Masters Degrees (Medical Physiology)

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    Molecular evaluation of tuberculosis agents on a H9c2 rat ventricular cardiomyoblast model.
    (Stellenbosch : Stellenbosch University, 2023-12) Februarie, Candice; Baatjies, Lucinda; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Tuberculosis (TB) remains a significant global health challenge, demanding ongoing assessment of the efficacy and safety of its therapeutic agents. This study focused on evaluating the potential cardiotoxicity of four anti-tuberculosis drugs, namely isoniazid, ethambutol, pyrazinamide, and rifampicin, on H9c2 rat ventricular cardiomyoblasts. Cardiotoxicity is a critical concern since these drugs are administered for extended periods. This prospective in vitro study encompassed three key phases. In the first phase, the study assessed the metabolic activity of first-line anti-TB drugs, secondary metabolites of Mycobacterium smegmatis mc2155, and M. smegmatis mc2155- infected H9c2 cells via MTT assays. The second phase aimed to determine the extent and mode of cell death induced by these substances through flow cytometry. Finally, the third phase involved an evaluation of mitochondrial oxidative phosphorylation (OXPHOS) using the Oroboros Oxygraph-2K. The first-line anti-TB drugs exhibited no cytotoxic effect on the cells. In contrast, the metabolites of M. smegmatis mc2155 displayed a concentration-dependent decrease in cell viability, with higher concentrations resulting in lower viability. Notably, the 25mg/mL concentration exhibited cytotoxic effects on the cells. When M. smegmatis mc2155-infected H9c2 cells were treated with the first-line anti-TB drugs, rifampicin showed a reduced cell viability of 57% compared to the control, indicating the cells' susceptibility to infection. Notably, infected cells consistently displayed lower viability across all drug treatments compared to their uninfected counterparts. Furthermore, the study revealed that treatment with rifampicin and M. smegmatis mc2155 secondary metabolites led to an increase in early apoptotic cells. This suggests efficient activation of apoptosis pathways compared to the untreated control (0.08%). Additionally, DMSO (10%) demonstrated a statistically significant decrease in the number of viable cells (29.07%) compared to the untreated control (99.58%), coupled with an increase in necrosis (70.93%) in contrast to the 0.14% observed in the untreated control. Variations in basal respiration rates were observed among the treatment groups compared to the untreated control. Leak respiration, associated with proton leak across the inner mitochondrial Stellenbosch University https://scholar.sun.ac.za iii membrane, increased when cells were treated with rifampicin and M. smegmatis mc2155, indicating a disturbance in mitochondrial function and the inner mitochondrial membrane. OXPHOS CI+CII activity, involving both Complexes I and II, exhibited a significant reduction in the oxygen consumption of the cells in all the treatment groups compared to the untreated control. This reduction points to a potential disruption in this specific segment of the electron transport chain. In summary, this study emphasises the impact of first-line anti-TB drugs on cardiac cells, considering the potential for drug-induced cardiotoxicity. The results suggest that H9c2 cardiomyoblasts are susceptible to infection with M. smegmatis mc2155. Moreover, M. smegmatis mc2155secondary metabolites significantly affect metabolic activity, induce early apoptosis, and disrupt electron flow in the electron transport chain. These findings warrant further investigations, particularly exploring the effects of first-line drugs in a 3D or animal model.
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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.
