Doctoral Degrees (Biochemistry)
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- ItemThe metabolism and receptor interactions of C11-oxy steroids: implications for hormonal homeostasis and pathophysiology(Stellenbosch : Stellenbosch University, 2024-03) Gent, Rachelle; Swart, Amanda C. ; Snoep, Jacky L. ; Kaschula, Catherine H.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: This study describes: • the determination of the kinetic parameters of the 11,B-hydroxysteroid dehydrogenase type 1 (11,BHSD1) and type 2 (11,BHSD2) isozyme conversion of C11-oxy C21 steroids, 11,B- hydroxyprogesterone (11,BOHP4) and 11-ketoprogesterone (11KP4), and C11-oxy C19 steroids, 11,B-hydroxyandrostenedione (11OHA4) and 11-ketoandrostenedione (11KA4), and 11,B- hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT). • the in vitro conversion of C11-oxy C21 steroids to C11-oxy C19 steroids by cytochrome P450 17a-hydroxylase (CYP17A1) present novel mechanisms whereby metabolites of the C11-oxy C21 backdoor pathway may contribute to androgen excess. The conversion of 11,BOHP4 to 21- deoxycortisol (21dF) and 11OHA4 as well as the conversion of 11KP4 to 21-deoxycortisone (21dE) and 11KA4 are catalysed by the 17a-hydroxylation and 17,20-lyase activity of CYP17A1 in vitro. • the utilisation of cell models (PNT2, VCaP, LNCaP and C4-2B) to demonstrate the possible role of C11-oxy steroids in prostate cancer (PCa) pathophysiology. Assays showed an increase in 11,BHSD2 activity in LNCaP (androgen-dependent castration resistant prostate cancer (CRPC)) and C4-2B (metastatic CRPC) cells compared to PNT2 (normal prostate) and VCaP (PCa model) cell lines. This demonstrates the role of 11,BHSD2 in PCa directing flux to the biosynthesis of 11KT and 11KDHT, potentially contributing to CRPC. In addition, an increase in 11KT production in LNCaP cells is directly associated with an increase in prostate specific antigen. • the in vitro metabolism of the 11,BHSD2 inhibitor, 11a-hydroxyprogesterone (11aOHP4), by CYP17A1 which resulted in the biosynthesis of novel C11a-metabolites, 11a,17a- dihydroxyprogesterone (11a,17a-diOHP4) and 11a-hydroxyandrostenedione (11aOHA4), suggesting the potential presence of novel in vivo C11-oxy pathways. • the pre-receptor regulation of the androgen receptor (AR), progesterone receptor A (PRA) and progesterone receptor B (PRB) by the 11,BHSD catalysed interconversion of the C11-oxy steroids. Specific C11-oxy metabolites act as potential agonists or antagonists of these steroid receptors while also suggesting regulatory roles for 5a-reductase (SRD5A), 17,B-hydroxysteroid dehydrogenase (17,BHSD), 3a-hydroxysteroid dehydrogenase (3aHSD) as well as CYP17A1. • the development and validation of three novel ultra-high performance supercritical fluid chromatography tandem mass spectrometry (UHPSFC-MS/MS) methods for the separation and quantification of C11-oxy C19, C11-oxy C21 and adrenal steroids. The application of these methods in the comprehensive steroid analyses of southern white rhinoceros (Ceratotherium simum simum) faecal samples and of type 2 diabetes (T2D) patient serum samples implicate homeostatic dysregulation of the C11-oxy steroid metabolites in pathophysiologies.
- ItemKinetic modelling of the peroxiredoxin system(Stellenbosch : Stellenbosch University, 2024-03) Barry, Christopher James; Rohwer, J. M.; Pillay, C. S.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Hydrogen peroxide is a cellular oxidant that disrupts numerous cellular systems by being readily converted to the hydroxyl radical, which indiscriminately reacts with biomolecules. However, in many contexts, it also serves crucial cellular functions and is intricately connected to maintaining normal functioning. As a result, antioxidant sys- tems find themselves in a delicate balance between two opposing roles: they must defend cells against this cellular toxin without being so effective as to disrupt the normal cellular functions of hydrogen peroxide. The peroxiredoxin protein forms the core of one such antioxidant system and, belying its significance, is found across all domains of life. This versatile and vital protein can switch between several levels of hydrogen peroxide metabolism and possesses its own signalling and chaperone properties. The switch between roles is effectuated through a combination of redox configuration transitions and quaternary structure transformations. The launching-point for this project was a 100-fold change in the ability of peroxire- doxin to neutralise hydrogen peroxide when transitioning from a dimer to a decamer. While this relationship between oligomeric form and peroxidase activity had already been thoroughly established, it had yet to be explored in any dynamic sense. The first major outcome of the research in this dissertation was the formulation of a theoretical framework for the transition between dimers and decamers. This was closely followed by a characterisation of the kinetics for the association and dissociation of a re- duced peroxiredoxin decamer. Armed with these data, the dimer-decamer transition was explored through simulation and found to have an inhibition-like effect on peroxiredoxin activity. Impressively, integrating this process into an established in vivo model resolved an outstanding discrepancy between the experimental and simulated responses of the peroxiredoxin oxidation state to hydrogen peroxide insult. In the course of researching the kinetics of peroxiredoxin, a bias was discovered in the data of multiple independent reports of horse radish peroxidase competition assays. Investigating this bias led to the discovery of a flaw in the data analysis methodology of this assay. This issue was resolved by developing a method of fitting the target rate constant directly to assay trace data instead of through the established fractional inhibition method. We provide tools for researchers to easily apply the improved analysis method in their own work. The final major outcome of this project was the formulation of a model of hydrogen peroxide neutralisation in a Saccharomyces cerevisiae cell culture. The model was parame- terised by deriving several new parameters and the parameter sensitivities of the model were assessed using a Fourier amplitude sensitivity test and metabolic control analysis. This model is capable of simulating the dynamic responses of the peroxidase systems of a S. cerevisiae culture in media exposed to hydrogen peroxide stimulation—a widely used experimental system in redox biology. This dissertation is focused on the exploration of the peroxiredoxin protein using a systems biology approach. This work has culminated in multiple novel findings which contribute significantly to our understanding of the dynamics of hydrogen peroxide metabolism by peroxiredoxin and to redox biology more broadly.
