Masters Degrees (Physiological Sciences)

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    The detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins
    (Stellenbosch : Stellenbosch University, 2023-12) Smith, Logan Jason; Essop, M. Faadiel ; Joseph, Danzil; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Psychosocial stress has strong links to numerous chronic diseases related to the dysregulated activation of the physiological stress system. This heightens the burden of mortality as there is a robust relationship between chronic psychosocial stress and non- communicable diseases. Hence there is a robust impetus for the identification of novel, circulating biomarkers to earlier detect stress-related chronic diseases. Although protein post-translational modifications such as glycosylation and phosphorylation can act as putative markers of pathophysiology, their relatively low abundance complicates extraction and identification from samples with a high dynamic range. The main aim of this study was therefore to establish two unique enrichment methods for circulatory glycoprotein and phosphoprotein extraction that would then be applied in a preclinical model of chronic psychosocial stress. Methods: Phosphoprotein enrichment was performed using functionalized magnetic particles while glycoprotein enrichment occurred using lectin-bound magnetic particles. Both these methods were tested using a known purified phosphorylated and glycosylated protein and compared to bottom-up proteomics methodology using rat serum. The latter was obtained from a rat model of chronic stress that is well-established in our laboratory (n = 16 controls versus n = 16 stressed rats). These were randomly selected for proteomics analysis to assess the efficiency of retrieval in enriched versus unenriched samples. Fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and proteins visualized using Coomassie and specific fluorescent staining. Here, the relevant Pro-Q stains were employed for the identification of glycosylation and phosphorylation, respectively. The coupling of such enrichment methods to LC – tandem mass spectrometry (MS/MS) was enabled by employing various preparation steps such as deglycosylation and digestion. An exogenous protein was also included as part of the sample preparation to ensure quality control analysis of the LC-MS/MS experiment. Results: SDS-PAGE analyses and staining methods revealed non-specific enrichment with regards to intact protein retrieval. In addition, LC-MS/MS data demonstrated that enrichment using the current set of affinity materials was inadequate for glycopeptide and phosphopeptide retrieval in serum. Conclusion: A lack of enrichment indicates that stringent sample preparation is needed for biological materials with a high dynamic range. This may also be due to the porous nature of both materials employed for phospho- and glycoprotein/peptide enrichment respectively. A combination of enrichment and/or depletion methods may therefore be beneficial for deeper analysis of the blood proteome. These enrichment techniques and the subsequent sample preparation still require further optimization to derive more definitive conclusions in the chronic stress context.
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    The tumour microenvironment: the effect of breast cancer cell conditioned medium on the endothelium
    (Stellenbosch : Stellenbosch University, 2023-03) Rass, Atarah Melanie Rose; Engelbrecht, Anna-Mart; Fourie, Carla; Marais, Erna; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Background: Breast cancer is the most common cancer diagnosed in women and the most common cancer globally. The human mammary gland is comprised of epithelium and vascular rich stroma. It has been established that breast cancer cells interact with and alter their stroma and neighbouring cells, to establish a tumour microenvironment (TME). Mammary endothelial cells are key targets to be transformed into tumour endothelial cells (TECs). These cells are genetically and phenotypically distinct from their normal, healthy counterparts and provide various pro-tumourigenic effects. These effects are modulated by the expression of various molecules that have been classified as TEC markers based on their expression in TECs compared to normal endothelial cells. As central role players in angiogenesis, TECs are key to tumour angiogenesis. Anti-angiogenic agents have proven to be effective, yet act as a double-edged sword, as a result of downstream complications and side effects. TECs therefore serve as potential targets for therapeutic intervention. Various role players in the tumour microenvironment have been investigated, but the effect of breast cancer cells on the tumour endothelial phenotype is not well established. The aims of this study were to evaluate a TEC phenotype in breast cancer and investigate how breast cancer impacts angiogenesis. Methods: Conditioned medium (CM) was harvested from non-malignant (MCF-12A) breast epithelial cells and from malignant (MCF-7 and MDA-MB-231) breast cancer cells starved of supplements and growth factors for 24 hours. Endothelial cells (HUVECs) were then treated with CM for 24 hours. To evaluate a TEC phenotype in breast cancer, cell viability (WST-1 assay), cell morphology (phase contrast imaging), and gene (reverse transcriptase-quantitative polymerase chain reaction) and protein (Western blots) expression of markers associated with a TEC phenotype were assessed. To assess angiogenesis in breast cancer, cell migration (scratch assay) and tube formation (tube formation assay) assays were utilised. A comparative model of non-malignant versus malignant signalling was used throughout the study. Results: Breast cell CM significantly increased HUVEC cell viability in all treatment groups. Changes in morphology were observed, which included elongation and branching, and occurred to a greater degree in malignant CM groups. TEC markers were significantly upregulated in response to non- malignant signalling and tumour endothelial marker 8 was observed to contribute to the TEC phenotype in breast cancer. Significant changes in cell migration were observed in the MCF-7 CM group. Furthermore, clear qualitative differences in the tube formation of HUVECs were noted in malignant groups compared to the non-malignant group. Conclusion: Our results highlight the fact that endothelial cells are highly responsive to interactions with nutrient deprived breast cells but the interaction with non-malignant breast cells compared to malignant breast cells is significantly different. Breast cancer cells therefore do alter endothelial cells, but the characteristic TEC phenotype is not specific to a malignant response. Breast cancer cells alter the angiogenic process but the degree of hyperactivation is influenced by the breast cancer phenotype. It is therefore evident that endothelial cells and angiogenesis are altered and key to breast cancer progression, yet a TEC phenotype specific to breast cancer remains to be defined.
