Masters Degrees (Biochemistry)

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The age-old problem of pollution, its role in endocrine disruption and the current analytical technologies that can be employed to monitor and assess waste water treatment plants
(Stellenbosch : Stellenbosch University, 2017-03) Olivier, Daniel Wilhelm; Wolfaardt, Gideon M.; Swart, Pieter; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Water scarcity is a global problem and pollution of this valuable resource is a growing concern. South Africa is no exception. As part of an on-going study aimed at developing decentralized water treatment systems based on biomimicry design, this project aimed to evaluate and optimize analytical methods that can be used to evaluate the efficiency of these systems in removing compounds with endocrine disrupting properties. In addition, this project also aimed to show the possible consequences if pollutants are not removed by investigating the effects of a number of endocrine disrupting chemicals in combination with one another and in combination with natural human hormones. This should provide a more realistic view of how a combination of pollutants that typically end up in the environment due to pollution can adversely affect organisms, and by extension, the ecosystem. It should also contribute to our understanding of how common pollutants that gets applied on the skin as personal care products (PCPs) can possibly influence human health and be linked to diseases such as breast cancer. The first aim resulted in a method that can be used to isolate, identify and quantify 11 endocrine disrupting chemicals (3 hormones, 1 synthetic hormone analog, 4 PCPs, 2 plasticizers and 1 anticonvulsant) and one human indicator. The method uses solid phase extraction to isolate compounds, dansyl chloride derivatization of compounds to enhance mass spectrometry detection and a novel super-critical fluid chromatography system, called an ultra-performance convergence chromatography (UPC2) system, coupled to tandem mass spectrometry for detection and quantification of each compound. This method can be used to evaluate the removal efficiency of waste water treatment systems. However, method validation revealed additional optimization and simplification should be considered. The second aim yielded data that showed the combined effect four common PCP pollutants as either being additive, antagonistic or synergistic. The data highlights how these PCPs can possibly interfere with the endocrine system of humans and animals if used as PCPs or are found in environment as pollutants. Finally, the data also suggest how common PCPs can influence diseases such as breast cancer.
Antibody production against Staphylococcus aureus CoA biosynthesis enzymes and their application in protein level quantification
(Stellenbosch : Stellenbosch University, 2021-04) Bothma, Karli; De Villiers, Marianne; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Antimicrobial resistance has become an increased burden worldwide as more and more human pathogens are becoming resistant to current antimicrobials. Therefore, the identification of novel drug targets and development of new antimicrobial drugs are currently of high priority. A drug target that has gained increased attention is the coenzyme A (CoA) biosynthesis pathway. CoA is an essential cofactor that is necessary for life in all organisms, including human pathogens, making it an attractive target for the development of new antimicrobial drugs. The CoA biosynthesis pathway of Staphylococcus aureus, which is the leading cause of hospital-associated infections, was the focus of this study. Although various studies have investigated this pathway in S. aureus as a possible drug target, there is still a lot that needs to be elucidated in this regard. One gap in our knowledge is that the levels of the CoA biosynthesis enzymes (PanK, CoaBC, PPAT and DPCK) under physiological conditions are currently unknown. This study therefore aimed to develop immunological techniques which could be implemented as tools to quantify the levels of these enzymes at different growth phases of S. aureus cultivated under physiological growth conditions. To achieve this aim, the four CoA biosynthesis enzymes of S. aureus were recombinantly expressed and purified using established methods. Polyclonal antibodies were raised in rabbits by immunising the animals with the respective enzymes adsorbed to acid-treated, naked Salmonella minnesota R595. This method has been used to successfully produce antibodies to a wide variety of antigens, especially in cases where only small amounts of the antigen were available. With these antibodies, indirect competition enzyme-linked immunosorbent assays (ELISAs) with excellent standard curves were obtained for the quantification of each of the respective enzymes. Furthermore, cross-reactivity studies performed with ELISA and western blot revealed that the anti-SaPanK, anti-SaPPAT and anti-SaDPCK antibodies showed limited cross-reactivity. In an attempt to quantify the amount of cross-reactivity of each antibody-antigen pair, however, it was found that only the cross-reactivity of the anti-SaPPAT antibodies had an effect on the final optimised assay. In this study an original, highly sensitive ELISA method for the detection and quantification of each of the enzymes of the CoA biosynthesis pathway of S. aureus was developed. These assays provide a cost-effective method for enzyme level quantification that have the potential to provide better insight on the levels of the different enzymes under physiological conditions and ultimately aid in the development of new antimicrobial drugs.