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    Exploring features of oxidative stress and senescence in the H9c2 cardiomyoblast cell line
    (Stellenbosch : Stellenbosch University, 2023-03) Harries, Sarah; Huisamen, Barbara; Sadie-Van Gijsen, Hanél; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Background: Age-related cardiovascular disease is one of the largest causes of mortality globally. Senescent cells in the heart have emerged as a possible contributor to cardiac disease progression; however, studies elucidating the mechanisms explaining this phenomenon are scarce. The protein kinase Ataxia Telangiectasia Mutated (ATM) has been shown to be involved in DNA damage repair, cellular redox homeostasis, and mitochondrial function, and therefore may play a role in regulating senescence in heart cells. Cardiomyocytes are the most common cell-type in the heart; however, due to logistical and ethical considerations, cardiomyocytes are not always a feasible model for cardiac cell research. Therefore, the aim of this study was to determine if the H9c2 cardiomyoblast cell-line can exhibit features of induced senescence as a foundation to study the role of ATM in cardiac cell senescence. Methods: Early and late passage H9c2 cardiomyoblast cells (EP and LP, respectively) were compared in terms of growth rate, secretome and responsiveness to exogenous hydrogen peroxide and retinoic acid, and were then assessed for markers of senescence and changes in oxidative stress status. Markers measured included intracellular reactive oxygen species (ROS), senescence-associated betagalactosidase (SA-βgal) staining, gene expression levels of p16, p21 and ATM, and protein levels of total and phosphorylated ATM, p53 and H2AX. The anti-senescent properties of the antioxidant polyphenol resveratrol were also explored in EP and LP H9c2 cells. Results: EP and LP cells exhibited little evidence of oxidative stress and senescence and displayed high proliferative capacity, no increase in intracellular ROS and no increase in senescent markers. However, LP cells exhibited upregulation of the DNA damage marker γH2AX and the DNA repair protein ATM. Unexpectedly, a high concentration (25 µM) of resveratrol had a detrimental effect on EP and LP cell culture density and significantly upregulated p21 gene expression in EP cells. Discussion: Despite an increase in DNA damage markers and DNA repair proteins in LP H9c2 cells, other markers of senescence were absent, suggesting that the cells were able to repair the damage without committing to senescence. Intriguingly, a high concentration of resveratrol was detrimental to both EP and LP cultures, and consequently, the use of resveratrol as a cardioprotective antioxidant should be https://scholar.sun.ac.za iv questioned and may be dose-dependent. In conclusion, the H9c2 cells overall showed a resistance to respond to various damaging stimuli. Therefore, this cardiomyoblast cell line may not be suitable for research within the context of senescence owing to its limited ability to develop features of senescence.
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    Does sexual violence alter social behvaiour via a maladaptive HPA-axis?
    (Stellenbosch : Stellenbosch University, 2023-03) Fortuin, Chelsi Shante; Qulu, Lihle; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
    ENGLISH ABSTRACT: Background and Aim: South Africa was identified as the rape capital of the world, recording one of the highest rates of sexual violence worldwide with 72.1 reported cases per 100 000 in the 2019/2020 year. The mental health and well-being of sexual assault survivors exhibit a dysregulated HPA-axis stress response, which may be a primary source of structural and functional alterations leading to PTSD symptoms, but they don’t always co-exist. Chronic stress and psychiatric diseases cause dysregulation of the HPA axis. A cortisol imbalance brought on by the dysregulation of the HPA-axis leads to a decrease in oxytocin secretion, which eliminates oxytocin's potential to attenuate HPA-axis activity. A lack of oxytocin has been linked to broken social and maternal bonds as well as losing relationship attachments as a result of trauma exposure such as sexual violence / rape. Oxytocin dysregulation has been associated in a large variety of dysregulated social behaviour and PTSD diagnosis. Additionally, when bound to cortisol, glucocorticoid receptor sensitivity modifies the stress response's equilibrium point, which in turn regulates the HPA axis's negative feedback loop. As a result, they have been linked to anticipating the effects of stress. Therefore, the aim of this study is to determine whether being subjected to sexual violence alters social behavior via a dysregulated HPA-axis. Methods: Data analysis was completed using PTSD, perceived stress and social support scores to investigate the effects of rape on mental health trajectory. Additionally, enzyme-linked immunosorbent assays were used, to explore the role of oxytocin and cortisol as indicators of PTSD in rape-exposed women. Both cortisol and oxytocin found within plasma samples were analysed over a one-year period at four time points (baseline [± twenty days], three months, six months and twelve months post rape) to assess whether these hormone levels, that are activated by sexual violence, were transient or long lasting, and whether these hormone levels may lead to the formation of PTSD. Mixed effect regression analysis were done to determine if cortisol and oxytocin concentrations predict PTSD outcomes. Results: PTSD symptoms significantly decreased overtime, p = 0.000. Similarly, perceived stress, p = 0.0023, and cortisol concentrations, p = 0.004, significantly decreased overtime. Whereas social support scores, p = 0.5851, and oxytocin concentrations, p = 0.995, had no significant changes. Additionally, cortisol concentrations, p = 0.1660, and social support, p = 0.827, were not able to predict PTSD outcome, however oxytocin concentrations, slope = 9.32, p = 0.002 and perceived stress scores, slope = -5.654, p = 0.033, could predict PTSD. Conclusion: These findings demonstrate a relationship between PTSD symptom severity and HPA-axis maladaptation, via augmented oxytocin responses in victims of rape. We conclude that these findings may have significant clinical ramifications for women who have been subjected to rape. Particularly, offering scientific based PTSD interventions to rape exposed victims may show promise in alleviating symptoms and "normalizing" HPA axis receptivity to stress related stimuli.
