http://hdl.handle.net/10386/69 2026-04-05T15:27:11Z http://hdl.handle.net/10386/5387 A low flow-high flow investigation into the effect of selected micronutrients on pre-determined innate immune biomarkers in two freshwater fish species from Witbank dam, Mpumalanga Radzuma, Murendeni Unarine Sandra The aquatic environment is constantly in jeopardy since it is the ultimate recipient of waste disposal. Mining, industrial and agricultural developments have led to a significant increase in, amongst others, heavy metal contamination in aquatic systems. The aim of this study was to use seasonal surveys to investigate the impact of Fe, Cu, and Zn levels on immune modulation in Cyprinus carpio and Micropterus salmoides collected from Witbank Dam, Mpumalanga. This study consisted of two surveys that were conducted during low flow periods in June and high flow periods in November. Temperature, pH, dissolved oxygen, conductivity, total dissolved solutes (TDS) and salinity were measured using YSI multiparameter instrument. A litre of water and sediment samples were collected from three different sites, while 26 common carp and 12 largemouth bass were sampled using conventional fishing gear. For each fish, the weight and length were measured. Furthermore, epidermal mucus and blood samples were collected. Serum and mucus samples were divided, half was treated with PBS and the other with protease inhibitor cocktail and stored at -20°C until further analysis. Gills, liver, and muscle tissue were dissected, weighed, rinsed, and frozen at -85°C for later analysis of Fe, Cu, and Zn levels by Waterlab (Pty) Ltd, Pretoria. From the results, it was discovered that Witbank dam is polluted by Cu and Zn. Furthermore, Cu concentrations were only detected in the liver of both common carp and largemouth bass. Iron and zinc were detected in varying concentrations across tissues, with Fe levels being highest in the liver and gills, particularly in largemouth bass, and Zn levels being highest in the gills of common carp. The use of the protease inhibitor cocktail was found to significantly preserve CRP levels, with treated samples showing higher concentrations compared to untreated samples, indicating its effectiveness in preventing protein degradation. The study highlights the need for pre-analytical care in feral studies to ensure accurate biomarker measurements and highlights the seasonal fluctuations in metal exposure, which could influence immune responses in fish. The recommendations of the study include implementing regular monitoring of metal concentrations in aquatic ecosystems and considering the use of protease inhibitors in future studies involving immune biomarkers. The study was limited to a small sample size and one sampling area. Thesis (M.Sc. (Physiology)) -- University of Limpopo, 2025 2025-01-01T00:00:00Z http://hdl.handle.net/10386/5347 Geology, geochemistry and occurrence of rare earth elements in coal from the Soutbansberg West Coalfield (Mopane Sector). Limpopo Province, South Africa Ntake, Mahlatse Manthepeng The supply security of rare earth elements (REE) is a global key concern due to their scarcity and increasing demand. Consequently, the quest for alternative sources of REE is pivotal for countries that are reliant on REE imports, including South Africa. This search for new alternative non-conventional sources of these critical elements has sparked a heightened interest in coal deposits as the new alternative source. The Soutpansberg Coalfield of South Africa hosts substantial coal deposits. However, there is a gap in understanding the geology and geochemistry of the coals as well as the rare earth-bearing potential of this coalfield. Therefore, this study aims to investigate the geology, geochemistry and occurrence of rare earth elements in the coal stockpiles within the Mopane Sector of the Soutpansberg Coalfield. To fulfil the purpose of this study, coal petrography, proximate analysis, ultimate analysis, x-ray diffraction, scanning electron microscopy-energy dispersive spectrometry and inductively coupled plasma-mass spectrometry were used. Most of the coal stockpiles were ranked as high-volatile bituminous B-A coals distinguished by their high vitrinite, moderate to low liptinite and low inertinite macerals. Geochemically, the coals contained low moisture content with high ash yields and moderate volatile matter. Additionally, the coals contained low sulphur with high oxygen and carbon contents, corroborating their coke properties. The mineralogical analysis of the coals confirmed that the coals were predominantly composed of organic carbon (24% to 73%), quartz, kaolinite, plagioclase, siderite and other minerals. These coal stockpiles were further confirmed to contain the renowned rare-earth bearing minerals, including xenotime and monazite, associated with clay, quartz and other minerals. Several REE were detected within the samples, including Er, Tm, Ho, Gd, Yb, Lu, Sm, Eu, Tb, Pr, Dy, Nd, Ce, La, Sc, and Y, with the predominant REE being Ce. The total REE within each stockpile varied between 230 ppm and 670 ppm, with the stockpiles being enriched in light REE (2166.67 ppm) and depleted in heavy REE. Furthermore, the coal stockpiles exhibited high yields of excessive REE compared to the critical and uncritical REE. The stockpiles are