Doctoral Degrees (Physiological Sciences)

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    The effects of kirrel1 isoform expression on C2C12 differentiation and fusion in vitro
    (Stellenbosch : Stellenbosch University, 2023-03) McColl, Rhys Stewart; Myburgh, Kathryn H.; Durcan, Peter J. ; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Adult skeletal muscle myogenesis involves the fusion of muscle progenitor cells into multi-nucleated myofibers, a process crucial for the growth and repair of muscle tissue. Vertebrate myoblast fusion is a relatively poorly understood process that involves a multitude of cell adhesion molecules, actin regulators and fusion proteins. A more comprehensive understanding of myogenesis is essential to better assess muscle myopathies and for the development of improved interventions. The kirrel family of mammalian cell adhesion molecules are highly involved in the production and maintenance of complex tissue structures such as the slit diaphragm in the kidney. The Drosophila paralogs of the kirrel proteins are known to be vital for actin regulation during myoblast fusion with the mechanisms of this regulation being mostly understood. However, these same findings have not been confirmed with regards to mammalian myoblast fusion; an arguably more complex process than that in the fly. It has been demonstrated that kirrel1A and its associated splice variant, kirrel1B, are differentially expressed in regenerating mouse muscle tissue; although, the exact roles of these molecules during this process are not clear. More recently, kirrel3 has been shown to be required for the successful fusion of mouse myoblasts. The aim of this study was to determine the effects of kirrel1A and kirrel1B expression levels on C2C12 differentiation and fusion in vitro. Three genetic strategies were employed to assess kirrel1 activity during C2C12 myogenesis, these being; CRISPR/Cas9 modification, shRNA knockdown and retroviral overexpression. CRISPR/Cas9 was used to disrupt kirrel1 expression by modifying genomic regulatory regions between exons 1 and 2 of the gene. The individual knockdown/inhibition of kirrel1A and kirrel1B mRNA activity was achieved using shRNAs. Overexpression was carried out by wild-type kirrel1A and kirrel1B gene-cloning followed by retroviral transduction. Additionally, a kirrel1A-mCherry mutant was overexpressed in the C2C12s. The differentiation of the various cell lines was assessed via western blotting, PCR analysis and phase-contrast microscopy. The experiments suggest that although the moderate overexpression of kirrel1A or kirrel1B has little effect on myotube production, the gross overexpression of kirrel1 variants leads to a drastic reduction in myogenesis, potentially due to increased steric hindrance at the cell surface. Moreover, our findings demonstrate the requirement for kirrel1A during myotube formation as no tubes were seen in kirrel1A-knockdown myoblasts. This inhibition appeared to be unrelated to the expression of the myogenic regulatory factors. However, it is still unclear whether there is a similar requirement for kirrel1B during fusion. The expression of a mutant form of kirrel1A with an mCherry tag inserted close to an intracellular cleavage site resulted in a complete lack of myotubes; seemingly due to altered early MRF expression. In each case where cell lines produced myotubes, non-reducing western blotting revealed large kirrel1-containing complexes that accumulated as fusion progressed. These complexes were not seen in any of the non-fusing cell lines. From the results it is apparent that kirrel1 is required for healthy myogenesis and that further research is required to fully understand the mechanisms of this regulation.
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    Investigating systemic inflammation and hypercoagulability in psoriasis: implications for cardiovascular disease
    (Stellenbosch : Stellenbosch University, 2022-12) Visser, Maria Johanna Elizabeth; Pretorius, Etheresia; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Psoriasis (PsO) is a common immune-mediated inflammatory disease of the skin, typically presenting as erythematous plaques covered with silvery scales. The condition is of multifactorial aetiology, encompassing interactions between environmental factors, genetic susceptibility, and dysregulated immune responses. The pathogenesis of PsO is predominantly driven by interleukin (IL)-17. In addition, inflammatory mediators associated with T helper (TH) 1, TH17, and TH22 subsets are also overexpressed in psoriatic skin. Moreover, these inflammatory molecules may also be detected in the systemic circulation of patients with PsO. The disease is not solely limited to cutaneous sites, as multiple comorbidities have been linked to the condition. Notably, patients with PsO have been reported to have a significantly increased risk of cardiovascular disease (CVD). A potential mechanism that might contribute to this association is the presence of a hypercoagulable state – driven by persistent systemic inflammation – in these individuals. A pro-inflammatory milieu may favour coagulation, while suppressing natural anticoagulant mechanisms. In addition, inflammation may also alter – albeit