Antifungal activity of antimicrobial polymers created with peptides from the tyrothricin complex
(Stellenbosch : Stellenbosch University, 2024-03) Mitha, Priyata; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The global burden posed by the lack of effective antifungal agents in addressing conditions like vulvovaginal candidiasis (VVC), diaper dermatitis, and nosocomial infections, caused by Candida species, has prompted an urgent need for innovative interventions. The increasing resistance to current antifungal agents further highlights the urgency of this issue. To address these challenges, the exploration of novel solutions or modifications to existing applications is paramount. In this context, antifungal peptides (AFPs) have emerged as promising candidates. This study specifically focuses on investigating anti-Candida activity, with a particular emphasis on the tyrocidines (Trcs), a natural cyclodecapeptide antibiotic complex. These peptides are considered good potential candidates for further development due to their broad-spectrum activity against bacteria, fungi, viruses, and parasites, as well as their inherent ability to adhere to various materials. Recent studies have indicated that the peptide adopts two major self-assembly modes depending on its surrounding environment, and the potent antibacterial activity of the peptide is relatively non- selective in terms of the type of material being functionalised. This study aimed to explore the effect of different solvent systems and Trc peptide complexes on anti-Candida activity of Trc-functionalised materials, as well as the biophysical tracking of peptide self-assembly and material binding. The investigated materials include polystyrene plastic and cellulose filter paper (used as controls), viscose and polypropylene sheets/wipes. The Trc mixture, Phe-rich and Trp-rich peptide complexes were formulated in four solvents: acetonitrile (ACN), methanol (MeOH), ethanol (EtOH) and isopropanol (IPA). Optimisation studies by assessing the target cell metabolism over time revealed that EtOH promotes good material adherence and effective antifungal activity for the Trc mixture and Phe-rich complex, while MeOH and IPA have similar effects on the Phe-rich and Trp-rich peptide complexes. The robustness and stability of the functionalised materials were assessed through a series of washing steps. These findings highlighted the significance of amino acid composition in the optimal peptide deposition onto the materials. Testing an industry-derived method for material treatment led to the conclusion that the average polarity of the peptide complex, solvent system, and the material each play a role. The Trc mixture and Phe-rich peptides associated more readily with the hydrophobic polystyrene, while the more polar Trp-rich peptide complex associated readily to the more polar cellulose and viscose. Furthermore, the viscose material allows for even distribution of the peptide, resulting in potent antifungal activity. Furthermore, the link between surface-derived antifungal activity and biophysical properties of the peptides in the different ethanolic solutions during deposition on materials was assessed by observing changes in Trp and Tyr fluorescence. This provided insight into the impact of peptide conformation in solution and its binding to a specific material on antifungal activity. This study indicated that peptide oligomers, driven by hydrophobic interactions and aromatic stacking, lead to the assembly of metastable oligomers that are crucial for material association and antifungal activity. In aqueous solvents, water propelled the formation of hydrophobic interaction-driven oligomers, represented by fluorescence quenching/loss. In these types of peptide structures, the polar amino acids decorate the outside of the oligomers with the Orn⁹/Lys⁹ residues, previously found to be essential for activity, interacting with the electronegative target cell wall. Active peptide moieties, such as amphipathic dimers, are then released from the peptide layers on the material to elicit antifungal activity. After the washing steps, only peptides strongly associated to the material were retained, and if in the correct conformation, could elicit the antifungal response. Organic solvents at higher concentrations resulted in a decreased hydrophobic effect on the peptides, represented by fluorescence dequenching/gain and poor antifungal activity. Here, peptides may either be in the incorrect conformation, stacked too tightly, or deposited as inactive stable oligomers. The potential of the tyrocidines in material functionalisation and the results in this study pave way towards the development of effective antifungal materials. With little to no anticipation of resistance emergence, and by prevention of chronic fungal infections, this study shows the potential of the Trc-functionalised materials for female and baby hygiene products, as well as surface sterilisation.
Applications of generalised supply-demand analysis
(Stellenbosch : Stellenbosch University, 2013-03) Christensen, Carl David; Rohwer, J. M.; Hofmeyr, J-H. S.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Supply-demand analysis (SDA) is a tool that allows for the control, regulation and behaviour of metabolic pathways to be understood. In this framework, reactions are grouped into reaction blocks that represent the supply and demand of a metabolic product. The elasticities of these supply and demand blocks can be used to determine the degree of control either block has over the flux in the pathway and the degree of homoeostasis of the metabolic product that links the blocks. Rate characteristic plots, on which the rates of supply and demand blocks are plotted as functions of the concentration of the linking metabolite, represent a powerful visual tool in this framework. Generalised supply-demand analysis (GSDA) allows for the analysis of metabolic models of arbitrary size and complexity without prior knowledge of the regulatory structure of the pathway. This is achieved by performing SDA on each variable metabolite in a pathway instead of choosing a single linking metabolite. GSDA also provides other benefits over SDA as it allows for potential sites of regulation and regulatory metabolites to be identified. Additionally it allows for the identification and quantification of the relative contribution of di erent routes of regulation from an intermediate to a reaction block. Moiety-conserved cycles present a challenge in performing in silico SDA or GSDA, as the total concentration of a moiety must remain constant, thereby limiting the range of possible concentrations of the metabolites between which it cycles. The first goal of this thesis was to develop methods to perform GSDA on two-membered and interlinked moiety-conserved cycles. We showed that by expressing the members of a moiety-conserved cycle as a ratio, rather than individual metabolite concentrations, we can freely vary the ratio without breaking moiety conservation in a GSDA. Furthermore, we showed that by linking the concentrations of the members of two interlinked two-membered moiety-conserved cycles to a “linking metabolite”, we could vary the concentration of this metabolite, within constraints, without breaking moiety conservation. The Python Simulator for Cellular Systems (PySCeS) is a software package developed within our group that provides a variety of tools for the analysis of cellular systems. The RateChar module for PySCeS was previously developed as a tool to perform GSDA on kinetic models of metabolic pathways by automatically generating rate characteristic plots for each variable metabolite in a pathway. The plots generated by RateChar, however, were at times unclear when the models analysed were too complex. Additionally, invalid results where steady-states could not be reached were not filtered out, and therefore appeared together with valid results on the rate characteristic plots generated by RateChar. We therefore set out to improve upon RateChar by building plotting interface that produces clear and error-free rate characteristics. The resulting RCFigure class allows users to interactively change the composition of a rate characteristic plot and it includes automatic error checking. It also provides clearer rate characteristics with e ective use of colour. Using these tools two case studies were undertaken. In the first, GSDA was used to investigate the regulation of aspartate-derived amino acid synthesis in Arabidopsis thaliana. A central result was that the direct interaction of aspartate-semialdehyde (ASA), a metabolite at a branch point in the pathway, with the enzyme that produces it only accounts for 7% of the total response in the flux of supply. Instead, 89% of the observed flux response was due to ASA interacting with of the downstream enzymes for which it is a substrate. This result was unexpected as the ASA producing enzyme had a high elasticity towards ASA. In a second case study moiety-conserved cycles in a model of the pyruvate branches in lactic acid bacteria were linearised using the above mentioned method. This served to illustrate how multiple reaction blocks are connected by these conserved moieties. By performing GSDA on this model, we demonstrated that the interactions of these conserved moieties with the various reaction blocks in the pathway, led to non-monotonic behaviour of the rate characteristics of the supply and demand for the moiety ratios. An example of this is that flux would increase in response to an increase in product for certain ranges. This thesis illustrates the power of GSDA as an entry point in studying metabolic pathways, as it can potentially reveal properties of the regulation and behaviour of metabolic pathways that were not previously known, even if these pathways were subjected to previous analysis and a kinetic model is available. In general it also demonstrates how e ective analysis tools and metabolic models are vital for the study of metabolism.