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    A comparison of compounded-bioidentical hormone formulations versus FDA-approved hormone formulations in breast cancer progression
    (Stellenbosch : Stellenbosch University, 2023-03) Mochoele, Kamano Angela; Engelbrecht, Anna-Mart; Africander, Donita; Du Plessis, Manisha; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Oestrogen and oestrogen receptor-induced signalling plays an important role in breast cancer development and progression. Studies have shown that certain menopausal hormone therapies {MHTs} containing oestrogens and oestrogens in combination with progestogens, increase the risk of invasive breast cancer. Compounded-bioidentical hormone therapies {cBHTs}, not FDA-approved or regulated by the Medicines Control Council of South Africa, have become a popular MHT and are advertised as safer efficient alternatives. Oestrogen alone and in combination with progestogens such as medroxyprogesterone acetate {MPA} and norethindrone {NET} enhance breast cell proliferation, migration and invasion. lt is therefore important to determine the effects of compounded oestrogen formulations in the development and progression of breast cancer. This study aims to provide a comparative profile of the effects of traditional menopausal therapies {estrone + MPA and estrone + NETA}, an FDA-approved bioidentical formulation {oestradiol + progesterone {bE2+bP4}} with the compounded bioidentical biest hormone formulation E2 + estriol {bE2+bE3} on the progression of breast cancer. Methods: Human ER+ mammary adenocarcinoma cells {MCF7} were used. Proliferation was assessed by determining the cell viability through water-soluble tetrazolium salt {WST-1} assays. The cell cycle was analysed with flow cytometry. Western blot analyses were performed to assess the proliferation marker MCM2, the Pl3K/Akt signalling pathway and epithelial-to-mesenchymal transition {EMT} markers; E-cadherin, N-cadherin, Snail and β-catenin. Migration was measured through a wound healing assay. Results and discussion: All treatment combinations significantly increased cancer cell viability. The cell cycle analysis shows that FDA-approved estrone + MPA and estrone + NETA treatments induced the accumulation of MCF7 cells in the GO/Gl phase of the cell cycle. Western blot analysis revealed that all hormone treatments did not activate the Pl3K/Akt pathway. Furthermore, treatment of BE2 + BP4 indicated mesenchymal characteristics of EMT. The wound closure assay showed that the hormone treatments did not induce migration. Conclusion: According to our findings, there are both similarities and differences among the compounded biest combinations and FDA-approved hormone formulations. Concerningly, cBHT increases cell viability in a manner consistent with the FDA-approved formulations. Similar to FDA- approved therapies, they did not cause migration or activate the Akt pathway for cell proliferation. In contrast, when compared to their FDA-approved counterparts, cBHT formulations exhibited different effects on EMT and the cell cycle. All together these results demonstrate that cBHT treatments did not stimulate the pathways associated with breast cancer progression that was stimulated by the FDA-approved formulations. Future recommendations include investigating the effects of cBHT preparations on other pathways involved in breast cancer initiation and progression in comparison to the FDA-approved formulations.
