Theses and Dissertations (Physiology)

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Investigation of vasculogenic mimicry in melanoma tissue
(University of Pretoria, 2025-01) Mabeta, Peaceful Lucy; Flepisi, Brian Thabile; Bida, Meshack; u10199544@tuks.co.za; Matlala, Rakolote Bruce
Melanoma is an invasive and aggressive form of skin cancer developing from melanocytes in the basal layer of the epidermis. Worldwide, the incidence of melanoma continues to increase, accounting for approximately 80% of all skin cancer-related deaths in 2020. In South Africa, the overall incidence of melanoma is approximately 2.7 per 100,000 people. In comparison, melanoma accounted for a similar proportion of approximately 80% of all skin cancer-related deaths in 2012, with an estimated 55,000 deaths globally. The rapid proliferation of melanocytes increases oxygen and nutrient requirements and results in increased vascularisation as a coping mechanism to meet the metabolic requirements of the tumour. Angiogenesis, the formation of new blood vessels from existing micro-vessels, is an important mechanism of vascularisation in melanoma. Various anti-angiogenic drugs such as bevacizumab have been employed to inhibit blood vessel formation in melanoma. However, resistance develops partly due to alternative pathways of vascularisation, mainly vasculogenic mimicry (VM). Vasculogenic mimicry is the process of forming highly patterned matrix and tubular channels lined with tumour cells, evidently known to conduct fluid movement. The process entails melanoma cell proliferation and their migration, and their subsequent coalescing into vessel-like structures. The formation of VM is regulated by multiple factors including, vascular cell adhesion molecule -1 (VCAM1), vascular endothelial growth factor-A (VEGF-A), and nodal. Therefore, the aim of this study was to investigate the vasculogenic mimicry in melanoma tissue by evaluating cell proliferation, migration, and the expression of molecular regulators of vasculogenic mimicry. For in vivo studies, immunohistochemistry (IHC) was utilised to determine the expression of the proteins VCAM1 and VEGF-A in tissue samples of melanoma patients. As challenges were encountered with optimising western blotting technique, further studies were undertaken using in vitro models . The endothelioma (sEnd.2) and melanoma (B16-F10) cell lines, employed in this study as in vitro models for benign and malignant skin tumours, were maintained in an incubator at 37 °C in a humidified atmosphere containing 5% CO2. Melanoma and endothelioma viability and growth patterns were studied in vitro using the crystal violet assay. Furthermore, cell morphology characteristics were studied using polarisation-optical interference contrast and light microscopy to augment cell growth data. Since vascular endothelial growth factor receptor-1 (VEGFR-1) is known to signal vasculogenic mimicry, western blot was employed to investigate the expression of the key receptor which promotes vasculogenic mimicry, VEGFR-1. The in vitro scratch migration assay and real-time, label-free xCELLigence technologies were used to study the effect of VEGFR-1 blockade on the migration of cells as well as cell invasion and migration respectively. In vivo studies showed that VCAM1 and VEGF-A in melanoma tissue were positively expressed intratumorally, suggesting their potential involvement in tumour growth and vasculogenic mimicry. This expression may contribute to melanoma progression and survival by promoting the formation of vessel-like structures, thereby facilitating nutrient and oxygen supply to the tumour cells. The results from in vitro studies demonstrated significant attenuation of melanoma cell growth. Morphology study results showed significant changes including cell rounding, membrane blebbing and reduction in cell density, which indicate cytoskeletal disruption and apoptosis. These observations, along with decreased cell density, suggest a reduction in cell proliferation and an increase in cell death, confirming the apoptotic effects of sunitinib malate. Western blot studies revealed that melanoma cells express VEGFR-1. Cell migration plays a critical role in vasculogenic mimicry by allowing tumour cells to create vessel-like structures that mimic blood vessels, promoting tumour growth, invasion, and the bypass of conventional angiogenesis, which in turn aids tumour progression and therapy resistance. In this regard, cell migration studies showed that VEGFR-1 blockade significantly decreased the rate of cell migration and invasion, highlighting the crucial role of VEGFR-1 in these processes and its potential as a therapeutic target. Therefore, taken together, these outcomes further support the evidence for the presence of vasculogenic mimicry in melanoma and thereby provide an opportunity for further investigation on the use of combination therapies to improve patient outcome.