An assessment of the mutation patterns in South African isolates of Potato leafroll virus and the expression of recombinant viral coat protein genes in Escherichia coli
(Stellenbosch : Stellenbosch University, 2007-03) Rothmann, Adri Hilda; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Presently, the observed variation in symptoms of Potato leafroll virus (PLRV) infection in potato cultivars in South Africa cannot be reconciled with PLRV symptoms obtained 10-15 years ago, even if the different interactions between the pathogen and the cultivar are taken into account. In an effort to analyze this variation, mutations in the coat protein (CP) gene of South African isolates of PLRV were assessed. The CP gene of PLRV isolates from different areas within South Africa was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. Significant sequence variation in the CP gene was found within the analyzed South African isolates of PLRV. Phylogenetic analysis revealed two major clades with most South African isolates and an Australian and North American isolate grouped together and the remainder grouped with isolates from diverse countries worldwide. The deduced amino acid sequences from representatives of these two clades indicated differences in CP threedimensional structure. In an effort to produce recombinant PLRV CP for the production of antibodies specific for South African isolates of PLRV for use in enzyme-linked immunosorbent assay (ELISA), the CP gene of a South African isolate of PLRV was subcloned into a bacterial expression vector (pET14-b). Expression of full length recombinant PLRV CP was attempted in Escherichia coli strains BL21(DE3)pLysS, Rosetta-gami B(DE3)pLysS and Rosetta-2(DE3)pLysS. As this was not successful, the PLRV CP gene was subcloned in another expression vector (pGEX) for expression as an N-terminal fusion protein with glutathione-S-transferase (GST) in E. coli strains BL21(DE3)pLysS and Rosetta-2(DE3)pLysS. The recombinant GST-PLRV CP fusion protein was purified and used for antibody production in rabbits. Using western blots, the effectiveness of antibodies produced to recombinant GST-PLRV CP fusion protein was assessed for PLRV recognition. It was found that antibodies to the recombinant GST-PLRV CP fusion protein were more effective for the detection of GST than PLRV CP and that production of antibodies to the cleaved PLRV CP product would be necessary if antibodies are required for ELISA applications.
Atomic force microscopy : a novel tool for the analysis of the mechanism of action of antimicrobial peptides on target membranes
(Stellenbosch : Stellenbosch University, 2003-03) Holroyd, Dale; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry .
ENGLISH ABSTRACT: Nanoscale visualisation of live cells and cellular components under physiological conditions has long been a goal in microscopy. The objective of this study was to validate the use of Atomic Force Microscopy (AFM) as a new tool in unravelling the mysteries of antimicrobial peptide mechanism of action. Using the simplest AFM imaging technique, we were able to analyse the influence of haemolytic melittin and anti-bacterial magainin 2 on different target membranes at nanometer resolution, without using fixing agents. First, magainin 2 was synthesised and purified by gel permeation chromatography and high performance liquid chromatography (HPLC). The purity of magainin 2 and melittin, isolated from bee venom (Sigma-Aldrich), was verified with electro spray ionisation mass spectrometry (ESI-MS). Second, dose-response experiments were used to determine the optimum peptide/target cell ratio that would allow interaction with the membrane without causing lysis. Third, peptide/target-cell samples were placed on silica plates and visualised using contact mode AFM. Images obtained of the cells before and after peptide treatment, showed distinct changes in cell membrane surface topology. We observed grooves, lesions, membrane collapse and vesiculation depending on the concentration, type of peptide and target-cell used, allowing us to make conclusions regarding the mechanism of action of melittin and magainin 2. In comparison with model membrane studies, our AFM results show that a peptide can function by more than one mechanism of action depending on the structural composition of the membrane, which appears to have specific segregated lateral organisation. Magainin 2 (non-toxic) selectively targets cell membranes using different mechanisms of action. In this way it can lyse bacterial membranes (anti-bacterial agent) using one mechanism, while using another mechanism to interact with mammalian cells at physiological concentrations, without destroying them. In contrast, melittin (toxic) is non-selective, and uses the same mechanism of interaction with bacterial and mammalian cells. In conclusion, we propose a new holistic model for the mechanism of action of antimicrobial peptides.
Biocatalytic preparation and characterization of alternative substrate of MshB, a mycothiol pathway enzyme
(Stellenbosch : Stellenbosch University, 2012-12) Muneri, Ndivhuwo Olga; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, utilizes mycothiol (MSH) as the major low molecular weight thiol to protect itself against oxidative stress and thereby to ensure its growth and survival. MSH is a pseudo-disaccharide molecule that contains an α(1→1) glycosidic bond, and is biosynthesised in five enzymatic steps involving the enzymes MshA, MshA2, MshB, MshC and MshD. Owing to the essentiality of MSH to M. tuberculosis, various studies have focused on the MSH biosynthetic and other MSH-dependent enzymes viewed as potential drug targets for the development of antituberculosis agents. In the course of this study two practical challenges affecting the development of inhibitors of one the MSH biosynthesis pathway enzyme, MshB, were addressed. These challenges entail the lack of a high-throughput continuous assay to determine MshB activity, and the poor availability of the natural and alternative MshB substrates. In this study an alternate MshB substrate was characterized and shown to undergo a rearrangement reaction upon deactylation, which allowed the development of a new continuous assay for MshB activity that uses DNTB (Ellman’s reagent). In addition, three new α-thioglycoligases were created from the α-Nacetylglucosaminidase of Clostridium perfringens. These enzymes showed potential as biocatalysts that can be used for the enzymatic synthesis of thioglycoside-based alternative substrates of MshB.