of a sedimentary source with felsic and terrigenous associations. Ce and Eu anomalies suggest that the enrichment of the coals is of a detritic origin, which might be from the Limpopo Mobile Belt and the Beit Bridge Complex. The normalized REE concentrations of the samples were compared with those of Chinese, USA, UCC and MKB coals and they were found to be higher than all the aforementioned deposits. In addition, the coals fall within the promising regions for the economic extraction of the REE. Thesis (M. Sc. (Geology)) -- University of Limpopo, 2025 2024-01-01T00:00:00Z http://hdl.handle.net/10386/5198 Examination of the effects of HIV protease inhibitors (HIV-PIs) on the function of the novel s-DAPK-1 in Human Papilloma Virus (HPV)-related cervical cancers Makgoo, Lillian Cervical cancer treatment continues to be every country’s nightmare due to ineffectiveness and non-specificity of the current therapeutic options, late diagnosis and chemo drug resistance. An escalating resistance of cervical cancer cells to chemotherapy coupled with severe side effects of commonly used cytotoxic drugs has intensified the need to search for new anti-cancer agents. Several drugs initially approved for non-cancerous conditions have recently been found to possess cytostatic effects on cancer cells. Thus, these drugs could be expediently repurposed for use as anti-cancer agents because they have already been tested for safety in animals and humans. In light of this, this study sought to investigate the possibility of adapting pure HIV protease inhibitors (HIV-PIs) and their over-the counter tablets for anti-cervical cancer therapeutic purposes. Additionally, since cervical cancer is viewed as a pathology that is partly driven by genes, it was of interest to understand the expression of short-DAPK-1 known as s-DAPK-1, which remains unexplored in cervical cancer, and the HIV-PI s' mechanisms of action. Therefore, this study was aimed at investigating the effect of HIV-PIs on s-DAPK-1 and other cancer-related genes in HPV-induced cervical cancer cells. To address the aim of this study, the MTT viability and Muse™ Count & Viability assays were used to evaluate the effect of the pure HIV protease inhibitors and their tablet forms on the viability of CaSki and HeLa cervical cancer cell lines, as well as on the non-cancerous cells (HEK-293). To detect the mode of death induced by pure HIV-PIs (lopinavir and atazanavir) and their tablet forms (Aluvia and Ritoataz) in HPV-associated cervical cancer cells, apoptosis was assessed using the Annexin V Assay. Apoptosis-related proteins regulated by HIV-PIs and their tablet forms were detected using the Human Apoptosis Array profiler. In addition, the Muse™ Cell Cycle assay was used to assess the effect of HIV-PIs and their tablet forms on cell cycle progression of the cervical cancer cells. The Polymerase Chain Reaction (PCR) was used to determine the expression of s-DAPK-1 in HIV-PIs-treated and untreated cervical cancer cells, and to elucidate the effect of pure HIV protease inhibitors and HIV protease inhibitor tablets on its expression. In addition to the use of PCR, the proteome profiler human apoptosis array kit was used to detect other cellular targets of pure HIV-PIs and their over-the counter tablets in HPV-associated cervical cancer cells. Furthermore, various bioinformatics tools such as ProtScale, ProteinPrompt, I-TASSER, PSIPRED, ProtParam, ScooP, Hawkdock, Phyre2, SAVES and PROCHECK along with user-friendly databases such as NCBI, TarBase and Protein Data Bank (PDB) were used to understand s-DAPK-1 regulation, 3D structure, physicochemical and thermodynamic properties. This study demonstrated that lopinavir and atazanavir pure HIV-PIs, as well as Aluvia and Ritoataz tablets did not affect the viability of non-cancerous cells (HEK-293), confirming the safety of the HIV-PIs. However, they have significantly decreased the viability of the CaSki and HeLa cervical cancer cells in a dose- and time-dependent manner. It is important to note that, relative to HeLa cells, a higher concentration of lopinavir (IC50=150 μM) and Ritoataz (IC50=180 μM) was required to reduce the viability of CaSki cells by 50%. All the HIV-PIs triggered apoptosis in CaSki and HeLa cervical cancer cells. However, lopinavir (39.925±1.483 in CaSki and 41.583±1.001 in HeLa) and atazanavir (49.092±0.9376 in CaSki and 36.717±1.729 in HeLa) significantly (p ≤ 0.001) exhibited the highest induction of apoptosis. In contrast, the corresponding tablets of Aluvia (24.418± 2.346 in CaSki and 26.795±0.6805 in HeLa) and Ritoataz (25.310±1.323 in CaSki and 28.432±2.374 in HeLa) induced a significantly (p ≤ 0.001) lower levels of apoptosis. In addition, pure HIV protease inhibitors along with their tablet forms significantly (p ≤ 0.05) regulated the activity of various apoptosis-related proteins, including phosphorylation p53 (S392) and Rad17 (S635). The HIV-PIs upregulated SMAC/Diablo, and Bcl-2, suggesting induction of an intrinsic apoptosis pathway, with cervical cells resisting cell death by upregulating Bcl-2. The s-DAPK-1 variant was significantly downregulated in HeLa cells relative to non-cancerous HEK-293 cells, suggesting that it may be a tumour suppressor. In addition, pure HIV protease inhibitors and the HIV protease inhibitor tablets did not