and spanned 1260.1 cM with a mean interval of 1.1 cM between markers. Sixteen phenotypic traits were evaluated in both populations, and three, 11 and 46 putative QTLs were discovered after mapping on the 15 linkage groups, associated with tea quality from Gas Chromatography-Mass Spectrometry (GC-MS), Nuclear Magnetic Resonance (1H-NMR) and Ultra-Performance Liquid Chromatography (UPLC) data respectively. The variance explained by the QTLs varied from 4.6 to 96.3%, with an average of 28%. Using the KEGG database, the putative QTLs linked to yield, drought tolerance and quality were secondary metabolites associated with tea phenolic biomolecules and abiotic stress. Principal Component Analysis was performed on the GC-MS, 1H-NMR and UPLC data, and from these, the UPLC data showed clearer separation and clustering between the Commercial and NonCommercial cultivars. With focus on the UPLC data, it was narrowed down to the five catechins, four theaflavins and caffeine; these were used to develop several logistic regression models. The model based on only the fresh leaf catechins classified over 90% of the 310 genotypes as either Commercial or NonCommercial cultivars. This model may be useful in predicting the suitability for commercialization of promising selections from mature seedling fields, based on the analysis of their dried green leaves. Last, 20 Commercial and 20 NonCommercial cultivars were analysed using UPLC-MS. New metabolites were identified as contributing to drought tolerance, yield and higher quality of the Commercial as compared to the NonCommercial cultivars. / Thesis (PhD (Biochemistry))--University of Pretoria, 2021. / Biochemistry / PhD (Biochemistry) / Unrestricted

No description available.

No description available.

The prevalence of diabetes mellitus is increasing dramatically worldwide. Approximately three out of four diabetic patients will die of cerebro- and cardiovascular disease (CVD). Atherosclerosis, a chronic inflammatory disease of the medium-large arteries, is the major underlying cause of most CVDs. Despite the established progressive relationship between diabetes and CVD, the molecular mechanisms by which diabetes promotes atherosclerosis are not well understood. This has impeded the development of strategies to block or slow atherogenesis in diabetic patients. The objective of my thesis is to investigate the molecular alterations by which diabetes accelerates the development of atherosclerosis using comprehensive metabolomics techniques. We first investigated the development and progression of atherosclerosis at the molecular level in apolipoprotein E-deficient mice. We identified specific changes in plasma-borne metabolites that are associated with the pathogenesis and progression of atherosclerosis. In addition, glycerophospholipid and sphingolipid metabolism were found to be the most significantly altered pathways. Using comprehensive metabolomics techniques, we were able to differentiate atherosclerotic plasma metabolome from healthy control and delineate different stages of atherosclerotic progression. Next, we characterized multiple mouse models of hyperglycemia-induced accelerated atherosclerosis. We showed that the vascular effects of glucosamine supplementation are comparable to streptozotocin-induced and genetically-induced (Ins2Akita) hyperglycemia in terms of lesional glucosamine, endoplasmic reticulum (ER) stress levels and atherosclerotic burden. In addition, we showed that a chemical chaperone (4-phenylbutyric acid) reduces ER stress levels and attenuates accelerated atherogenesis in each of these models. Together these findings support the mechanism involving glucosamine-induced ER stress in hyperglycemia-induced accelerated atherosclerosis. Lastly, metabolomics techniques were used to investigate the molecular alterations by which hyperglycemia promotes the accelerated development of atherosclerosis in several disease models. The three mouse models induced both unique and common changes in the plasma metabolome. Identification of the commonly altered metabolite features revealed alterations in glycerophospholipid and sphingolipid metabolisms, and key atherosclerosis-associated processes including inflammation and oxidative stress. Together, we showed that comprehensive metabolomics techniques can be used to identify specific alterations in the metabolome that are associated with a particular disease genotype and phenotype. These data highlight the important roles of the glycerophospholipid and sphingolipid metabolisms in the pathogenesis of atherosclerosis and diabetic atherosclerosis. The clear difference in the level of several metabolites supports the use of plasma lipid profiling as a diagnostic tool of atherogenesis. / Thesis / Doctor of Science (PhD)