- ItemTo target or not to target: strategies for the aroma analysis of alcoholic beverages(Stellenbosch : Stellenbosch University, 2023-12) Williams, Cody; Buica, Astrid; Stander, Marietjie A.; Medvedovici, Andrei V.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Classically, targeted analysis has dominated analytical chemistry research, whereby analytical methods are required for separation, identification and quantitation of specific analytes of interest. The complex wine, gin and beer matrices contain several hundreds of compounds, each with varying concentration levels and unique chemistries. Furthermore, comprehensive analysis of these alcoholic beverages are challenging, as suitable instrumentation and data handling strategies are required to effectively profile these matrices. The use of non-targeted methodologies emerge as an alternative tool used for profiling the aroma space of alcoholic beverages. Non-targeted analysis is an information rich technique which focuses on profiling a sample in its entirety. More data can equal more information, however more noise is also generated; thus, suitable strategies are highly desired to overcome this caveat. The question is now, are these strategies complementary or is the generated information redundant? The aim of this dissertation was to compare targeted and non-targeted strategies for the aroma analysis of wine, beer and gin. This dissertation is comprised of seven chapters showing the evolution of both targeted and non- targeted strategies starting with wine and culminating in the application of these strategies in craft gin and beer, together with the evaluation of the results using appropriate (and sometimes complex) statistical tools. The first part of the study is a comprehensive literature review, with specific focus on terpenoid analysis. Terpenoids are an integral component to the sensory profile in wine, beer and gin. These compounds are present in various concentrations and require advanced analytical tools to profile and quantify. The origin of terpenoids as derived from the raw materials or production practices, common concentration ranges, and sample preparation methods are reported. The use of non-targeted methods is also summarised in this review for wine, beer and gin, with particular focus on the use of hyphenated instrumentation, software processing tools, and multivariate statistical analysis. The first objective (chapter 3) focused on targeted strategies in wine. Terpenoids were selected due to their importance to the aroma profile and inherent challenges associated with the analysis of these compounds. The complexity associated with the analyses stimulated interest and prompted further investigation in the quantitation of terpenoids in wine. Two sample preparation strategies, namely offline-SPE and online-HS-SPME were explored for the quantitation of 20 terpenoids in red and white wine. This study documented which sample preparation was best for the quantitation of terpenoids in wine and reported on various method performance parameters. The second objective (chapter 4) expanded on the quantitation of terpenoids in wine, whereby the method was expanded from 20 to 53 terpenoids in a single method. This was accomplished through method development, optimisation and method performance characterisation. In addition, terpenoid stability was evaluated over a period of forty days. As this extended method includes compounds not found in wine, it was applied to craft gin (n=21) and craft beer (n=34) samples, and it documents for the first time the most terpenoids quantified in a single method using authentic standards. It also constitutes the first report on terpenoids quantified in South African craft gin and beer. In the non-targeted space, the third objective (chapter 5) highlights the use of various sample prepration strategies (LLE, SPE and HS-SPME) applied to wine of different cultivar (Chenin Blanc, Sauvignon Blanc and Chardonnay) and winemaking style (wooded and unwooded). The data was acquired using a non-targeted profiling method and processed using cloud metabolomics software, XCMS online, followed by multivariate analysis. Importantly, the pipeline methodology for this approach was established to characterise both qualitative and quantitative information on the wine samples. This strategy enabled the identification of the most sensitive and best profiling sample preparation method. In addition, this application conceptualises a pipeline methodology approach, whereby sample preparation, instrument analysis, cloud data processing and multivariate analysis is applied in order to obtain meaningful relationships in complex data sets. As an extension of the targeted and non-targeted strategies reported, the fourth and final objective (chapter 6) showcases an application of this pipeline methodology to craft beer (n=91) and gin (n=67). Craft beer and gin samples were analysed using the targeted terpenoid and non-targeted profiling strategies. Subsequent data processing and curation allowed for application to multivariate statistical analysis. The configurational similarity was compared to reveal differences between the targeted and non-targeted strategies. Further insight was made into identifying characteristic biomarkers for distinguishing between lager-style and ale-style craft beers.
