Masters Degrees (Medical Physiology)

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    Exploring the interactions of hyperglycaemia, SARS-CoV-2 spike protein & iron in HepG2 cells
    (Stellenbosch : Stellenbosch University, 2024-03) Breytenbach, Jacomi Heidi; Nell, Theo A. ; Joseph, Danzil; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Background: Diabetic COVID-19 patients have increased severity, disease progression and mortality, as well as an increased risk to develop persisting symptoms. Hyperglycaemia could be a central driving force of the pathophysiology in diabetic COVID-19 patients, demonstrating a multifactorial interaction with metabolic abnormalities, immune dysfunction, and dysregulated iron metabolism. An intricate relationship between hypoxia inducible factor 1, iron and cellular metabolism was also uncovered. Considering the need for a greater mechanistic understanding regarding this interplay, an in vitro investigation into the interaction between glucose and SARS-CoV2 Spike protein (protein involved in viral entry) and the effect of this on HIF-1 activation, iron and inflammatory parameters was therefore undertaken. Methods: HepG2 cells were cultured, and the following treatment groups were allocated: Low glucose (LG), High glucose (HG), Vehicle control (DMSO), LG + Spike protein, HG + Spike protein, HG + Prolyl Hydroxylase Inhibitor (PHI) (HIF-1α stabilizer), and HG + PHI + Spike protein. Various assays were used to assess cell viability, and production of reactive oxygen species (ROS). Western Blots were employed to measure iron, inflammatory, metabolic, and viral entry factor expression. Results: Significant decreases in ferritin levels were observed in HG + Spike, and HG + PHI treatment groups, indicating an interesting interaction between glucose and SARS-CoV-2 Spike that could underly attenuated circulating iron in COVID-19 patients. No significant differences in ROS, or inflammatory parameters were noted between different groups, indicating a lack of an inflammatory response, potentially mediated by low levels of intracellular iron. Lastly, stabilization of HIF-1α, as well as protein expression in different groups, could not be determined due to protein fragmentation. Conclusion: A remarkable interaction between glucose and Spike was associated with lower intracellular ferritin levels, independent of inflammation, necessitating more research.
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    Evaluating the effect of synthetic curcumin derivatives on skeletal muscle metabolism
    (Stellenbosch : Stellenbosch University, 2024-02) Ramashia, Rudzani; Jack, Babalwa; Pheiffer, Carmen; Titinchi, Salam; Windvogel, Shantal; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Background: Curcumin, a polyphenolic compound derived from the rhizomes of turmeric (Curcuma longa L.), exhibits an extensive range of beneficial health effects, including the modulation of skeletal muscle disorders. Despite its promising therapeutic potential, the poor bioavailability of curcumin has prompted the development of synthetic derivatives to enhance its efficacy. The aim of this study was to evaluate the effects of curcumin and three synthetic curcumin derivatives on skeletal muscle metabolism using in silico and in vitro approaches. Methods: Two chloro-curcumin derivatives (1A6 and 1A8) and an asymmetric curcumin derivative (1B8) were synthesized through a chemical reaction of 3-chloroacetylacetone (1A6 and 1A8) or acetylacetone (1B8) with boric oxide. High resolution - mass spectrometry - electrospray ionization (HR-MS-ESI), fourier transform - infrared (FT-IR) spectroscopy, and proton nuclear magnetic resonance (1H-NMR) spectroscopy were used to characterize the curcumin derivatives. In silico computational tools, Protox II, SwissADME, and SwissTargetPrediction predicted the toxicity profile, absorption, distribution, metabolism, and excretion (ADME) properties, and the biological targets of curcumin, 1A6, 1A8, and 1B8. Molecular docking was conducted to predict the molecular interactions and binding affinities of curcumin, 1A6, 1A8, and 1B8 with the predicted biological targets. The bioactivity of curcumin, 1A6, 1A8, and 1B8 was assessed in vitro in differentiating C2C12 myoblasts and in tumor necrosis factor alpha (TNFα)-treated C2C12 myotubes. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the adenosine triphosphate (ATP) assays. Oxidative stress was assessed by quantifying total antioxidant capacity (TAC) and lipid peroxidation. The expression of genes involved in insulin signaling, oxidative stress, inflammation, myogenesis and muscle atrophy was measured using quantitative real-time polymerase chain reaction. Results: Curcumin derivatives were synthesized with high purity and the chemical structures were elucidated by HR-MS-ESI, FTIR, and 1H-NMR spectroscopy. In silico prediction showed that curcumin and the curcumin derivatives exhibited favourable pharmacokinetic profiles, low toxicity, and improved drug-likeness. 