Doctoral Degrees (Biochemistry)
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- 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. .
- ItemQuantifying the insulin response in mouse C2C12 skeletal muscle: a minimal modelling approach(Stellenbosch : Stellenbosch University, 2021-12) Kuhn, Stefan; Snoep, Jacky L.; Westerhoff, Hans V.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The insulin signalling cascade is one of the most important regulatory and signalling pathways in humans. Dysregulation or dysfunction of the insulin signalling path- ways often underlies the molecular ætiology of diseases such as diabetes, obesity, and Alzheimer’s. In turn, these diseases are the harbingers of various co-morbidities such as cardio-vascular disease, chronic inflammation, and dementia. The healthcare, eco- nomic, personal, and mortality burden of these diseases cannot be overstated. Mathematical modelling of insulin signalling is indispensable in the effort to un- derstand the dynamics of the insulin signalling cascade and how malfunctions therein lead to disease. However, despite the availability and complexity of existing models, few have explicitly connected the signalling cascade, glucose transporter activity, and metabolism with one another. In order to study these interactions, a ‘three-module’ approach was adopted that defined the signalling cascade, glucose transporter activ- ity, and metabolism as core, ‘input-output’ modules. The present work is limited to the signalling cascade and glucose transporter activity modules whereas work by Dr. Cobus van Dyk is concerned with the metabolic module. With this in mind, this thesis sets forth three aims. Firstly, to establish standard- ised culturing conditions which can be used to determine the basal state of insulin signalling and glucose transporter activity. Secondly, to develop a core, mathemati- cal model based on Western blotting and radio-labelled glucose -assay data which is able to describe the concentration- and time-dependence of the signalling cascade and glucose transporter activity in response to insulin. Thirdly, to determine the clustering behaviour of GFP-tagged GLUT4 molecules in response to insulin. The first goal was to standardise culturing conditions. Herein, the ability of high (25mM), medium (15mM), and low (5mM) glucose culturing conditions were evalu- ated with regards to their ability to sensitise or desensitise the insulin signalling cascade as well as the degree to which they are able to induce the differentiation of C2C12 my- oblasts into myocytes. The glucose and lactate concentrations in the external media were used to determine the glucose-lactate flux of the C2C12 cells. This served as a proxy for the induction of insulin-dependent glucose transport and metabolism. A modified Ladd staining protocol was used to assess the degree to which C2C12 cells could differentiate under the culturing protocols. The second goal was to construct a core, mathematical model of insulin signalling and glucose transporter activity. The time-dependent phosphorylation and dephos- phorylation of the insulin receptor and the serine 473 and threonine 308 sites of Akt in response to varying insulin concentrations was investigated using Western blotting techniques. The glucose transporter (GLUT4) activity was assayed using radio-carbon glucose. The data were used to optimise parameters for a core, ODE-based model of the signalling and glucose transporter modules. The third goal, to investigate the clustering behaviour of GLUT4 in response to insulin, was investigated by using confocal microscopy to image GFP-tagged GLUT4 molecules before and after being stimulated with insulin. A hierarchical clustering algorithm as well as further geometric and statistical analyses were used to determine the number, size, density, and distribution of GLUT4 clusters pre and post insulin exposure. Of the remaining chapters, Chapter 1 discusses the background, context, scope, and aims of this study as well as further elaborating on the ‘three module’ approach. The literature review in Chapter 2 provides an overview of the relevant literature as delineated by the scope and aims of this study. The materials and methods are provided in Chapter 3, with specific alterations or methodologies being further discussed in the relevant experimental chapters. The final chapter, Chapter 7, provides the reader with general discussions, limitations, and final thoughts concerning this work.