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    The impact of SARS-CoV-2 on respiratory function
    (Stellenbosch : Stellenbosch University, 2023-02) Van Heerden, Jacques; Strijdom, Hans; Koegelenberg, Coenraad; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
    ENGLISH ABSTRACT: The iliocapsularis (IC) is a deep skeletal muscle that overlies and attaches to the anteromedial hip joint capsule and is an important anatomical landmark in anterior approaches to hip replacement surgery. Researchers have proposed the IC functions to stabilise the anterior hip joint and limit impingement of the hip capsule, between the femoral head and acetabulum, in hip flexion. However, a conclusive description of the function of the IC is not yet known. This study, therefore, aims to determine the skeletal muscle properties of the IC muscle and to compare these to that of the iliacus (IL) and vastus lateralis (VL). A cross-sectional observational study was conducted on 11 recently deceased unembalmed bodies with a mean age of 83 ± 9 years (range 69 - 95 years). Muscle samples, harvested from the IC, IL, and VL, were analysed for muscle fibre type distribution and fibre cross-sectional area (CSA) using fluorescent immunohistochemistry, while relative mitochondrial density was visualised histochemically using the NADH stain. IC had predominantly type I fibres (63 ± 12%), followed by type IIA (32 ± 13%) and IIX (5 ± 3%) fibres. IL comprised of a similar high distribution of type I fibres (61 ± 8%), compared to type IIA (31 ± 7%) and IIX (8 ± 8%) fibres. Conversely, VL had equal amounts of type I (47 ± 12%) and IIA (40 ± 11%) fibres, with lower proportions of type IIX (13 ± 10%) fibres. No difference in fibre type distributions were found between the IC and IL, whereas VL had less type I fibres compared to the IC and IL. The latter two muscles observed higher relative mitochondrial density (darker fibres) and, therefore, oxidative capacity, compared to the VL with a more equal proportion of light and dark stained fibres. The IC had larger (p < 0.0001) type I fibres (3607 ± 1422 μm2) compared to its type IIA (1849 ± 1306 μm2) and IIX (1379 ± 900 μm2) fibres. Similarly, the IL and VL had larger (p < 0.0001) type I fibres (3320 ± 1182 μm2 and 4235 ± 882 μm2, respectively) compared to type IIA (1790 ± 987 μm2 and 2738 ± 1650 μm2, respectively) and IIX (1428 ± 769 μm2 and 2170 ± 1355 μm2, respectively) fibres. No difference in the CSA of fibre types were found when the IC was compared with the IL and VL. However, the VL reported larger CSA compared to IL for type I and IIA fibres. Mean fibre CSA of the IC and IL were similar in size, while the VL had larger fibres. Fibre type distribution and fibre CSA showed no association with age. Therefore, the predominant oxidative type I fibre distribution of the IC may supports its proposed function to stabilise the hip joint and limit impingement of the hip capsule in hip flexion. Therefore, conclusive knowledge of the function of the IC will allow for more informed decisions regarding patient care and rehabilitation following anterior approaches for hip-replacement surgery.