Biochemical and genetic characterization of bacteria isolated from diseased rainbow trout (Oncorhynchus mykiss) farmed in Lesotho and Mpumalanga province of South Africa
(Stellenbosch : Stellenbosch University, 2017-12) Kutu, Vuyokazi; Bellstedt, D. U.; Macey, B. M.; Mouton, A.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Rainbow trout farms in Mpumalanga Province, South Africa and Lesotho, have periodically suffered significant losses from infections caused by Gram-positive bacteria. Such outbreaks have hampered the development of this industry in both South Africa and Lesotho. A total of 55 bacterial strains had been isolated between 2006-2012 from infected trout farmed in Lesotho and Mpumalanga Province and had been stored for long term by freeze drying. Some isolate identification had been performed and a few were used for vaccine development. Vaccines were however only effective for one or two seasons, highlighting the need to properly characterize these Gram-positive bacteria. The aims of the study were therefore to: (i) investigate the genetic diversity of these bacterial isolates by their phenotype; antimicrobial susceptibility and 16S ribosomal RNA (rRNA) sequencing and phylogenetic analysis, (ii) investigate the different antigenic epitopes that exist within this group of bacterial isolates by development of an enzyme-linked immunosorbent assay (ELISA) utilizing six rabbit produced polyclonal antibodies, produced against six selected bacterial isolates from the 55 isolates investigated in this study. Phenotypic analysis showed that fifty of the isolates were Gram-positive cocci and five were Gram-positive rods. Their growth characterists and antimicrobial susceptibility were extensively characterized. The 16S rRNA analysis indicated the following isolate composition: 49 Lactococcus garvieae, one Lactococcus lactis, three Carnobacterium maltaromaticum and two Weissella species, which is the first report of Weissella from diseased trout from South Africa. Antigenicity analysis showed that there were highly specific epitopes that were limited to very few isolates, but also common epitopes that were shared between isolates of the same genus, but even some epitopes that were shared between different bacterial genera. The patterns of epitope sharing broadly correlated with the 16S rRNA phylogeny, but not entirely which was not unexpected as phylogeny does not indicate the presence or absence of bacterial epitopes. These results address the importance and accuracy of molecular identification of disease causing species and the need to investigate the antigenic differences expressed by these pathogenic bacteria to assist in generating correct information needed for the development of vaccines of high efficacy.
A biochemical and immunochemical study of ovine adrenal cytochrome P-450 reductase
(Stellenbosch : Stellenbosch University, 1992-03) Petersen, Carmen J.; Swart, P.; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: This study describes: (a) the isolation · of ovine adrenal cytochrome P-450 reductase, (b) the preparation of , antibodies against this enzyme and (c) comparitive immunochemical studies with ovine' liver and .bovine. adrenal cytochrome P-450 'reductases
A biochemical and immunological study of horseradish peroxidase
(Stellenbosch : University of Stellenbosch, 2009-12) Odendaal, Ruenda; Swart, P.; Lombard, N.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: This study describes: a) the isolation and purification of horseradish peroxidase isoenzymes from horseradish roots, b) the characterization of various forms and components of the enzyme by cation-exchange and reversed-phase high performance liquid chromatography, c) the preparation of antibodies against horseradish peroxidase isoenzymes, d) immunological studies for the development of an isoenzyme quantification method and e) the formation of an enzyme-melamine conjugate for use in a melamine quantification immunoassay.
A biochemical investigation into the compounds involved in pigmentation of apple and pear skin and the manipulation thereof
(Stellenbosch : Stellenbosch University, 1998) Arends, Arrie Paul; Bellstedt, D. U.; Swart, P.; Stellenbosch University. Faculty of Science. Department of Biochemistry.
ENGLISH ABSTRACT: This study describes (a) Identification of the main anthocyanin pigment in the skin of the 'Fuji' apple cultivar and in the pear cultivars 'Bon Rouge' 'Forelle', Red d'Anjou', 'Rosemarie' and 'Flamingo', (b) an investigation of the effect of on-tree bagging on anthocyanin pigment accumulation in the skin of the 'Fuji' apple cultivar by means of high performance liquid chromatography (HPLC) technology. (c) an investigation of the influence of cold storage and ripening on anthocyanin concentration and accumulation in the skin of the pear cultivars, 'Forelle', 'Rosemarie', 'Flamingo', 'Bon Rouge' and Red d'Anjou' pear cultivars by means of HPLC technology and (d) an investigation of the level of induction of dihydroflavonol 4-reductase (DFR) gene in 'Fuji' apple cultivar during bagging trial, through mRNA studies.