T V Gale

The lesser scaup (Aythya affinis, henceforth scaup) population decreased during the 1980s and has remained below conservation objectives. With two-thirds of the breeding population nesting in boreal forests, it is imperative to understand how changes in habitat features and landscape changes could negatively impact breeding scaup; this information could also help to guide waterfowl conservation and management programs. It has been hypothesized that landscape modifications, such as agricultural and oil exploitation activities, could adversely affect scaup reproductive performance. Female scaup were collected by shooting in the boreal forest fringe of central Alberta during within the first two weeks of June 2008 and 2009, corresponding to the early laying period. Collection location was determined using a hand-held GPS device and body mass (BM, g) was recorded immediately. When scaup carcasses were dissected, ovaries were removed and weighed. Rapidly growing follicles (RGF) and oviductal follicles (OF) were subjected to radioimmunoassay to quantify amount of ovarian corticosterone (OCORT) deposited into follicles. Nest initiation date (NID) was determined by counting and subtracting the number of postovulatory follicles (POF) from collection date. Natural landscape features and habitat disturbances within 100 m, 250 m, 500 m, 1500 m, and 5000 m buffers of each scaup collection location were quantified, and then normalized using z-score transformation. General linear modeling was applied to BM, OCORT and NID across each of the five buffer sizes using a priori candidate models. Natural landscape and habitat disturbance parameters were model-averaged to obtain 85% confidence intervals to determine which habitat features best predicted BM, NID, and OCORT. Model-averaging revealed that the amount of natural water body habitats were negatively correlated to scaup OCORT at the 100 m and 500 m buffer zones, and negatively correlated to scaup BM at 1500 m and 5000 m buffer zones. A combination of natural water bodies (BOGSFENS, SWAMP, WATER, and WETLAND) were negatively correlated to NID across all buffer sizes tested, AGRIC disturbance was positively correlated to NID at the 100 m and 1500 m buffer zones, WELLS were negatively correlated to NID at the 1500 m buffer zone, while TRANSPORT showed varied responses at the 1500 m and 5000 m buffer zones. Post-hoc exploratory analyses were conducted to assess whether variation in OCORT, BM, and NID was related to habitat features operating at more than one spatial scale. This analysis revealed models incorporating more than one spatial scale were competitive when compared to the original a priori model sets at the 250 m buffer zone, with post-hoc models performing better than a priori models. This study highlights the need for a holistic approach to conservation management, considering not only local habitat characteristics and disturbances in the immediate vicinity of breeding waterfowl, but extending beyond and incorporating regional landscape attributes.

In 2001, the WHO declared tuberculosis (TB) a global emergency, as one third of the world‟s population suffered from latent M. tuberculosis infection. Today, a decade later, millions of people still die worldwide as a result of this disease. This growing TB incidence may be ascribed to a variety of reasons, including, amongst others, the inadequacies associated with the currently available diagnostic methods and TB treatment regimes, especially when considering the growing MDR-TB and HIV epidemics. This study investigated the potential of metabolomics as a tool for characterising TB and various TB-causing bacteria, allowing for a better understanding of TB disease mechanisms, which may ultimately lead to improved diagnostic and treatment regimens. Firstly, we investigated the potential of a fatty acid, metabolomics approach to characterise various cultured Mycobacterium species. For this exploration, three fatty acid extraction procedures, prior to GC-MS analyses, were compared based on their respective repeatability and extraction capacities. Using the data obtained from the analyses done with the most optimal extraction approach (the modified Bligh-Dyer method), multivariate statistical analyses were able to differentiate between the various Mycobacterium species at a detection limit of 1 x 103 bacterial mL-1, in 16 hours. Subsequently, the compounds best describing the variation between the sample groups were identified as potential metabolite markers and were discussed in the light of previous studies. The most optimal GC-MS, fatty acid metabolomics approach, mentioned above, was then applied to analyse and characterise a wild-type M. tuberculosis parent strain and two rifampicinresistant conferring rpoB mutants (S522L and S531L). Due to the variation in their fatty acid profiles, a clear differentiation was achieved between these M. tuberculosis sample groups, and those metabolites contributing most to this variation were identified as metabolite markers characteristic for rifampicin-resistance. The altered metabolite markers detected in the rpoB mutants propose a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids and alkanes as alternative carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 50% of all clinical rifampicin-resistant M. tuberculosis strains, showed a better capacity for using these alternative energy sources, in comparison to the less frequently detected rpoB S522L mutant. The developed fatty acid GC-MS metabolomics approach was then successfully adapted in order to improve its speed, cost and complexity. This improved fatty acid extraction method was furthermore compared to another, similar approach (total metabolome extraction method), developed for the extraction of a much wider variety of compounds, prior to GC-MS and statistical data analyses. Although both these methods show promise for bacterial characterisation using matabolomics, the total metabolome extraction method proved the better of the two methods because it is comparatively simpler, faster (taking less than 4 hours), more repeatable, better differentiates between sample groups due to less within group variation, has a lower detection limit, and isolates a wider variety of biologically relevant metabolites (as opposed to fatty acids alone). We, furthermore, identified and described the occurrence of those compounds, extracted by both methods, which contribute most to the variation between the bacterial groups, in order to validate these methods for metabolomic applications and the isolation of compounds with biological relevance. In order to evaluate the potential of this developed metabolomics approach for application to biological samples other than bacteriological cultures, it was adapted for the direct analyses of complex sputum samples. For this application, four sputum pre-extraction preparation methods, including three standard Mycobacterium cell isolation procedures (Sputolysin, NALC-NaOH, and NaOH) and a fourth, applying only a simple ethanol homogenisation step, prior to direct sputum extraction, were compared. Of these methods, the ethanol homogenisation method proved to have the best comparative extraction efficiency, repeatability and differentiation capacity, when used in combination with the previously developed metabolomics methods. Subsequently, when applying this approach to patient collected sputum samples, a set of metabolite markers, differentiating the TB-positive from the TB-negative samples, were identified. These markers could directly be linked to: 1) the physical presence of the M. tuberculosis in these samples; 2) changes in the bacterial metabolome due to in vivo growth conditions and; 3) changes in the human metabolome due to pulmonary M. tuberculosis infection. In addition to the proposal of a number of new hypotheses, explaining various mechanisms of TB and drug-resistant TB, the mapping of the newly identified metabolite markers to known metabolic pathways led to the confirmation of various previously suggested metabolic pathways and alterations thereof due to an assortment of perturbations. Therefore, this study significantly contributes to the characterisation of various TB causing bacteria, rifampicin-resistant M. tuberculosis strains and the TB disease state, which may in future lead to the development of innovative TB vaccination, diagnostic and treatment protocols. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

