- ItemInvestigation into the assimilation of free amino nitrogen from bread waste during yeast proliferation(Stellenbosch : Stellenbosch University, 2023-12) Botha, Dewald Du Plessis; Stefan, Hayward; Strauss, Eric; Tait, Timo; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Bread is a staple food in many economies. It is nutrient rich, cheap, and modern bakeries provide it in excess to be able to meet supply within stringent quality control parameters. Overproduction and consumer underutilisation, however, often result in accumulated bread going to waste if suitable valorisation methods are not available. Yet, unused bread is often returned to production facilities unaltered and may easily be processed to a dried commodity with a stable shelf life. Dried bread crumb is especially applicable to the agricultural industry as a feed additive, but it is also attractive in the biotechnology sector to sustain microbial growth for industrial and pharmaceutical applications. Ethanol is an attractive end-product due to its applicability in various industries, whether related to food and beverages, chemicals, or pharmaceutical sectors. This study therefore aims to establish a process to liberate sugars and free amino compounds from bread waste, relevant to alcoholic fermentation in yeast. Dried bread waste was processed enzymatically to hydrolyse polysaccharides to fermentable sugars and, following fermentation and distillation, alcohol yields of 10% alcohol by volume (ABV) was achieved. Moreover, high performance liquid chromatography coupled to UV/Vis detection was used to establish an analytical method for the quantification amino acids. Analysis of these nitrogen containing compounds revealed that a substantial amount of protein and essential amino acids are left following distillation to support a secondary fermentation. After proteolytic digestion of the dried wash, a nutrient rich mixture was obtained that promoted and supported the growth of yeast in the presence of a suitable carbohydrate source such as sucrose. This nutrient source was shown to be comparable to conventional commercial alternatives. Although the crude yeast nutrient resulted in slower exponential phase it ultimately proved to be superior in sustaining greater yeast biomass. This thesis describes the successful generation of a yeast nutrient from a bread waste product which was obtained after valorisation of bread waste in a bioethanol production process, thus creating a solution to bread waste that may with further development be waste free.
- ItemResisting the molecular evolution of fitness: an investigation into the role of ploidy in the perturbed adaptability observed in an HTZ1 knockout(Stellenbosch : Stellenbosch University, 2023-12) Reid, Jessica Laura; Patterton, Hugh; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Shifts in ploidy or gaining and or loss of a set of chromosomes, have occurred in the evolution of many fungi, plant, and animal species and is thought to contribute to speciation. Despite the important role of ploidy in evolution, little is known about how it contributes to adaptation and speciation. Previously it was shown that deletion of the histone variant Htz1p in S. cerevisiae resulted in perturbed evolvability under oxidative stress. Considering the histone variant’s documented roles in mechanisms such as genome integrity and chromosome segregation, this study sought to uncover if the perturbed evolvability of the strain might be caused by increased ploidy. To address this, we employ flow cytometry and ~300 generations of experimental evolution to analyse the DNA ploidy levels in several S. cerevisiae strains. Initial interest was given to two HTZ1 null mutants, one commercially available (Y11703) and another newly synthesized (PB1012). It was expected that both strains would exhibit similar, near-diploid phenotypes. However, it was found that only Y11703 exhibited the expected diploid phenotype. In parallel, we tested whether the reintroduction of the HTZ1 gene into a null mutant background would revert the cell to its parental ploidy state. To investigate this, two HTZ1 recovery strains were synthesised to observe the effects of introducing the gene in Y11703 under its native promoter and the inducible GAL1 promoter. These two strains presented little deviation from the flow cytometry behaviour exhibited by Y11703, presenting no indication of any reduction in its DNA ploidy level, but, unexpectedly, a slight increase in some cases. Although unexpected, this study adds to the intriguing relationship between Htz1p and ploidy alterations in S. cerevisiae. However, more research is needed to elucidate the mechanisms underlying these observations more completely. Future investigations may explore the molecular pathways connecting Htz1p to ploidy maintenance and its impact on evolvability. Understanding these processes may provide valuable insights into the broader context of eukaryote adaptation and speciation.
- 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.
- ItemInvestigating the effects of glucocorticoids and pro-inflammatory cytokines on oxidative stress in the HepG2 cell line(Stellenbosch : Stellenbosch University, 2023-03) Petersen, Ashlin Veruscka; Verhoog, Nicolette; Louw-Dutoit, Renate; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia, hyperinsulinemia, and the inability of insulin-target tissues (the liver, adipose tissue, and skeletal muscle) to respond to insulin. If target tissues are unable to respond to insulin to maintain glucose homeostasis, insulin resistance develops, resulting in the onset of T2DM. Furthermore, oxidative stress is defined as an imbalance of reactive oxygen species (ROS) and antioxidants linked to obesity, cancer, and T2DM. Organelles such as mitochondria and transmembrane proteins such as NADPH oxidases (NOX) generate ROS. NADPH oxidase 3 (NOX3)-derived ROS has been linked to insulin resistance in the liver, which was investigated further in this project. In contrast, the antioxidant system is important in eliminating ROS and nuclear factor erythroid- 2 related factor 2 (NRF-2) was investigated as it is the most abundant antioxidant transcription factor in the liver. Glucocorticoids, which are associated with the stress response, and inflammatory mediators such as pro-inflammatory cytokines impair insulin signalling. However, the underlying cause of this impairment is not fully understood and therefore the primary aim of this study was to examine if oxidative stress contributes to stress- and inflammation-induced insulin resistance, either via NOX3-derived ROS or by affecting the antioxidant system through NRF-2 regulation. The findings showed that the synthetic glucocorticoid dexamethasone and the pro-inflammatory cytokines tumour necrosis factor- alpha (TNF-α) and interleukin-6 (IL-6) impair insulin signalling at different exposure times. Dexamethasone and TNF-α both increased ROS production (in the absence and presence of insulin). While IL-6 only induced ROS production in the presence of insulin only. The increase in ROS production was not accompanied by an increase in NOX3 mRNA or protein levels. The downregulation of NOX3 mRNA expression was observed in response to TNF-α in the absence and presence of dexamethasone. Similarly, IL-6 together with dexamethasone in the presence of insulin upregulated NOX3 mRNA expression. Additionally, NRF-2 expression, which is inhibited by glycogen synthase kinase-3 beta (GSK-3β), was not affected by any of the treatment conditions. To conclude the findings of this study, indicate that although glucocorticoids and pro-inflammatory cytokines cause hepatic oxidative stress, whether this ROS is produced via NOX3 or if the antioxidant system via NRF-2 plays a role is unclear and remains to be elucidated.