A biochemical study of budbreak and plant growth regulators in table grapes
(Stellenbosch : Stellenbosch University, 2002-03) Lombard, Petrus Johannes; Bellstedt, D. U.; Cook, N. C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The cultivation of table grapes in the warmer areas of South Africa, indeed worldwide, is complicated by rest breaking problems in spring due to delayed budbreak. In order to overcome these problems rest breaking agents, mainly hydrogen cyanamide, are applied. However, instead of alleviating the problem, additional problems such as uneven budbreak and reduced production are often induced. This study was initiated to further understand the physiological processes occurring during budbreak and how the application of hydrogen cyanamide influences these processes. The following aspects were investigated in this study: a. The effect of hydrogen cyanamide on tissue cytokinin (specifically zeatin riboside) levels of Sultanina table grape vines after application at different times before natural budbreak was studied over two seasons. In 1997, hydrogen cyanamide was applied at three weeks before induced budbreak and in 1998 at six weeks before induced budbreak. One year-old canes were sampled weekly after hydrogen cyanamide application, divided into distal and proximal sections, then further divided into buds, bark and wood tissues and the zeatin riboside (ZR) levels determined. A relatively high amount of chilling coupled to late hydrogen cyanamide application in 1997 led to a large effect on ZR release, but did not lead to significant shifting of the budbreak pattern. Zeatin riboside peaks were observed in buds, internode wood and bark of treated vines compared to control vines. The peaks were higher in distal portions compared to proximal portions in all tissues. The relatively lower chilling and earlier application of hydrogen cyanamide in 1998 had a larger effect on the budbreak pattern while the bud ZR peak was shifted earlier. The distal portion bud ZR . peak was again higher than the proximal portion bud ZR peak. In 1997, as sampling was not initiated early enough, bud ZR peaks were only observed after budbreak, while in 1998 bud ZR peaks were observed before and after budbreak. The effect of these ZR increases on the development of inflorescence primordia, subsequent bunch development and ultimately production, are discussed. b. Free xylem sap was sampled at cane and spur pruned lengths from unpruned canes of Sultanina from budswell until after budbreak in 1999 and from three table grape cultivars, i.e Sultanina, Alphonse Lavalleé and Sunred Seedless, in 2001 and ZR levels determined. The ZR levels in the buds of these three table grape cultivars, pruned to different cane lengths were also determined. One year old canes of these cultivars, were each pruned to long canes (14 buds) and short spurs (2 buds). The ZR content in buds of these canes at distal and proximal positions were determined weekly from budswell until after budbreak in 1999. Xylary ZR peaks occurred before 50% budbreak. Spur xylary ZR levels of all three cultivars followed a similar pattern, although at lower ZR levels than that of the canes. This is similar to previous studies on xylary ZR levels of apple shoots. The high levels of free ZR found in xylem sap at the distal portions of canes support the hypothesis of a cumulative ZR build-up effect as cane length increases. Spur pruning resulted in earlier budbreak and a higher final budbreak than cane pruning. The proximal portions of shoots, whether spur pruned or the proximal portions of canes, showed elevated ZR levels in all cultivars. This difference in ZR levels in bud tissue of different portions of the cane would suggest a difference in ZR consumption or turnover. The results of this study have important management implications for the cultivation of vines in warmer areas in which hydrogen cyanamide is used to alleviate budbreak problems.
Bioinformatic characterisation of genes associated with coenzyme A biosynthesis in mycoplasmas and expression and isolation of dephospho-coenzyme A kinase from Mycoplasma sp. Ms02
(Stellenbosch : Stellenbosch University, 2018-03) Ras, Tertius Alwyn; Botes, Annelise; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The South African ostrich industry is internationally the leading provider of ostrich products. The increasing popularity of ostrich products has resulted in the adjustment of production strategies, which includes intensifying rearing conditions by using feedlot systems. However, this intensive rearing strategy creates an ideal environment for pathogens, such as mycoplasmas, to spread. There are three Mycoplasma species that infect ostriches, which are associated with respiratory diseases. These mycoplasma infections can result in production losses, which not only have an economic impact on the ostrich industry but also significant socio-economic implications. Hence, there is a need for specific and cost-effective treatment against these ostrich-infecting mycoplasmas. The enzymes involved in the biosynthesis pathway of coenzyme A have long been regarded as potential targets for drug development. These enzymes could, therefore, offer a solution to the control of mycoplasma infections in ostriches. Since the coenzyme A biosynthetic pathway is relatively unexplored in mycoplasmas, the first aim of this study was to determine the presence or absence of enzyme-encoding genes involved in this pathway in Mycoplasma species. This was done using a bioinformatics approach. Of the 62 Mycoplasma species investigated, there were eight species (13%) found to have none of the enzyme-encoding genes, while the remaining species had at least one. Additionally, twelve enzyme-encoding gene homologues were identified and their predicted identities confirmed by evaluating the conserved and functional motifs and domains. The enzyme-encoding gene found to be most common amongst the investigated species was that of dephosphocoenzyme A kinase (DPCK), the final enzyme in the biosynthesis pathway. Furthermore, there was no correlation between the number of identified coenzyme A biosynthetic pathway enzyme-encoding genes in a species and the phylogeny of the respective proteins. There was also no correlation with the 16S rRNA phylogenetic groupings. Given the common presence of the DPCK-encoding gene, the second aim of this study was to recombinantly express the DPCK of the ostrich-infecting Mycoplasma sp. Ms02 (Ms02) and isolate the protein using a His-tag. The Ms02 DPCK-encoding gene was successfully amplified, cloned and mutated by site-directed mutagenesis to allow for expression in a nonmycoplasma host. However, the soluble expression and isolation of the Ms02 DPCK protein proved to be challenging. Using a variation of methods, the protein was eventually solubilised using a sarkosyl treatment method. A pure isolate of the Ms02 DPCK protein could, however, not be attained when using immobilised metal affinity chromatography (IMAC) purification. Subsequent activity testing of the isolated DPCK enzyme, using an HPLC-based method, also showed no activity.
