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- ItemStudies of mitophagy in mouse and fish models using antibodies and PCR(Stellenbosch : Stellenbosch University, 2023-03) Leukes, Kay-Lynn Amber-Marie; Bellstedt, Dirk; Loos, Ben; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Parkinson’s Disease (PD) is a neurodegenerative progressive movement disorder that affects aging populations. Characterized by various motor and non-motor symptoms, it affects the daily lives of approximately 7-10 million people globally. It has significant socio-economic and mental effects on patients, and although various treatment and management methods exist and are available, PD is incurable, emphasising a strong need for further and extensive investigation into the underlying molecular pathways that cause it. Mitophagy is a process crucial for the regulation of degradation of dysfunctional mitochondria through autophagy. It upholds cellular homeostasis; however, its dysfunction has been linked to the development of PD and other neurodegenerative diseases. Various model organisms exist for the study of aging, Nothobranchius fish being one of the more recently emerging models due to their short lifespan in addition to the fact that these fish begin to display Parkinson’s-like Disease symptoms as they age. Numerous genes have been identified in these fish that have been linked to the development of these symptoms. This project therefore aimed to produce antibodies against two well-known proteins that have previously been used as molecular markers to study autophagy and mitophagy – LC3 and p62. The production of these antibodies allows for possible future studies of mitophagy and its role in PD- development. With aging being a major risk factor for the onset of neurodegenerative diseases, this project also aimed to investigate mitochondrial DNA (mtDNA) degradation in two aging groups of Nothobranchius species by DNA isolation and PCR analysis to observe mtDNA degradation with age. In this study, recombinant His6-LC3 and p62-KLH conjugates were used to immunize rabbits. Antigen specific enzyme-linked immunosorbent assays were used to confirm the production of anti-LC3 and anti-p62 antibodies. The characterization of the anti-LC3 antibodies was accomplished by means of western blotting and immunofluorescence and they were found to recognize the unlipidated, or unactivated form of LC3, LC3-I and not the lipidated form, LC3-II, which limits their usage in mitophagy activation studies. In spite of this, through affinity chromatography, and MagReSyn magnetic bead purification with these anti-LC3 antibodies, native LC3 from mitophagy activated mouse fibroblast (MEF) cells lines could be isolated for future potential antibody production against activated LC3-II. Characterization of anti-p62 antibodies was accomplished by means of western blotting and immunofluorescence, which yielded positive results. The antibodies raised against p62 possessed the same specificity as commercially available anti-p62 antibodies and can therefore be used as valuable tools in future studies of mitophagy in MEF cells. Due to the fact that these antibodies, based on the similarity of epitopes, were specific for p62 Nothobranchius epitopes, these antibodies may be of particular value for immunofluorescence studies of Nothobranchius mitophagy in future. The investigation into mtDNA degradation in Nothobranchius fish was attempted by PCR analyses with a variety of primer pairs of aging Nothobranchius korthausae and Nothobranchius guentheri in comparison with appropriate control samples. Mitochondrial degradation based on a reduction in amplification of large fragments of mtDNA from aging fish was not observed and calls for further and more in-depth investigation.
- ItemMetal and tyrocidine nano-assemblies to create broadspectrum metal-peptide formulations(Stellenbosch : Stellenbosch University, 2023-03) De Villiers, Carmen; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Without the discovery or development of novel drugs, the phenomena of antimicrobial resistance (AMR) will continue to threaten the effective treatment of pathogenic microbes and diseases across the globe. Combinational therapies of antimicrobial peptides (AMPs) with metal nanoparticles (MNPs) have shown promise for the creation of potent nano- drugs with broad spectrum antimicrobial activity, alternative applications, and lowered risk of resistance development. In this study the combinational formulation of biologically relevant metals (magnesium, calcium, iron, copper, silver, gold, and zinc) with a group of natural antimicrobial cyclodecapeptides (CDPs) was investigated for the fabrication of potent AMP-MNP nanodrugs. The CDPs selected for this study include an aromatic residue rich peptide complex (tyrocidine mixture, Trc mix) and tryptophan rich purified analogues (tyrocidine C, TrcC and tryptocidine C, TpcC). Mass spectrophotometric studies revealed that these peptides form peptide-metal complexes with certain metals, and that the absence or presence of such complexes in formulations altered the peptides oligomerisation behaviour. Although formulations with group 11 metals lacked peptide- metal complexes, changes in peptide oligomerisation and analogue-specific prevalence was observed. Since the aromatic rich structure of these CDPs holds potential for synthesis of MNPs, it was hypothesised that the absence of peptide-metal complexes in group 11 formulations is likely due to the reduction of metal ions and formation of MNPs. This hypothesis was confirmed by spectrophotometric and spectrofluorometric studies which reported the formation of silver nanoparticles (AgNPs) in Trc mix, TpcC and TrcC formulations with silver. These studies also indicated alterations in peptide conformation when in formulation and highlighted the critical role of tryptophan for successful CDP- AgNP fabrication. Scanning transmission microscopy revealed that the peptide- synthesised spherical AgNPs were encapsulated by CDP nanostructures, a promising conjugate structure for drug delivery. Solid surface antimicrobial assays and reported additive and synergistic antimicrobial actions between CDPs and MNPs against model organisms, Gram positive Staphylococcus aureus and Gram-negative Escherichia coli, respectively. The innate self-assembly of these aromatic amino acid rich CDPs therefore holds potential to streamline the synthesis of potent yet versatile CDP-MNP nanoformulations for topical or antimicrobial surface treatments against Gram-positive and Gram-negative bacteria. Moreover, unprecedented antibacterial activity was also reported for TpcC, which could have applications in future therapies.