In 2001, the WHO declared tuberculosis (TB) a global emergency, as one third of the world‟s population suffered from latent M. tuberculosis infection. Today, a decade later, millions of people still die worldwide as a result of this disease. This growing TB incidence may be ascribed to a variety of reasons, including, amongst others, the inadequacies associated with the currently available diagnostic methods and TB treatment regimes, especially when considering the growing MDR-TB and HIV epidemics. This study investigated the potential of metabolomics as a tool for characterising TB and various TB-causing bacteria, allowing for a better understanding of TB disease mechanisms, which may ultimately lead to improved diagnostic and treatment regimens. Firstly, we investigated the potential of a fatty acid, metabolomics approach to characterise various cultured Mycobacterium species. For this exploration, three fatty acid extraction procedures, prior to GC-MS analyses, were compared based on their respective repeatability and extraction capacities. Using the data obtained from the analyses done with the most optimal extraction approach (the modified Bligh-Dyer method), multivariate statistical analyses were able to differentiate between the various Mycobacterium species at a detection limit of 1 x 103 bacterial mL-1, in 16 hours. Subsequently, the compounds best describing the variation between the sample groups were identified as potential metabolite markers and were discussed in the light of previous studies. The most optimal GC-MS, fatty acid metabolomics approach, mentioned above, was then applied to analyse and characterise a wild-type M. tuberculosis parent strain and two rifampicinresistant conferring rpoB mutants (S522L and S531L). Due to the variation in their fatty acid profiles, a clear differentiation was achieved between these M. tuberculosis sample groups, and those metabolites contributing most to this variation were identified as metabolite markers characteristic for rifampicin-resistance. The altered metabolite markers detected in the rpoB mutants propose a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids and alkanes as alternative carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 50% of all clinical rifampicin-resistant M. tuberculosis strains, showed a better capacity for using these alternative energy sources, in comparison to the less frequently detected rpoB S522L mutant. The developed fatty acid GC-MS metabolomics approach was then successfully adapted in order to improve its speed, cost and complexity. This improved fatty acid extraction method was furthermore compared to another, similar approach (total metabolome extraction method), developed for the extraction of a much wider variety of compounds, prior to GC-MS and statistical data analyses. Although both these methods show promise for bacterial characterisation using matabolomics, the total metabolome extraction method proved the better of the two methods because it is comparatively simpler, faster (taking less than 4 hours), more repeatable, better differentiates between sample groups due to less within group variation, has a lower detection limit, and isolates a wider variety of biologically relevant metabolites (as opposed to fatty acids alone). We, furthermore, identified and described the occurrence of those compounds, extracted by both methods, which contribute most to the variation between the bacterial groups, in order to validate these methods for metabolomic applications and the isolation of compounds with biological relevance. In order to evaluate the potential of this developed metabolomics approach for application to biological samples other than bacteriological cultures, it was adapted for the direct analyses of complex sputum samples. For this application, four sputum pre-extraction preparation methods, including three standard Mycobacterium cell isolation procedures (Sputolysin, NALC-NaOH, and NaOH) and a fourth, applying only a simple ethanol homogenisation step, prior to direct sputum extraction, were compared. Of these methods, the ethanol homogenisation method proved to have the best comparative extraction efficiency, repeatability and differentiation capacity, when used in combination with the previously developed metabolomics methods. Subsequently, when applying this approach to patient collected sputum samples, a set of metabolite markers, differentiating the TB-positive from the TB-negative samples, were identified. These markers could directly be linked to: 1) the physical presence of the M. tuberculosis in these samples; 2) changes in the bacterial metabolome due to in vivo growth conditions and; 3) changes in the human metabolome due to pulmonary M. tuberculosis infection. In addition to the proposal of a number of new hypotheses, explaining various mechanisms of TB and drug-resistant TB, the mapping of the newly identified metabolite markers to known metabolic pathways led to the confirmation of various previously suggested metabolic pathways and alterations thereof due to an assortment of perturbations. Therefore, this study significantly contributes to the characterisation of various TB causing bacteria, rifampicin-resistant M. tuberculosis strains and the TB disease state, which may in future lead to the development of innovative TB vaccination, diagnostic and treatment protocols. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012