A bioinformatics tool to detect physical gene clusters in functional genomics data
(Stellenbosch : Stellenbosch University, 2021-03) Conradie, Louis Timoteus; Patterton, Hugh-G.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Many studies have investigated the biological effects of the external environment on the genetic expression of a living cell. This particular study looks at gene clusters that are switched on at a series of discrete time points when Saccharomyces cerevisiae comes out of stationary phase, when the cell is fed a carbon source after a starvation period. To achieve this, Pyxis2 was developed, a bioinformatics tool that is able to detect gene clusters in functional genomics data from any organism. The program detects physical clusters of genes that share a defined functional genomic property, such as transcriptional activity, that may be interpreted in terms of a biological functionality. Pyxis2 provides several options to the user that may be adjusted for appropriate levels of sensitivity. Pyxis2 is available at https://github.com/Louis-Conradie/Pyxis2_classes_2020. Following the identification of gene clusters that are induced during the exit of stationary phase in yeast, the study is extended to also investigate the biological relationship between the identified genes and possible regulatory mechanisms. These mechanisms may include transcriptional activators and co-activators, epigenetic modifications such as histone H3K9ac acetylation, or the effect of the domain wide change of the spatial structure of chromatin. I show that the mega-Dalton transcriptional activation complex SAGA is associated with some of the transcriptionally induced clusters at early times during stationary phase exit, and that acetylation of lysine 9 of histone H3 is not a detectable property of active clusters. I finally show some association between spatial proximity of parts of the genome and cluster gene induction but find this association in cycling cells as opposed to in cells re-engaging passage through the cell-cycle. I finally discuss the implications of my findings in the context of the current literature and identify future avenues of enquiry.
The biosynthesis of adrenal C 11-oxy C21 steroids, implicated in 21-hydroxylase deficiency-21-desoxycortisol and 21 desoxycortisone and their downstream metabolism
(Stellenbosch : Stellenbosch University, 2017-03) Barnard, Lise; Swart, Amanda C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Adrenal C19 steroids are often implicated in numerous androgen dependent disease conditions. Androgen excess is the hallmark of 21-hydroxylase deficiency (21OHD) fuelled by an increase in the production of adrenal androgens and androgen precursors. In addition, increased levels of progesterone (P4) and 17α-hydroxyprogesterone (17OHP4), the substrates of the defective cytochrome P450 steroid 21-hydroxylase, have also been reported. Adrenal steroids are secreted into circulation, for further downstream metabolism. Conversion of adrenal steroids to active androgens in peripheral target tissue is dependent on the tissue specific expression of key enzymes, which significantly influence steroid profiles at cellular level, ultimately influencing homeostasis in androgen responsive tissue. In 21OHD, the conversion of P4 and 17OHP4 in an alternative metabolic pathway to the potent androgen, dihydrotestosterone, is reported, which has been described as the backdoor pathway. The accumulation of P4 and 17OHP4 furthermore present substrates for 11β-hydroxylation by the cytochrome P450 11β-hydroxylase (CYP11B) isozymes. In this study the catalytic activity of the CYP11B isozymes towards P4 and 17OHP4 are presented. CYP11B1 and CYP11B2, also termed cytochrome P450 aldosterone synthase, transiently expressed in HEK293 cells, effectively utilised P4 and 17OHP4 as substrates, yielding 11β-hydroxyprogesterone (11OHP4) and 21-desoxycortisol (DOF). Catalytic conversions of DOF and 21-desoxycortisone (DOE), the C11-keto derivative of DOF, together with P4 and 17OHP4, were assayed in HEK293 cells transiently transfected with steroid 5α-reductase type 1 or type 2 (SRD5A1 or SRD5A2). Conversion of DOF, DOE, P4 and 17OHP4 by the SRD5A isozymes, yielded 5α-pregnan-11β, 17α-diol-3, 20-dione (11OHPdione) and 5α-pregnan-17α-ol-3, 11, 20-trione (11KPdione), dihydroprogesterone and 5α-pregnan-17α-ol-3, 20-dione (Pdione), respectively. We identified these novel steroids, 11OHPdione and 11KPdione, which were converted to the novel products 5α-pregnan-3α, 11β, 17α-triol-20-one and 5α-pregnane-3α, 17α-diol-11, 20-dione by 3α-hydroxysteroid dehydrogenase type 3 (AKR1C2) catalysed conversions in transiently transfected HEK293 cells by accurate mass determination. Metabolism of DOF in LNCaP cells, yielded DOE, 11β-hydroxyandrosterone and 11-ketoandrosterone, indicative of the conversion by endogenous 11β-hydroxysteroid dehydrogenase type 2, SRD5A, AKR1C2 and cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17A1) enzymes. These findings shows that DOF, produced in 21OHD, can be metabolised via the C11-oxy Pdione en Pdiol (5α-pregnan-3α, 17α-diol-20-one) intermediates to suitable substrates for the lyase activity of CYP17A1 thus leading to the production of C11-oxy C19 steroids. Taken together, the biosynthesis of C11-oxy C21 steroids, together with their metabolism by the enzymes in the backdoor pathway, yielded novel steroid metabolites contributing to the pool of potent androgens in 21OHD, with said steroids also presenting possible biomarkers in disease identification.