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    Establishing and validating an in vivo rodent model of chronic restraint stress
    (Stellenbosch : Stellenbosch University, 2022-12) Van Wyk, Minette; Essop, M. Faadiel; Joseph, Danzil; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction. Psychological stress has emerged as one of the health epidemics of the 21st century and provides an impetus for increased investigation into the effects of a dysregulated stress response on whole body physiology. Although previous studies helped to clarify the association between chronic psychological stress and the onset and progression of cardiovascular diseases, a paucity of mechanistic insights underlying this association remain. Considering the complex nature of the stress system and the similarities that exist between humans and animals, it is therefore ideal to use rodent models to investigate stress-related disorders. Although the incidence and onset of various disorders in humans are gender-specific, clinical, and preclinical research using male subjects still far outnumber those using females. This study therefore aimed to establish and validate an in vivo model of chronic restraint stress in male and female Wistar rats. Materials and Methods. Male and female Wistar rats were subjected to a 4-week restraint stress protocol versus matched controls. Following this, behavioral tests (elevated plus maze [EPM] and tail flick task) were performed together with an assessment of body weight changes and biochemical biomarkers to ascertain whether the model was successfully established. Results & Findings. Our data revealed that male stressed rats displayed a decreased percentage change in body weight over time versus controls (p<0.01). Furthermore, the male stressed group exhibited increased plasma corticosterone levels compared to controls (p<0.01), while no significant differences were detected for plasma adrenocorticotropic hormone (ACTH) concentrations. Male brain-derived neurotrophic factor levels (biomarker for neuronal survival and growth) were lower in the stress group versus controls (p<0.05). Stressed males also displayed a reduced number of attempts into the open arms of the EPM versus controls (p<0.05). There were no significant weight changes for female rats. However, stressed females exhibited lowered plasma corticosterone levels versus controls (p<0.05), while also displaying higher plasma ACTH concentrations compared to the control group (p<0.05). Stressed females also displayed increased rears (as assessed by EPM test) versus matched controls (p<0.01). Our findings reveal intriguing sex-based differences in response to a chronic restraint stress protocol, with males displaying a depressive-type phenotype while females exhibited a post-traumatic stress disorder phenotype. Sex-specific preclinical research can provide unique insights into the various mechanisms driving stress-related diseases and should eventually lead to the identification of novel diagnostic and therapeutic targets.
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    Generation of a transgenic zebrafish with a fluorescent TNF-alpha reporter gene
    (Stellenbosch : Stellenbosch University, 2022-12) Ambrone, Wade; Smith, Carine; Van Staden, Anton du Preez; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Given the high degree of innate immune system conservation between zebrafish and humans, researchers have taken a keen interest in utilising zebrafish to unpack innate immunity related mechanisms. Notably, transgenic zebrafish expressing reporter genes are routinely employed for monitoring the spatio-temporal dynamics of innate immunity related proteins in vivo, and thus, are an invaluable tool to the zebrafish research community. While a number of alternative protein detection methods are available, they are subject to inference from tissue samples, often acquired through invasive techniques, which are incapable of accurately monitoring the spatio- temporal dynamics of protein expression in vivo. Given our research group’s interest in innate immune processes, as well as the advantageous applications of transgenic zebrafish, this study aimed to develop a stable transgenic zebrafish line, expressing a reporter gene under the transcriptional control of the pro-inflammatory cytokine TNF-α. For this, the transgene construct, pRSF-zTNFα-mCherry – expressing the fluorescent protein mCherry under the transcriptional control of the zebrafish TNF-α1 promoter – was designed and synthesized. Zebrafish embryos were transfected with the transgene construct by microinjection at the single-cell stage. Following microinjection, an acute inflammatory response was induced in transfected zebrafish larvae, coupled with fluorescence microscopy, to access the number of zebrafish in which the transgene was successfully integrated into the genome. Likewise, a genotyping method, using PCR, was developed to assess the rate of successful transgene integration. The data presented in this study illustrates the successful synthesis of the pRSF-zTNFα- mCherry transgene construct. Transfection of zebrafish larvae with the construct yielded zero zebrafish with detectable mCherry expression, using a stereomicroscope fitted with a fluorescent light source, both before and after the induction of an acute inflammatory response. Genotyping, however, revealed that the transgene was successfully integrated into the genome of the majority of transfected zebrafish, despite the inability to visually illustrate expression of the transgene. The transgene integration rate observed in this study was considerably higher than those previously seen in literature, in which similar transfection techniques were used. Potential reasons for the lack of detectable transgene expression were discussed throughout this study, namely: insufficient equipment sensitivity; insufficient transgene stimulation; integration of a dysfunctional transgene. While the study at hand was unable to demonstrate a TNF-α response spatio-temporally, many of the methodologies needed for the development of transgenic zebrafish were introduced and explored within the research group for the first time. Likewise, the means of both accessing and improving on future transgenic zebrafish development were investigated. In conclusion, this study lays the foundation for prospective transgenic zebrafish development – a model that will be utilized within the research group moving forward.