1A8 had the highest binding affinity and 1A6 had the lowest binding affinity for the majority of predicted biological targets when compared to curcumin. In differentiating C2C12 myoblasts, curcumin, 1A6, 1A8, and 1B8 reduced cell viability at higher doses (10 and 20 µM), while treatment had no effect on TNFα-induced C2C12 myotubes. In TNFα-treated myotubes, 1A6 at 5 µM increased TAC. The expression of phosphoinositide-3-kinase regulatory subunit 1 (Pi3kr1) was upregulated in C2C12 myoblasts treated with curcumin, 1A6, 1A8, and 1B8 at 5 µM. In contrast, myogenic factor 6 (Myf6) expression was downregulated by curcumin, 1A6, 1A8, and 1B8 (5 µM) in https://scholar.sun.ac.za ii differentiating C2C12 myoblasts, while curcumin and 1A8 also decreased the expression of myogenic differentiation 1 (Myod1) at 5 µM. Conclusion: This study showed that curcumin and the three curcumin derivatives had favourable pharmacokinetic profiles and improved molecular interactions with their biological targets. Lower doses did not affect cell viability, while 5 µM increased TAC and Pi3kr1 expression, and reduced Myf6 and Myod1 expression, demonstrating their potential effects to modulate skeletal muscle metabolism, particularly targeting myogenesis. Further exploration of the therapeutic potential of synthetic curcumin derivatives on pathological conditions associated with muscle dysfunction is warranted.
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    Obesity, cardiometabolic diseases and aging-the role of Ataxia Telangiectasia Mutated protein kinase: optimization of in vitro lipotoxic H9c2 cardiomyoblasts and rat aortic endothelial cell models for cell signalling analysis
    (Stellenbosch : Stellenbosch University, 2024-02) Rabela, Vuyisane Michael; Huisamen, Barbara; Blignaut, Marguarite; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Background and aim: There has been an interest to establish optimised and standardized protocols for in vitro cell culture models with free fatty acid (FFA) overload to study the effects of obesityinduced cardiovascular diseases (CVDs). However, the disadvantages, such as solvent-induced cytotoxicity in these existing culture models, have been well-established. As such, robust, wellcharacterized cellular models that can fully represent the cardiac and endothelial tissue in vitro are required to study the effects of lipotoxicity on the signalling pathways involved in obesity-induced CVDs. This study aimed to establish and optimise insulin-resistant cardiac (H9c2 cardiomyoblasts) and vascular endothelial (rat aortic endothelial) cell models with palmitic acid (PA) and oleic acid (OA) to determine whether Ataxia Telangiectasia Mutated protein kinase (ATM) is decreased in these models and can contribute to increased markers of senescence as a measure of aging. Experimental design: H9c2 cardiomyoblasts and rat aortic endothelial cells were subjected to 24-hour treatments with increasing amount of PA (100 to 400 µM) and a fixed amount of 100 µM OA (n=3), and in a separate experiment, with 100 nM insulin (n=1 and n=2) for 15 minutes. FFA accumulation in these cell lines were determined with oil red O staining, followed by the assessment of cell viability and oxidative stress using MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and dichloro-dihydrofluorescein (H2DCFDA) for oxidative stress, respectively. Total and phosphorylated ATM, together with antioxidants and other metabolic proteins, were subjected to gel electrophoresis and Western blotting. The mRNA expression of senescence markers (p21Cip1 and p16INK4) together with ATM were determined with RT-qPCR. Nitric oxide was determined with flow cytometry in the endothelial cell model. Results: In H9c2, FFAs induced the upregulation of p16INK4 but not ATM, whereas FFAs did not affect these markers in endothelial cells. Total ATM, but not the phosphorylation (p-ATM-ser1981) was influenced by FFAs in endothelial cells. Metabolic proteins were not influenced by FFAs, whereas antioxidants were decreased under the same conditions in endothelial cells. Our results also showed that FFA treatment attenuate eNOS in endothelial cells, with a concomitant decrease in NO production, indicative of endothelial dysfunction. Our results also showed an attenuated response to insulin in endothelial cells suggesting an insulin resistance model; however, these observations were absent in H9c2 model. Instead, we observed an attenuated mTORC 1 phosphorylation, suggesting that this protein reacted to insulin through other mechanisms other than