influence the expression of s-DAPK-1 in cervical cancer cells. Furthermore, the in-silico approach, to determine s-DAPK-1 regulation, successfully identified several s-DAPK-1-specific microRNAs. In addition, phyre2 database demonstrated that the s-DAPK-1 isoform possesses 40% alpha helices and 4% beta strands, forming a stable 3D structure. Moreover, s-DAPK-1 was discovered to withstand high temperatures and to interact with a variety of proteins involved in tumour progression and gene regulation, including Prion protein and Histone H2B type 2-E (H2B2E). The findings of this study highlight the HIV protease inhibitors as promising anticancer agents, demonstrating significant effects on inducing cell death and suppressing proliferation. Furthermore, this work has discovered more anticancer drug targets that should be exploited for drug development. Moreover, since this is the first study to explore the expression and regulation of s-DAPK-1 by therapeutic agents, there is a pressing need to identify novel compounds that can modulates s-DAPK-1 and to explore its potential tumour suppressor function in cancer cells. Thesis (Ph. D. (Biochemistry)) -- University of Limpopo, 2025 2025-01-01T00:00:00Z http://hdl.handle.net/10386/5150 Exploration of the regulation of immunomodulatory genes by commelina benghalensis aqueous extra in breast cancer cells Mnisi, Hellen Laura Cancer is a noteworthy global health issue, and according to predictions by the World Health Organization, the number of new cases was projected to increase to approximately 29-37 million by 2040, worldwide (WHO, 2020, Sung et al., 2021). Female breast cancer has been shown to be the leading cause of global cancer incidence in 2020, with reported 2.3 million new cases, representing 11.7% of all cancer cases (Reyes-Monasterio et al., 2022). Treatments such as surgery, radiotherapy, and chemotherapy are used to counteract breast cancer; however, these treatment approaches are now ineffective, which has led to the shift of interest towards the use of medicinal plants such as Commelina benghalensis (Cb), which has been traditionally used in folk medicine to treat various ailments, including inflammation-related conditions. Studies reveal that the plant contains bioactive compounds, such as flavonoids, tannins and phenolics, which possess anti-inflammatory, antioxidant, and antitumor properties (Islam et al., 2018; Islam et al., 2017; Islam et al., 2016). Thus, the aim of the study was to determine the antioxidant and anticancer properties of Cb aqueous extract in breast cancer cells. Breast cancer cell lines, MDA-MB 231 and MCF-7, were cultured and maintained in appropriate growth media. The Cb was extracted using only water, and this was done to mimic what the traditional healers do, when they prepare medicinal plants concoctions for medicinal purposes. The profiling of the bioactive compounds was done using Thin Layer Chromatography (TLC), phytochemical screening, and the Liquid Chromatography-Mass Spectrometry (LC-MS). The total amount of phenols, flavonoids and tannin in the Cb aq-extract was quantified using the Folin-Ciocalteu Colorimetric Assay. Additionally, the antioxidant activity was done using 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and free radical antioxidant power (FRAP) assays. Furthermore, the cytotoxicity effect of Cb aqueous extract was determined against the breast cancer using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The Muse® Cell Count and Viability assay was done to confirm the MTT results. Morphological changes were observed using light microscopy and the images were captured using computer connected to microscope. Moreover, Annexin V assay was conducted to investigate the potential cell death that may be induced by the Cb leaf-aq extract. The TLC plates demonstrated that the Cb root, leaf and stem-aq extract have bioactive molecules. The quantitative phytochemical analysis using Folin-Ciocalteu Colorimetric Assays, showed that the Cb aq extracts had high phenolic, flavonoid and low tannin contents Folin-Ciocalteu Colorimetric Assays. Moreover, the Cb aq-extracts demonstrated antioxidant properties, and this was mostly observed in the leaf and the root parts of the plant. The LC-MS results of Cb leaf-aq extract demonstrated the presence of bioactive compounds that have been previously shown to exhibit anticancer cancer immunomodulatory activities. In this study, three compounds with immunomodulatory effects; namely, Abietic acid, Vorinostat and Lauramide, were identified. These compounds regulate a number of cytokine genes, and these include TGF-b1, IGF1R, IFN, IL-2, IL-4, IL-10, IL-13. Additionally, the Cb leaf-aq significantly (**P ˂ 0.01, ***P ˂0.001 and ****P ˂0.0001) reduced the viability of MDA-MB 231 cells after 24- and 48-hours treatment with 500 and 1000μg/ml compared to other parts of the plant. Furthermore, the Cb leaf aqueous extract significantly (****P ˂0.0001) induced apoptosis of MDA-MB-231 cells after their treatment with the IC50 concentration (750μg/mL). These findings collectively support the conclusion that the phytocompounds identified in Cb have anticancer properties and a potential to modulate immunological responses in breast cancer cells. Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2025 2025-01-01T00:00:00Z