- ItemThe development of standardized ion mobility and mass spectrometry metabolomics methods for the characterization of plant phenolics(Stellenbosch : Stellenbosch University, 2021-04) Masike, Keabetswe; Stander, Marietjie; De Villiers, Andre; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Plants and plant-derived products contain a plethora of phenolic compounds, with a broad range of health benefits and useful applications such as in drug design. These phenolic compounds are often amplified by conjugation and rearrangements, thus producing isobaric and isomeric species. The standard analytical method for the identification and characterization of plant phenolics, liquid chromatography (LC) hyphenated to photo diode array (PDA) and/or high-resolution mass spectrometry (HR-MS) detectors, is not able to discriminate isomeric species in complex plant samples. The incorporation of ion mobility spectrometry (IMS) to LC-PDA-HR-MS workflows is being recognized as an additional orthogonal dimension of separation to HR-MS; where ions are separated through a drift region, filled with gas, based on their size, shape, and charge. The attractive component of IMS is the determination of the collisional cross section (CCS/Ω) values, which describes the unique rotationally averaged surface area of the ion, as it interacts and travels through the gas-filled drift region. The CCS as a feature, can be beneficial in the development of an in-house phenolics compound library in analytical laboratories, which can help expedite the characterization of phenolic compounds in varying research fields, such as in plant metabolomics and food science. The goal of the work reported in this thesis was, therefore, to develop LC-PDA-IM-HR-MS methods capable of structurally characterizing plant phenolics, found in South African indigenous herbal teas and plant species, based on a range of structural descriptors (e.g., tR, spectroscopic data, mass spectral information (including high resolution and MS/MS data), and CCS value). In the first part of the study, the phenolic profiles of Protea pure and hybrid cultivars were characterised by LC-PDA-IM-HR-MS for the first time in detail. Whereby, IMS in conjunction with other structure elucidation techniques, namely tandem MS data, UV-vis spectroscopy, nuclear magnetic resonance (NMR) were used for characterization of 67 metabolites. With the aid of NMR an undescribed hydroxycinnamic acid-polygalatol ester, caffeoyl-O-polygalatol (1,5-anhydro-[6-O-caffeoyl]-sorbitol(glucitol)) was isolated and characterised for the first time. Furthermore, positional isomers with similar MS/MS profiles were resolved by the IMS-dimension and consequently could be distinguished by their differences in CCS values. The CCS values obtained using two IMS platforms (drift-time ion mobility spectrometry (DTIMS) and travelling wave IMS (TWIMS)) were compared, and it was observed that the CCS values obtained with a TWIMS instrument were underestimated for compounds with CCS values below 200 Å2. Conversely, good agreement was obtained between both instruments for compounds with higher CCS values. Poor calibration of the TWIMS platform was attributed to the underestimation of the CCS values below 200 Å2. In the second part of the study, a detailed comparison of phytochemical profiles of a much larger set of Protea species, selections, and cultivars was reported. Using metabolomics tools and the data collected and documented (in the previous study) of phenolic compounds characterised based on their UPLC-PDA-IM-HR-MS profiles, plant metabolites associated with a post-harvesting disorder, leaf blackening, in Protea were identified/and annotated. Species, selections, and cultivars susceptible to leaf blackening contained features identified as benzenetriol- and/or hydroquinone-glycoside derivatives. On the other hand, stems not prone to blackening were linked to phenolic compounds with known protective properties against biotic and abiotic stressors. CCS values of the metabolites with protective features against leaf blackening, and those for compounds that instigate the process, were determined. Such observations serve as preliminary insights that can help accelerate plant improvement and aid in the selection of trait-specific markers in plant metabolomics. In the final portion of the study, using direct injection-IM-MS (DI-IM-MS) and descriptive chemometrics, differences between adulterated herbal teas (rooibos and honeybush) were observed. The diagnostic value of marker compounds in distinguishing the three commercialised honeybush species (Cyclopia intermedia, C. genistoides and C. subternata) for the quality control purpose were observed. To derive CCS values using the TWIMS instrument two calibrants, poly-DL-alanine and poly-L-malic acid, were compared. Poly-DL- alanine is a widely used TWIMS calibrant which results in an underestimation for compounds with CCS values below 200 Å2 (initial study), when compared to poly-L-malic acid an improvement in the underestimation of CCS values below 200 Å2 was observed. DI-IM-MS proved to be a useful tool for quality control purposes, particularly considering the analysis time is 1 minute per sample.