Akt under these conditions. Conclusion: Collectively, even though we could not confirm insulin resistance in H9c2, we observed an increased p16INK4 but not ATM, suggesting that FFAs induce aging (senescence) independently of https://scholar.sun.ac.za iii | P a g e ATM in this cell lines. As a proof of principle, we observed an attenuated response to insulin stimulation in ROAECs, suggesting insulin resistance. This was also characterised by disrupted metabolism due to a decrease in eNOS phosphorylation together with decrease NO production, indicative of endothelial dysfunction. We also observed a decrease in total ATM in these cell lines, supporting the evidence that ATM is decreased in obese conditions. Altogether, our data suggest that the FFAs overload models have been established
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    Characterising pathways that contribute to post-TB lung disease
    (Stellenbosch : Stellenbosch University, 2024-02) Jacobs, Steve; Maarman, Gerald; Windvogel, Shantal; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Tuberculosis (TB) is a major global health challenge, especially in low- and middle-income countries (LMICs). Post-tuberculosis lung disease (PTLD) is a common and debilitating sequela of TB, which can lead to chronic respiratory symptoms and impaired lung function. The pathogenesis of PTLD is not well understood, and much remains to be discovered, although some other authors have hypothesized that inflammation may be a key contribution, despite successful completion of TB treatment. Moreover, accumulating data suggests that PTLD might also include pulmonary hypertension (PH). The latter is a multi-organ disease and highly morbid clinical condition, with a broad range of clinical presentations and is caused by a large spectrum of underlying conditions. The relationship between PTLD and PH is complex and multifactorial, involving host, environmental, and pathogenic factors. Given the inflammatory nature of TB, it is likely that pro-inflammation may contribute to the development of PH post-TB, however, there is currently no data on this topic. This is concerning, as millions of people live with TB, close to 60 000 people die of TB in South Africa per annum, while every year almost 600 new cases of TB are reported. Given this context, it is worrying that TB patients (whether previous or current, or treated) are at risk of developing debilitating PTLD and fatal PH. This project, aimed to delineate the involvement of pathwaysthat may contribute to the development of PH in a post-TB context, and to explore the pathways that specifically contribute to PH in the same context. In our pursuit, we managed to highlight the instrumental roles of inflammatory pathways as part of PTLD pathogenesis. Our novel findings suggest that there is a pro-inflammatory state in active TB patients on treatment that persists post-TB, regardless of being successfully treated for TB. The unusual circulation pattern of inflammatory cytokines could be ascribed to mitochondrial dysfunction in immune cells. Considering the destructive nature of these cytokines, there is a need for further research to explore the implications of a persistent pro-inflammatory state, as it may predispose patients to PTLD. In terms of PH, we explored myriad pathways that may cause PH, now a new key feature of PTLD. Our review of the literature demonstrated a link between melatonin and PH as part of PTLD. Our findings demonstrate that melatonin is an important link between the gut microbiota and the development of PH (where suppressed melatonin-crosstalk between the gut and lungs could promote the development of PH). More studies are needed to investigate the link between the gut microbiota, melatonin and PH. Studies could also investigate whether microbiota genes play a role in the epigenetic aspects of PH. This is relevant because, e.g., dysbiosis (caused by epigenetic factors) could reduce melatonin signalling between the gut and lungs, reduce subcellular melatonin concentrations in the gut/lungs, or reduce melatonin serum levels secondary to epigenetic factors. PH is a fatal disease, and this essentially means that despite successful TB treatment thousands of patients are at risk of developing PH. Yet, there is no cure for PH and most developing countries do not have specialised PH drugs. Therefore, there is a need for research on better treatments for PH, particularly, in the post-TB context. We explored the potential of the repurposing of drugs for the treatment of PH especially in countries with resource limitations. Studies have demonstrated the benefits of medicinal plants against PH, most of which are of Indian or Asian descent. Africa is a rich source of multiple medicinal plants scientifically proven to counteract