Characterisation & purification of class IIa bacteriocins from lactiplantibacillus plantarum
(Stellenbosch : Stellenbosch University, 2024-03) Beukes, Cheyenne Tamika; Beukes, Mervyn; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The rapid emergence of antibiotic resistance, coupled with the scarcity in the development of new drugs to combat resistance are threatening the food, medical and agricultural industries. This demands the development of novel alternative treatments, such as antimicrobial substances to target specific pathogenic micro-organisms. Lactic acid bacteria (LAB) have attracted attention in this research field, as LAB secrete antimicrobial peptides bacteriocins. Bacteriocins are ribosomally synthesized antimicrobial peptides that display promising potential as natural food preservatives and source of microbial inhibitors. The producer organism chosen for further research was Lactiplantibacillus plantarum (L. plantarum), a member of the LAB known to commonly produce multiple bacteriocins. The goal of this study was to perform the in-silico gene mining in conjunction with the in vitro molecular screening with the aim to isolate, and characterise bacteriocins from the producer organism, L. plantarum. The results obtained from the various assays and characterisation of bacteriocins will contribute to expanding the knowledge in the field. With potential application of such bacteriocins, specifically in the food industry as food preservative or health care sector, as further encouragement in this field. A combinatorial approach was adopted for the in silico gene mining of organisms of interest, alongside the in vitro studies. In silico screening methods included the use of bacteriocin genome mining tool version 4 (BAGEL4), National Centre for Biotechnology Information (NCBI) and Basic Local Alignment Search Tool (BLAST) for rapid identification of putative operons within the genomes of selected producer organisms. Initial in vitro screening of class IIa LAB from our local culture collection was conducted through bioassays. This included the colony-spot, spot-on-lawn, and microtiter plate growth inhibition assays, to monitor inhibitory activity. A producer organism was selected for subsequent isolation and purification studies. Inhibition zones surrounding colony spots were compared for a broad range of indicator organisms, including Listeria monocytogenes (L. monocytogenes), L. plantarum Ta10c, Micrococcus luteus (M. luteus), Staphylococcus aureus (S. aureus), Streptococcus milleri (S. milleri), and Enterococcus faecalis (E. faecalis). The highest sensitivity was observed towards L. monocytogenes. Further, another bioassay was used to confirm initial colony-spot and spot-on-lawn assays results. This included the microtiter plate growth inhibition assay providing inhibition results from continuous monitoring, instead of an end-point result. Production of bacteriocins was optimized with regards to type of media, pH and temperature. The optimal conditions were found to be in Man, Rogosa and Sharpe (MRS) medium (Neogen, USA), pH of 7, and at a temperature of 37°C. Genomic DNA (gDNA) of the producer organism was extracted, amplified through PCR with universal 16S rDNA primers, and the amplicons sequenced. A BLAST search was conducted for homology comparison to the NCBI DNA database, to confirm the producer organism as L. plantarum. Purification of bacteriocins was initiated with the use of Amberlite XAD-16N, for separation based on hydrophobic interactions; followed by cation exchange chromatography, for separation based on ionic charge; and, reversed-phase high-performance liquid chromatography (RP-HPLC), for separation based on hydrophobicity. Mass Spectrometry analysis at each purification step revealed three prominent peaks at 789, 1124.6282 and 2185 Daltons (Da). These display potential masses of bacteriocins as they fall within the range of molecular masses linked to inhibitory activity observed.
Characterisation and formulation of natural cyclodecapeptides with anti-Candida activity
(Stellenbosch : Stellenbosch University, 2021-03) Masoudi, Yasamin; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The increase in the number of fungal infections since the 1980s is a major concern globally. Fungal infections affect various medical and agricultural sectors. A limited number of available antifungal drugs in addition to the development of the antifungal drug resistance, urge the need for the development of new antifungal drugs. Candida albicans and Candida glabrata are the two most common causes of topical and systemic Candida infections, and specifically prone to drug resistance. Therefore, the next generation of antifungal drugs has to be compounds with a low probability to elicit resistance. The tyrocidines (Trcs) and analogues are cyclic decapeptides with a broad antimicrobial spectrum and proven anti-Candida activity. The development of resistance is less likely due to their rapid mode of action and multiple targets. The Trcs and analogues have agreat tendency to oligomerise and the antimicrobial activity of the Trcs may be dependent on their ability to form active oligomers. In this study, we aimed to manipulate the Trc oligomerisation by combining it with cellulose derivatives as formulants. The goal was to increase the anti-Candida activity as well as the stability of theses peptides in solution. Furthermore, if a concomitant decrease in the toxicity against human erythrocytes would alsobe beneficial. The oligomerisation profile of Trcs is driven by four main factors, namely, concentration, maturation time, peptide structure and the viscosity of the cellulose derivatives used in the formulation. The oligomerisation of Trcs is a dynamic arrangement and rearrangement of the peptide in the Trc mixture throughout the maturation period. For the more viscous cellulose-type formulants, this rearrangement resulted in the release of the more dimeric oligomers and increased the anti-Candida activity of the Trc mixture, However, for pure peptides such as TrcA and TpcC, both formulation and the maturationtime did not alter the anti-Candida activity. This could be that TrcA oligomers are highly stable and maturation time does not result in releasing of more active moieties. Tic is inherently less active, and it may not have the optimal structure to interact with the Candida target. It was observed that a high concentration of cellulose derivatives significantly increased the anti-Candida activity of the Tic mixture, as well as stabilising the tic peptides in solutions, as detected with fluorescence. Unfortunately, none of the cellulose formulations of try mix decreased the haemolytic activity against human erythrocytes. However, anti-Candida activity was maintained and/or improved, as the solution stability of the peptides in the Trc mixture was improved.
The characterisation of the catalytic activity of human steroid 5α-reductase towards novel C19 substrates
(Stellenbosch : Stellenbosch University, 2015-02-25) Quanson, Jonathan Luke; Storbeck, Karl-Heinz; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: This study describes: • The UPLC-MS/MS analyses and quantification of novel 5α-reduced steroids using response factors. • The kinetic characterisation of human steroid 5α-reductase type 1 (SRD5A1), expressed in HEK-293 cells, towards 11OHA4 and 11OHT and their keto derivatives by progress curve analysis. • The subcloning, transformation and functional expression of SRD5A1 in the yeast expression system, P. pastoris. • The conversion of 11OHA4 and 11OHT and their keto derivatives by SRD5A1 expressed in P. pastoris. • The endogenous enzymatic activity in P. pastoris towards the 5α-reduced metabolites in the 11OHA4- and alternate 5α-dione pathways. • The potential application of P. pastoris as a biocatalyst in the production of 5α- reduced C19 steroids.
