3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item An investigation into the regulation of peroxidasin gene expression by the SNAI1 transcription factor and its involvement in epithelial-to-mesenchymal transition in human cervical carcinoma cell lines(2018) Moleya, Boitumelo NonhlanhlaPeroxidasin (PXDN) is a unique member of the peroxidase - cyclooxygenase superfamily and differs from its counterparts in that in addition to an enzymatic peroxidase domain, PXDN has protein-binding domains characteristic of proteins found in the extracellular matrix. PXDN is integral to basement membrane consolidation and catalyses sulfilimine bonds in collagen IV and covalent cross-linking of dityrosine. PXDN also has microbicidal activity in plasma through the generation of hypochlorous acid and may also catalyse peroxidative reactions intracellularly. Additionally, PXDN is involved in processes where epithelial-to-mesenchymal transition (EMT) takes place, namely fibrosis, development and cancer. In cancer, PXDN has been found to be aberrantly expressed in a colorectal cancer cell line undergoing p53-dependent apoptosis, in malignant primary glial and metastatic brain tumours; and was identified as a candidate tumour suppressor gene silenced by DNA methylation in patients diagnosed with acute myeloid leukemias. PXDN has also been implicated in tumour invasion of choriocarcinoma and melanoma cells. Considering PXDN expression can be modified by transforming growth factor β1 (TGF-β1), a major signaling molecule involved in the initiation of EMT, in this study we aimed to investigate the role of PXDN in TGF-β1-induced EMT in human cervical cancer cells. This included i) identifying whether Snai1, the main transcription factor involved in EMT, regulates the expression of PXDN; and ii) whether PXDN contributes to the various aspects of EMT in cervical carcinoma cell lines. During TGF-β1-induced EMT, PXDN expression decreased in HeLa and SiHa cells, with concomitant increases in Snai1 and vimentin, and decrease in E-cadherin, as quantified by western blotting and visualised by immunofluorescence microscopy. We showed that TGF-β1 induced Snai1 binding to the PXDN promoter, as assessed by chromatin immunoprecipitation-PCR, and significantly repressed luciferase reporter gene expression, as did Snai1 over- expression. We also found that knocking down PXDN expression decreases proliferation, attachment and invasion of HeLa and SiHa cells and increases the migration rate of HeLa cells but decreases that of SiHa cells. In summary, our findings show that Snai1 mediates repression of PXDN and consolidates a role for this ECM-modifier during EMT, in particular during proliferation, attachment and migration of the cervical carcinoma cells, HeLa and SiHa.Item Evaluating the productivity of selected in vitro culture techniques for the production of a locally isolated entomopathogenic nematode(2018) Matlhabe, OfentseEntomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have gained interest as biocontrol agents of insect pests due to their ability to search and kill soil dwelling insects. Some members of the Oscheius genus have been shown to have insecticidal abilities as a result of their association with bacteria in the Serratia genus. This has led to the consideration of the Oscheius as an EPN genus. The biocontrol potential serves as an incentive for studying EPNs and various production methods for their commercial use as biopesticides. A putative EPN was isolated from soil samples collected at Brits in the North West Province of South Africa. 18S rDNA based identification indicated that it belonged to the Heterorhabditis genus however, phylogenetic analysis and symptoms on Galleria mellonella larvae suggested that the nematode may belong to the Oscheius genus. The bacterial symbiont associated with this nematode was found to be a strain of Serratia marcescens, through 16S rDNA based sequencing and phylogenetic analysis. Various in vitro production methods were evaluated for their effect in the production of Oscheius L8 MCB. These include monoxenic and axenic culturing, growth media supplementation, and production in solid and liquid state. Axenic cultures were found to produce high maximum yields of EPNs (78 600 EPNs/ml) compared to monoxenic cultures (53 833 EPNs/ml). It was concluded that axenic culturing was efficient for the production of this species. Oscheius L8 MCB was cultured in NB supplemented with varying concentrations of oil and glucose. Supplementation of NB with 2% canola produced a significant amount of EPNs in reduced culture times, but NB supplemented with 4% canola oil and 25mg/ml glucose increased nematode yield but prolonged the culture time. It is noted that media composition (with regards lipid and carbohydrate content) plays an important role in nematode yield and culture time and thus optimization of these components is critical for efficient nematode production. Solid state and liquid state production of Oscheius L8 MCB showed that solid state cultures allowed for early IJ production, whereas liquid state culturing produced the highest IJ yield (36 000 IJs/ml). The reduced culture period, makes solid state production more cost effective and preferable for mass production. However, liquid production can still be used as it offers the benefits of high nematode yeild and efficient recovery of nematodes from culture. The in vitro methods of EPN production have been reported to have an effect on the efficacy of the EPNs against insect hosts. Dose-response assays showed that in vivo produced EPNs resulted in high G. mellonella larvae mortality at lower concentrations compared to solid and liquid in vitro methods. Larvae infected with in vivo produced Oscheius L8 MCB produced a high number of emerging IJs compared to larvae infected with EPNs produced using axenic in vitro culturing methods. The differences between mortality and IJ emergence in larvae infected with solid state and liquid state cultured EPNs were marginal. Therefore, it is concluded that axenic culturing methods may reduce the efficacy of Oscheius L8 MCB against insect hosts.Item Efficiency of degrading packed bed bioreactors(2016) Botes, Anthin JohnIn South Africa, the need for water treatment is increasing, especially in the mining sector. As active water treatment technologies are expensive, the mining sector has an increasing need for passive water treatment technology, with low maintenance and operating costs, yet efficient water treatment ability. Literature on passive water treatment suggests that these systems only offer a narrow range of treatment capabilities. Therefore, hybrid water treatment systems could be a solution to low-cost water treatment in South Africa. The Degrading Packed Bed Reactor (DPBR) is one of the units comprising the hybrid treatment group. The DPBR’s main action is to convert sulfates into sulfides and alkalinity. In practice, the main drawback of the DPBR is clogging. Clogging lessens the amount of Acid Mine Drainage (AMD) that comes into contact with Sulfur Reducing Bacteria (SRB) in the DPBR, thereby reducing the efficiency of the bioreactor. In this study, six small-scale DPBRs were constructed. Each was classified according to its unique organic source (manure, straw, vegetable food processing waste, wood shavings, chicken litter and a combined sample with layers of all the carbon sources). Synthetic AMD was fed through the six bioreactors for a period of three months. From the small-scale DPBRs, the permeability, sulfate, iron and pH of the exit samples were measured. On average, the carbon sources removed 50 % of the sulfates and 98 % of the iron from the fed AMD. The different carbon sources showed no significant difference between each other in terms their sulfate and iron removal. The range between the best performing carbon source and the poorest performing carbon source, in terms of sulfate removal, was 17%. For iron removal, the range between the best and poorest performing carbon sources was only 2%. It was found that the permeability of the carbon sources played a larger role in the efficiency of the DPBR than the type of carbon source used. Manure is highly effective in terms of pH improvement, sulfate and iron removal. However, this is at the expense of permeability, as its packing clogs very rapidly. Compost and straw have excellent permeabilities which do not change significantly over long timeframes. This is, however, at the expense of the remedial ability of the packing materials. The combined reactor, in every instance, offers a good compromise between these different behaviours.