- ItemDiscovery of novel anti-Candida albicans compounds from environmental bacteria(Stellenbosch : Stellenbosch University, 2023-12) Barnard-Jenkins, Bernice; Rautenbach, Marina; Snoep, Jacky L. ; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The opportunistic fungal pathogen, Candida albicans (C. albicans), has the ability to colonize different epithelial tissues (e.g. vaginal, intestinal and oral tissues) causing moderate to severe and fatal infections. The fatal infections are due to fungal cells that enter the bloodstream and circulate after being dispersed from their resistant biofilms. Organisms that have adapted to form biofilms are associated with improved tolerance to drugs, which was also found for some drugs in this study. A medium-throughput multiplex assay system (MPAS) has been developed for the in-parallel study of several fungal life-cycle stages of different strains of C. albicans. MPAS was designed to use conventional 96-well plates and 96-pin lids in four different assay layouts. In MPAS, the metabolic activity measurements were used to determine the effect of antimicrobial compounds in assay targeting planktonic cell susceptibility, biofilm prevention, biofilm eradication, as well as susceptibility of cells released from biofilms. MPAS detection limits were further optimized by testing the activity of antimicrobial culture extracts from known bacterial producers on the different C. albicans life stages. The MPAS data showed which producer extracts were more active against certain strains of C. albicans, as well as highlighted which fungal forms are more sensitive to certain culture extracts. This led to the successful application of MPAS to identify active culture extracts of unknown soil microbes against planktonic and biofilm forms of C. albicans for further characterization. Ultra-performance liquid chromatography linked to high resolution tandem mass spectrometry (HR UPLC-MSᵉ) was used to identify and characterize known antifungal lipopeptides (such as surfactins, iturins and fengycins), rhamnolipids, as well as active small quorum sensing compounds in the most active culture extracts. The overall success of the MPAS together with HR UPLC-MSᵉ was confirmed by the successful identification of known and unknown compounds, from soil sample bacteria, with activity against C. albicans. The study showed the potential of the medium throughput MPAS together with HR UPLC-MSᵉ to discover and characterize potential antibiofilm and antifungal compounds for further development.
- ItemPhylogenetic analyses in the african fish genus nothobranchius in relation to landscape evolution(Stellenbosch : Stellenbosch University, 2023-03) Van der Merwe, Pieter De Wet; Bellstedt, Dirk U.; Cotterill, Fenton P. D. ; Schliewen, Ulrich Kurt; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Nothobranchius is a genus of short-lived annual to semi-annual African freshwater fishes that are found in smaller seasonal ephemeral wetlands or pools and not in larger open lakes or non-seasonal river systems. The waterbodies in which they are found are situated on vertisol-type soils that have a large capacity to absorb and retain water which is slowly released upon drying. Eggs are fertilized and are deposited into the vertisol-type substrate of the waterbody. As the soil dries during the dry season cracks are formed and the eggs, which up to that point have been in an anoxic environment, are exposed to oxygen which stimulates them to develop. Several complicated pathways exist through which the eggs develop, which can lead to full development, or to several types of delayed development called diapauses. Some will remain dormant for subsequent rainy seasons ensuring that the species can survive extended droughts. Nothobranchius hatch quickly after the rainy season starts and subsequently grow very rapidly to reach maturity to repeat the life cycle. Sequencing of two mitochondrial and three nuclear genes of the majority of the species in Nothobranchius and appropriate outgroups was used to generate suitable sequence matrices which were analysed extensively using maximum likelihood and Bayesian Inference to generate robust phylogenies and dated phylogenies of the genus. A dated phylogeny was used in a biogeographic analysis to determine dispersal routes and establish that the ancestral area of Nothobranchius is in the Nilo-Sudan. Using the recently developed technique, double digest RAD sequencing, an even greater sequence data matrix of the species in Nothobranchius and appropriate outgroups was generated and analysed using the same techniques as described above. This resulted in an even more robust phylogenetic hypothesis for the genus. From this dated phylogeny landscape evolution in a tectonically active area on south central Africa could be inferred. Four key landscape evolutionary events in this area could be inferred and dated: (i) landscape warping, faulting and rifting in the Luapula river drainage, (ii), formation of the Upemba rift in the Lufira-Lualaba river drainage system, and (iii) formation of the Congo-Zambezi Watershed between the Kafue and Luapula drainage systems disrupting the ancient Palaeo-Chambeshi river drainage system, (iv) drainage of Palaeo Lake Mweru, from a previous maximum level of 1200 masl to the present-day level of 917 masl. Lastly, this research resulted in the identification of 15 new Nothobranchius species which were described in the course of this study. .