myriad pathologies. When perusing these studies one can notice that African medicinal plants afford biological effects that counteract the same molecular pathways (e.g., proliferation, vasoconstriction, inflammation, oxidative stress, and mitochondrial dysfunction) also involved in the pathogenesis of PH. Viable options include Aspalathus linearis, Allium sativium, Trifolium pratense L, Mimosa pigra L, and Aloe ferox. However, most of these plants have never been tested in an experimental PH model, and https://scholar.sun.ac.za 4 therefore, our proposition is hypothetical at the most. Regardless, we believe that future studies should investigate these and other African medicinal plants in appropriate models of PH, to test their efficacy and effectiveness. The relationship between PTLD and PH is complex and still requires several puzzles to be placed to fully understand it. The pathophysiology of PTLD that leads to PH, epidemiological factors and potential treatment options remains largely unexplored and are key areas for future research. Research into alternative and novel therapies is especially crucial as this has the potential to improve patient’s quality of life and clinical outcomes. Ultimately, further research will hopefully pave the way for the development of a comprehensive approach to PH prevention, detection and diagnosis, and treatment strategies.
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    An in vivo model investigating the effect of stress on the mammalian blood-testis barrier
    (Stellenbosch : Stellenbosch University, 2023-12) Ramsunder, Yashthi; Skosana, Bongekile; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Introduction: Stress has profound effects on the reproductive system. These effects occur via the hypothalamic-pituitary-gonadal (HPG) axis, which is augmented by the release of glucocorticoids during stress. Glucocorticoids affect testicular function by directly modulating steroidogenesis and gametogenesis. The blood-testis barrier (BTB) is a physical barrier between the Sertoli cells of the seminiferous tubules and is made up of tight junctions (TJ), ectoplasmic specializations, gap junctions and desmosomes. Any threat to the functioning and integrity of the BTB may perturb the process of spermatogenesis. Stress has the potential to disrupt spermatogenesis via the altering of the BTB proteins, which may affect the permeability and integrity of the BTB. However, the effects of stress on the BTB have not been well elucidated and therefore warrant further investigation. Methods: Considering that rats are social animals, we used a social isolation stress model to elicit stress and anxiety. Male Wistar rats (n = 18) were used for the study, which were divided into two groups, a stressed group and a control group. The stressed group were socially isolated for 7 days and the control group were group-housed concomitantly. At 18 weeks the rats were sacrificed, blood plasma was collected for corticosterone and testosterone analysis and the testes were harvested for analysis of BTB proteins. Corticosterone was measured to confirm the induction of stress. Expression levels of BTB proteins were assessed, including: focal adhesion kinase (FAK) and two of its tyrosine phosphorylates (Tyrosine (Tyr) 397 and 407), occludin, zonula occludin-1 (ZO-1) as well as both total and phosphorylated p38 mitogen activated kinase (MAPK). The expression and localization of these proteins was assessed by western blotting and immunohistochemistry (IHC) techniques respectively. The effect of stress on the FAK-occludin immunocomplex was evaluated through coimmunoprecipitation (Co-IP). The integrity of the BTB was assessed through transmission electron microscopy (TEM). Results: Corticosterone levels were significantly increased (P < 0.0001) and testosterone levels remained unchanged in the stressed group in comparison to the controls. Western blot analysis demonstrated a trend towards a decrease in FAK, FAK-Tyr397, ZO-1 and p38, while a trend towards an increase in p38 phosphorylation was observed. Occludin levels remained unchanged and FAKTyr407 expression was inconclusive. IHC revealed a non-significant decrease in occludin, ZO-1 and p38, however, FAK expression was inconclusive. Co-IP results were additionally inconclusive. TEM evaluation revealed no physical damage to BTB integrity in the stressed group. Conclusion: No visible, significant compromise to the integrity of BTB was observed during stress. Nonetheless, we observed non-significant reductions in the expression of TJ proteins, which may indicate a potential loss of BTB integrity. These findings suggest that the BTB is at risk of being compromised during stress. The BTB serves as a potential avenue in delineating how stress may affect male fertility.