- ItemUnderstanding yeast glycolysis dynamics using HPLC analysis and mathematical modelling.(Stellenbosch : Stellenbosch University, 2023-03) Van Schalkwyk, Clara Elizabeth; Van Niekerk, David Douglas; Snoep, Jacob Leendert ; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Glycolysis is a universally conserved metabolic pathway that forms the backbone of carbon metabolism and energy generation. A comprehensive understanding of this pathway may lead to a more complete understanding of energy metabolism. A novel HPLC-based method was recently developed for the detection of metabolite, cofactor and coenzyme concentrations during metabolic studies. The purpose of the current study is to implement this novel method to generate data which can be used to eluci- date dynamics of glycolytic intermediates and cofactors in conjunction with a existing enzyme kinetic model of yeast glycolysis in Saccharomyces cerevisiae, a model organism for eukariotic biology. The HPLC method was used to study the dynamic behaviour of three key glycolytic intermediates in response to a glucose pulse in cell free extracts of S. cerevisiae. It was found that in response to a glucose pulse, glucose is consumed, fructose-1,6- bisphosphate accumulates prior to glucose depletion and ethanol accumulates. Sub- sequently, the dynamic behaviour of the adenine nucleotides in response to a glucose pulse was investigated. It was found that while glycolysis is active, AMP accumulates and ATP depletes while ADP remains at a constant low concentration. Once glucose is depleted ATP increases and AMP decreases. This is the result of the combined action of adenylate kinase, glycolytic enzymes and ATPases. Finally, the effect of phosphate on glycolytic intermediate dynamics and specific enzyme activity was investigated. It was found that phosphate influences the rate of glucose consumption and degree of F16BP accumulation. The inhibitory effect of phosphate on pyruvate kinase activity was char- acterised and incorporated into the model, however, the adapted model could not yet describe the observed phenomena and further investigation is required.
- ItemInvestigation of protein complexes involved in coenzyme A biosynthesis(Stellenbosch : Stellenbosch University, 2022-12) Sitzer, Warrick Lyle; Strauss, Erick; Woodward, Jeremy; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The biochemistry of the CoA biosynthetic pathway has been extensively studied and the differences in how the pathway functions between domains of life has led to its validation in recent years as target for drug development against many infectious disease-causing organisms. However, the structural organization of the CoA biosynthetic machinery remains to be investigated. Compelling evidence of a potential CoA-synthesizing protein complex (CoA-SPC) in yeast was recently demonstrated by pairwise interaction studies of the CoA biosynthetic enzymes from Saccharomyces cerevisiae which were found to interact with each other. In bacteria, the formation of a CoA biosynthetic enzyme complex has not been reported. However, the elucidation of the Mycobacterium smegmatis CoaBC (MsmCoaBC) structure and the recent findings showing CoaBC to be a vulnerable point in the CoA pathway suggested that it could form the basis for the formation of such complexes in bacteria. The first aim of this study focused on the isolation of the proposed CoA-SPC from S. cerevisiae and its characterisation by means of affinity pull-down assays. In this investigation, we were able to successfully overexpress yeast-related CoA biosynthetic proteins but unsuccessful in isolating the CoA-SPC from yeast lysate using pull-downs. High performance liquid chromatography (HPLC) was performed as an alternative means to assay for CoA formation by partially purified yeast protein samples. However, no detectable amounts of CoA were observed in the samples after repeated attempts. As CoA is readily detected within yeast lysates, we speculate that errors in either the preparation methods or assays procedure is a result that hindered our progress. Overall, as this complex has not yet been isolated before, the conditions that would lead to its formation could impact its overall isolation and therefore needs to be further explored. In the second aim, we explored the concept of a CoA biosynthetic complex in bacteria by investigating the structure of the bi-functional CoaBC of Staphylococcus aureus utilising single particle electron microscopy techniques. We were able to produce a low-resolution model of the S. aureus CoaBC (SaCoaBC) at ~20Å under negative stain conditions. Two distinct models were reconstructed with one accommodating the overall architecture of MsmCoaBC and the other significantly different along its vertices. 3D classifications were unable to clearly establish that the two models are a result of a true conformational change. A detailed single particle cryo-EM study is necessary to determine this, and it will also contribute greatly to the proposed idea of multiple conformations within the CoaBC structure.
- ItemModular control analysis and its application to glucose metabolism in plasmodium falciparum-infected erythrocytes(Stellenbosch : Stellenbosch University, 2021-12) Fisher, Joel Chanse; Snoep, Jacob Leendert; Van Niekerk, David; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Disease treatment is achieved through the administration of medication acting at the molecular level which results in changes at the physiological level. These changes are caused by molecular interactions which are usually complex and not well understood. Research often focuses on the mechanism of action of the drug as well as the specificity and binding affinity for the drug target. Such research is important, however, understanding and analysing the drug effects at the physiological level are equally important. The analysis and quantification of these effects can be difficult in large, complex systems. Consequently, such systems are mostly analysed from a broad perspective, often with the implementation of computational techniques including mathematical modelling. Analyses can then be performed using specialised mathematical frameworks, such as modular control analysis. These frameworks often focus on determining the control of groups of reactions (modules) and are usually complex, requiring detailed knowledge of the framework prior to implementation. To this end, modular control analysis was formulated in Mathematica and a software package was constructed to automate the application of the analysis framework. Use of the package was demonstrated on a model of glucose metabolism in Plasmodium falciparum- infected erythrocytes. The control of the whole parasite was determined, with the control of the parasite on the flux through itself and the infected erythrocyte determined to be near complete. Use of the package to analyse models with multiple modules was also demonstrated. In this way, use of the modular control analysis framework has been simplified, with only fundamental knowledge required to perform analyses with the software package.