indirectly – the fibrin clot structure and, by extension, the properties of the fibrin network. Therefore, the aim of this study was to assess the haemostatic profile and investigate potential alterations in fibrin clot structure in patients with PsO, compared to healthy individuals. Whole blood samples were collected from patients with PsO (n=20) and healthy control subjects (n=20). The concentrations of blood-based markers of inflammation and endothelial and platelet activation were determined using enzyme-linked immunosorbent assays. Coagulation status was assessed by thromboelastography. The fibrin network architecture was analysed by scanning electron microscopy. Fibrin secondary structure was assessed through the examination of formalin-fixed, paraffin-embedded plasma clot sections using fluorescence microscopy and Fourier transform infrared (FTIR) spectroscopy. In order to detect areas rich in β-sheet structures, Congo red staining was performed, and sections were examined with brightfield and fluorescence microscopy. To determine if there were quantitative differences in the distribution of specific secondary structural elements in fibrin clots, FTIR analysis was conducted. Elevated levels of inflammatory molecules (C-reactive protein, serum amyloid A, soluble intercellular adhesion molecule-1, and soluble P-selectin) were associated with PsO, thereby confirming the presence of systemic inflammation in patients with PsO. Thromboelastographic analysis revealed an increased tendency towards clot formation, that was also associated with disease presence. Moreover, the ultrastructure of fibrin clots from patients with PsO was altered – these clots were denser and consisted of thicker fibrin fibres, as compared to control subjects. Regarding the secondary structure of fibrin, the presence of β-sheet-rich areas, as identified by Congo red fluorescence, was detected in fibrin clots from both groups. Accordingly, FTIR analysis also did not show any significant differences between the secondary structure composition of fibrin clots from patients with PsO and those of healthy control subjects. Taken together, the results of this study indicate that a hypercoagulable state is present in patients with PsO. This hypercoagulability seems to be a result of persistent systemic inflammation, rather than alterations to the molecular structure of fibrin. The hypercoagulable state in PsO might have implications for the management of CVD risk for individuals living with the condition.
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    Comparative assessment of neurological vs metabolic allostasis as reflected in human skin fibroblasts
    (Stellenbosch : Stellenbosch University, 2022-12) Benecke, Rohan Meerholz; Smith, Carine; Van de Vyver, Mari; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Incidence of mental health disorders are rising in modernity. Many mental health disorders share molecular and genetic overlap as well as having high incidence of comorbidities. Stress and the compounded effect of multiple low-grade stressors may be contributing to a relative increase in the pro-inflammatory and oxidative state seen in mental health disorders and other complex diseases. This leads to allostatic changes that potentially contribute to disease aetiology and progression. Allostasis is the process of homeostatic equilibrium under stress. Inflammation, which is often used to measure allostatic load, is potentially the incorrect measure as inflammation is transient and the damage ascribed to chronic inflammation is due to increases in reactive oxygen species (ROS) and decreases in antioxidant capacity. Post-traumatic stress disorder (PTSD) is a mental health disorder that is characterised by severe stressors and a maladaptive response to these stressors. Although the role of inflammation and oxidative stress have been implicated in the disease aetiology it is still a relatively neglected aspect of PTSD research. Furthermore, despite the high rate of comorbidities associated with PTSD there is still a lack of understanding in terms of the peripheral effects of PTSD. PTSD and potentially comorbid obesity, present ideal health paradigms to assess this relative neglect of allostatic changes, in particular those in the periphery, that may be contributing to disease outcome in PTSD. A novel therapeutic target, namely the trace amine system, is investigated as a potential anxiolytic in zebrafish larvae that could address allostatic changes of chronic diseases such as PTSD and obesity. Patient derived fibroblasts are used as model cell type to investigate potential functional changes in the periphery of PTSD patients as result of allostatic load. Finally, the potential for peripheral signalling to influence central function is explored in astrocytes, that represent the ideal candidate cell to investigate allostatic load in the context of mental health. Changes in peripheral calcium function and central redox function indicate the allostatic load of PTSD can modulate the chemiosmotic potential of cells through longitudinal shifts in the homeostatic set point. As a result, low grade cumulative stressors may be damaging to cellular function without activating endogenous defence mechanisms.