Characterising the gut microbiome of ostrich chicks reared under intensive conditions
(Stellenbosch : Stellenbosch University, 2020-12) Heitmann, Sinjon; Botes, Annelise; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Every year the ostrich industry suffers severe losses from the high mortality rate of intensively farmed ostrich chicks during early post-hatch development. One of the major contributors to the high mortality is enteritis, an enteric disease that stems largely from microbial imbalance. Efforts to reduce and prevent enteric diseases in ostrich chicks requires in part an extensive understanding of the changes in microbial composition within gastrointestinal tract (GIT). This study characterises the successional development of the microbiota present in the GIT of ostrich chicks reared under intensive conditions within the first three months post-hatch. In targeting the microbiota present in the small intestine, caeca, colon and faeces, the changes in bacterial composition and abundance provide insights unique to its development in the gut region and the development of the GIT. To achieve this, samples were taken from three ostrich chicks at five time points (15 chicks). For each time point the samples per gut region were pooled, microbial genomic DNA extracted and used for 16S metagenomic sequencing on the Ion Torrent platform. To improve definition at lower taxonomic levels, seven of the nine hypervariable regions in the 16S rRNA gene were sequenced. The raw sequence data was processed, and the bioinformatic analyses performed using the Ion Reporter software. Analyses of the gut regions over time found a progressive increase in bacterial diversity and stability despite the presence of both colonisation and extinctions events. Initial colonization of the GIT by week 2 coincided with the change in nutritive source from yolk to feed, and with it the introduction of a wide range of taxa including members from the Firmicutes, Bacteroidetes, Proteobacteria and Tenericutes phyla. Yet the changes in bacterial composition and abundance over time were not uniform between gut regions. The small intestine and colon regions were found to have substantial dissimilarities to remaining gut regions from week 0 - 4 and week 6 - 12, respectively. The changeover from small intestine to the colon was marked by the chronological shift of some species such as C. butyricum, C. disporicum and T. sanguinis, and with them the localised proliferation of potentially pathogenic species. The movement of C. butyricum away from the small intestine may remove its protective influence and allow the opportunistic proliferation of pathogenic species. The changeover between the small intestine and colon correlated both with the change in diet, as a part of the intensive rearing system, and the development of the colon into a more efficient fermentation chamber. Furthermore, the developed colon did not present the greater abundance of fibrolytic species from the Ruminococcaceae or Bacteroidaceae families as anticipated. Rather, a greater abundance of fibrolytic species from the Clostridiaceae family were present such as C. butyricum, C. chartatabidum, C. disporicum and C. paraputrificum, which suggest an accumulation of resistant starches and starch components in the colon. Furthermore, differences in bacterial composition were established in the core microbiota of the different gut regions, which shows that faecal samples do not provide a complete representation of GIT microbiota. Ideally the gut regions should be examined individually and together to understand the full impact that changes in diet have on the GIT. An examination of the distribution of relative abundance data may serve as a reference in adapting feeding strategies and strategies to manage GIT infections in intensively reared ostrich chicks.
The characterization of 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2) activity towards novel C19 substrates.
(Stellenbosch : Stellenbosch University, 2017-03) Barnard, Monique; Storbeck, Karl-Heinz; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Castration resistant prostate cancer (CRPC) is an androgen dependent disease driven by the intratumoural metabolism of adrenal androgen precursors to potent androgens. The alternative 5α-dione pathway converts the adrenal steroids DHEA and androstenedione (A4) to the potent androgen DHT, while the recently identified 11β-hydroxyandrostenedione (11OHA4) pathway converts the adrenal steroid 11OHA4 into the potent androgens 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). Two 17β-hydroxysteroid dehydrogenase (17βHSD) enzymes catalyse vital reactions in both pathways. 17βHSD2 catalyses the oxidation of androgens to their less active form, while 17βHSD5, which is better known as AKR1C3, catalyses the reduction of weak androgens to more potent androgens. The relative activity and expression levels of these enzymes are therefore vital in regulating the amount of active androgen produced. The aim of this project was to characterise the activity of 17βHSD2 towards 11-oxygenated steroids from the 11OHA4 pathway and to investigate the effect of different ratios of AKR1C3 and 17βHSD2 on the flux through the alternative 5α-dione and 11OHA4 pathways. 17βHSD2 activity towards the 11-oxygenated steroids 11KT and 11KDHT were characterised for the first time using HEK293 cells transiently transfected to express 17βHSD2. The results showed that 17βHSD2 efficiently catalysed the conversion of 11KT and 11KDHT to their respective products. Interestingly the catalytic efficiency tended to be higher for the conversion of testosterone (T) to A4 than the conversion of 11KT to 11KA4, although this difference was not significant. The effect of increasing ratios of AKR1C3:17βHSD2, which occur during CRPC, were subsequently investigated. HEK293 cells, which do not endogenously express either AKR1C3 or 17βHSD2, were transiently transfected to express each enzyme, and the cells subsequently combined in different ratios. PC3 cells, which endogenously express 17βHSD2, were transfected with increasing amounts of AKR1C3 to obtain different AKR1C3:17βHSD2 ratios. Collectively the results showed that increased expression of AKR1C3 had a significant influence on the flux through the 11OHA4 pathway, leading to the production of more potent androgens, but had little or no effect on the classical pathways. Increasing AKR1C3:17βHSD2 expression in both HEK293 and PC3 cells lead to increased levels of 11KA4 being converted to 11KT while the conversion of A4 to T remained low. A mathematical model was subsequently constructed and confirmed the experimental findings. These results were further validated in three prostate cancer cell lines each expressing different AKR1C3:17βHSD2 ratios. Taken together, the results from this study show that 17βHSD2 likely plays an important role in regulating intratumoural androgen levels and that increased AKR1C3:17βHSD2 ratios favour the flux through the 11OHA4 pathway.

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