- ItemProgestins and breast cancer : significance of progesterone receptor isoforms and their altered ratios(Stellenbosch : Stellenbosch University, 2021-03) Cartwright, Meghan Carni; Africander, Donita; Louw-du Toit, Renate; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Progestins used in menopausal hormonal therapy have been associated with increased incidence of breast cancer. While these synthetic ligands were designed, in four consecutive generations, to mimic the activity of natural progesterone (P4) via the progesterone receptor (PR), the precise mechanism whereby some progestins and/or their metabolites may cause an increase in breast cancer incidence is still mostly unknown. Whether the PR, existing as two isoforms, PR-A and PR-B, plays a role in mediating the effects of progestins on breast cancer is unclear. As the metabolism of a progestin can ultimately influence effects via the PR, ultrahigh performance supercritical fluid chromatography-tandem mass spectrometry was used to investigate the metabolism of P4 and selected progestins in three breast cancer cell lines in the first part of this thesis. Unlike P4 that was rapidly metabolised in all three cell lines, promegestone (R5020), gestodene (GES) and nomegestrol acetate (NOMAC) were not metabolised, while only drospirenone (DRSP) was metabolised in the MDA-MB-231 and T47D cells. Additionally, we showed that P4 metabolism occurred at a similar rate in the MDAMB- 231 and T47D cells, but faster than its metabolism in the MCF-7 BUS cells. In the second part of this study, transactivation and transrepression transcriptional assays showed that the activities of a selected panel of progestins from all four generations are not all similar to each other, P4 or R5020, via PR-A and PR-B. For transactivation, most progestins were more efficacious via PR-B, but more potent via PR-A. We also showed that an increase in PR-A density and excess PR-A relative to PR-B, resulted in decreased efficacies of all progestins for transactivation. While an increase in PR-A density resulted in an increase in the activity of all progestins for transrepression, the activity of only a few progestins were influenced by excess expression of PR-A relative to PR-B. Realtime PCR showed progestin- and gene-specific regulation of endogenous genes known to play a role in breast cancer in T47D breast cancer cells. While the response of some progestins on the selected genes were PR-B mediated, some progestin effects were not mediated by either PR-A or PR-B. In the third part of this thesis, investigations into the effects of the progestins on proliferation, apoptosis, anchorageindependent growth, migration and invasion showed that these processes are differentially influenced by P4 and the selected progestins, and that the responses are also differentially mediated by PR-A or PR-B. Excess expression of PR-A resulted in both positive and/or negative ligand-independent, as well as progestin-induced, effects on these cancer hallmarks. Taken together, the findings of this thesis emphasize the fact that progestins do not always mimic the activities of P4 or each other. The results further highlight the complexity of progestin action via the PR, underscoring the importance of distinguishing progestin activities via PR-A and PR-B, and also considering the PR-A:PR-B ratio when investigating the mechanisms of progestins and the PR in breast cancer. Finally, our results suggest that a progestin such as medroxyprogesterone acetate (MPA) acting via PR-A and/or PR-B may indeed increase breast cancer risk, while others like DRSP may not.
- ItemIn vitro reconstitution of the coenzyme A biosynthesis pathway for the study of its regulation and disruption by possible inhibitors(Stellenbosch : Stellenbosch University, 2021-04) Mostert, Konrad Johannes; Strauss, Erick; Snoep, Jacky L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The coenzyme A (CoA) biosynthesis pathway has been validated as a novel target for antimicrobial drug development against numerous disease-causing organisms such as Mycobacterium tuberculosis (Mtb) and Staphylococcus aureus. It is therefore not surprising that various enzymes of the CoA biosynthesis pathways of these organisms have been the targets of multiple target-based drug discovery and development efforts. However, many of the inhibitors that have been identified and developed to target these enzymes have not translated into potent cell growth inhibitors, while the mechanisms of action of some of the most promising CoA-directed growth inhibitors, such as homopantothenate (HoPan) and the N-substituted pantothenamides (PanAms), are not completely understood. In this study we set out to address these knowledge gaps to provide better direction for CoA-directed antimicrobial drug development. We first show that the chemical modulator HoPan, which is used to reduce CoA levels in eukaryotic disease models, does not target PanK, but rather PPCS, after metabolic activation by PanK. Next, we considered the current approach to CoA-directed drug development and make the argument that potential inhibitors should be studied in a systems context that takes into account the flux control distribution among the pathway enzymes. To demonstrate the utility of such an approach, we developed a systems-based assay to identify and characterise CoaBC-targeted inhibitors that display different mechanisms of action. The assay was validated by using known inhibitors of the PPCS activities of the S. aureus and Mtb CoaBC proteins, and then used to investigate the potential of two promising CoaBC-targeted drug development strategies: one depending on metabolic activation by S. aureus PanK (SaPanK), and the other making use of prodrugs. For the PanAms, we found that these compounds can have different modes of action depending on their interaction with SaPanK. We show that the most potent PanAm growth inhibitors target CoA- utilising processes as their main mode of action, with PanAms that only inhibit the CoA biosynthesis pathway enzymes having reduced potency. Following this, we demonstrate the importance of determining the vulnerabilities of the CoA pathway enzymes in the specific organism being targeted as a key prerequisite for a drug development campaign as previously developed CoA-directed inhibitors that failed to show growth inhibition seem to target enzymes that were found to be less vulnerable to inhibition. We used an in vitro reconstituted S. aureus CoA biosynthesis pathway to determine the potential feedback regulation mechanisms of the S. aureus CoA biosynthesis pathway as well as the vulnerabilities of the individual pathway enzymes. We discovered that DPCK, the last enzyme in the pathway, is the most vulnerable target in the pathway, followed by PanK. CoaBC and PPAT, on the other hand, are not vulnerable targets. This study ultimately sheds new light on the mode of action of existing CoA-directed inhibitors while providing important new tools for directing future CoA-directed drug development efforts.