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    The effect of altered trace aminergic signalling and estrogen on intestinal inflammation, within an IBS context
    (Stellenbosch : Stellenbosch University, 2022-12) Pretorius, Lesha; Smith, Carine; Van Staden, Anton du Preez; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Irritable bowel syndrome (IBS) is a widespread (≈10% global prevalence) female predominant functional gastrointestinal (GI) disorder. While it is known that IBS is underpinned by relative microbial dysbiosis and chronic microinflammation, current therapeutic strategies often only provide transient symptomatic relief (with relative neglect of inflammation) and are thus unsatisfactory in many cases. As such, the development of targeted therapeutics to alleviate GI inflammation and consequential symptomologies are required. We suggest that the trace aminergic system, which connects several IBS risk factors (sex, dysbiosis, diet, inflammation and anxiety), may be a pretermitted regulatory system that could be manipulated as a therapeutic target. In addition, existing data supports an interpretation of sex dependence in trace aminergic signalling. As such, fluctuations of female reproductive hormones, such as 17β-estradiol (E2), may alter subsequent signalling cascades. Therefore, this thesis aimed to investigate the GI modulatory effects of selected trace amines (TAs), with consideration of the context of female predominance. To elucidate mechanisms at play, a multidisciplinary approach was necessitated. As such, multiple model systems were utilised, including both in vitro (microbial cultures and human tissue cultures) and in vivo (zebrafish larval) models. In this regard, microbial (probiotic and commensal strains) culturing techniques, coupled with the development of a novel multianalyte mass spectrometry methodology, allowed for the accurate assessment of microbial TA generation. Indeed, data generated in these studies highlighted firstly, the dependence of probiotic secretome profile on host hormonal status, and secondly, that specific rooibos supplementation strategies may be able to negate E2-induced alterations in secretome TA profiles, both of which have important implications in TA-associated symptom management in females with GI disorders. Data generated in vitro in HT-29 colon adenocarcinoma cells and in vivo in zebrafish larvae, in which the effects of increased TA load were assessed, demonstrated potential differences in the mechanisms of actions between TYR and AGM in particular. In this regard, extensive occludin redistribution was observed following TYR-exposure, which was associated with increased reactive oxygen species and pro-inflammatory cytokine levels, as well as tight junction disruption – an outcome prevented by E2 treatment. In contrast, AGM administration promoted the colocalization of ZO-1 and occludin to promote tight junction integrity but was also associated with risk of pro-oxidant damage when AGM metabolism was insufficient. In conclusion, this dissertation contributes significantly to our understanding of the role of TAs in GI physiology, consistently illustrating (across in vitro and in vivo models), that while some TAs may promote disease symptomology, others may have therapeutic benefit when responsibly administered. From a therapeutics standpoint, data presented here crucially highlights the importance of dosage and administration optimisation to achieve benefit and minimize adverse side effects when targeting TA signalling in the context of functional GI disease. In addition, potential mechanistic insights by which E2 - or rather the transient cyclic lack thereof - is associated with trace aminergic signalling, was elucidated.
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    The role of Serum Amyloid A in NLRP3 inflammasome signalling in breast and colon cancer
    (Stellenbosch : Stellenbosch University, 2022-04) Fourie, Carla; Engelbrecht, Anna-Mart; Davis, Tanja; De Villiers, Willem J. S.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Cancer is a complex disease with multiple interactions targeting the organism on cellular, tissue and systemic levels. The main research focus for the past decades has been on the genome and on the molecular level where signaling pathways were dissected for the development of targeted therapies. However, in order to develop more efficient therapeutic regimes, a better understanding on systemic level is required. Over the past few years, the role of serum amyloid A (SAA) has gathered significant evidence which highlights its role in the pathogenesis of several cancers, including breast and colorectum carcinomas. To date, SAA has been shown to bind to several pattern recognition receptors, which might suggest that inflammasomes play a role in the tumour-promoting properties of SAA. Inflammasomes are cytoplasmic multiprotein complexes characterized by a sensor protein, an adaptor protein, and inflammatory caspases. However, the role of inflammasomes in cancer remain controversial. The aim of this study was therefore to investigate the role of SAA in inflammasome signaling in breast and colon cancer. Methods: In this 3-part animal study, tissues were subjected to immunoblotting, real-time PCR, haematoxylin and eosin staining and immunohistochemistry. For the first inflammatory model, wild-type and SAA double knockout C57BL/6 mice received 2.5% dextran sulfate sodium, which was administered for a total of 5 days. To assess tumourigenesis, colitis-associated cancer and triple negative breast cancer models were used, respectively. For colitis- associated cancer, wild-type and SAA double knockout C57BL/6 mice received an intraperitoneal injection of 12.5 mg/kg azoxymethane. After one week, dextran sulfate sodium treatment was administered at a concentration of 2.5% for a total of 5 days, followed by a recovery period of 16 days. Dextran sulfate sodium treatment was administered for a total of 3 cycles. Triple negative breast tumours were established in wild-type and SAA double knockout C57BL/6 mice by injecting EO771 cells subcutaneously at the fourth mammary fat pad. The experimental endpoint was reached when tumour volumes reached 300-400 mm3. Results: In this study we have showed that in an in vivo model of dextran sulfate sodium induced colitis, SAA ablation exerted pro-inflammatory properties independent of the NLRP3 inflammasome. The ablation of serum amyloid A1/2 was associated with the increased expression of pro- inflammatory cytokines. In contrast, in an in vivo colitis-associated cancer and in a triple negative breast cancer model, the ablation of SAA suppressed canonical NLRP3 inflammasome activation, which was associated with anti-inflammatory properties. These findings suggest that during tumourigenesis, SAA functions as an endogenous damage associated molecular pattern in the tumour microenvironment. Conclusion: Here we show for the first time, in models of CAC and TNBC, the novel role of SAA in the activation of the NLRP3 inflammasome and the generation of pro-inflammatory cytokines, two mechanisms known to promote tumour development and metastasis. This study emphasizes the notion that the tumour-induced systemic environment acts as a critical regulator of cancer progression and metastasis. In conclusion, simultaneously targeting SAA and NLRP3 components could be beneficial for cancer treatments.