The effect of combination treatment of vascular endothelial growth factor receptor 3 inhibitor, MAZ-51, and zingerone on melanoma cell proliferation
(University of Pretoria, 2024-12) Hlophe, Yvette Nkondo; Nyakudya, Trevor Tapiwa ; u18067507@tuks.co.za; Letsoalo, Remmotile Kganya
Melanoma is an aggressive form of skin cancer that is characterised by the carcinogenic transformation of melanocytes. Globally approximately 132 000 new melanoma cases are recorded annually and South Africa has recorded an incidence of 2.7 per 100 000 individuals. Various treatment strategies such as chemotherapy, targeted therapy and surgical excision are currently employed to inhibit melanoma growth, survival and progression however, the use of current treatments often results in unintended side-effects and drug resistance. Therefore, to combat this negative effect alternative treatment strategies such as medicinal plants and their bioactive phytochemicals have been widely studied and accepted as an alternative treatment, suggesting that the combined use of phytochemicals and synthetic drugs may inhibit cancer growth and proliferation with limited toxicity observed to noncancerous cells. Therefore, this study aims to investigate the individual and combined effects of (3-(4-Dimethylamino-naphthelen-1-ylmethylene)-1, 3-hydroindol-2-one) (MAZ-51) and a derivative of ginger, zingerone, on melanoma cell proliferation and survival in the melanoma (B16-F10) and human keratinocyte (HaCaT) cell lines. The cytotoxic effects of MAZ-51 (0.002-0.005 mg/mL) and zingerone (0.5-2 mg/mL) at 24, 48 and 72 hours were investigated using crystal violet assay. Additionally, crystal violet analysis was used to investigate the effects of MAZ-51 and zingerone co-treated with vascular endothelial growth factor (VEGF) on cell numbers. The morphological changes induced by the compounds were investigated using polarization optical transmitted differential contrast (PlasDIC) and hematoxylin and eosin (H&E) staining. The effects of the compounds on cell cycle progression were determined using flow cytometry. The results indicate that MAZ-51 and zingerone significantly inhibited cell growth at 48 and 72 hours (p<0.05). Morphological changes included the formation of apoptotic bodies, cellular protrusions, cell swelling and cell rounding suggesting cell death. In addition, MAZ-51 and zingerone resulted in a cell cycle block. Our findings demonstrate that MAZ-51 and zingerone exhibit significant antiproliferative effects on melanoma cells, with zingerone showing potential in reducing melanoma cell viability.
In vitro influence of nutrient deprivation and papaverine exposure in MCF-7 and MDA-MB-231 breast cancer cell lines
(University of Pretoria, 2025-02-24) Visagie, M.H. (Michelle Helen); Lee, Yuhan; Joubert, Annie M.; mr.mcstark@gmail.com; Stark, Michael Christopher
Breast cancer treatment in South Africa is constrained by limited access to affordable targeted therapies, highlighting the urgent need for cost-effective solutions within the public health sector. Fasting, a free and accessible intervention, shows promise in reducing tumorigenic metabolism and chemotherapy side effects; however, the efficacy is frequently reduced by metabolic plasticity, where metabolic activity shifts from glycolysis to oxidative phosphorylation (OXPHOS) in order to resist nutrient deprivation (ND). Various studies have demonstrated that nutrient-deprived breast tumorigenic cells exhibit increased dependence on OXPHOS for optimal proliferation through augmented mitochondrial complex I expression, highlighting a notable metabolic vulnerability that could potentially be targeted by combining fasting with a mitochondrial complex I inhibitor. Papaverine (PPV), a repurposed mitochondrial complex I inhibitor, was selected for this study due to its low cost, established safety, and accessibility in South Africa. However, the combination of physiological ND and PPV remains to be elucidated. Thus, the aim of this study was to investigate the combined effects of fasting-mimetic glucose and glutamine deprivation and PPV exposure, on proliferation, morphology, cell cycle progression, oxidative stress, superoxide dismutase (SOD) activity, mitochondrial membrane potential (ΔΨM), and the activity of 5′ adenosine monophosphate–activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) in metabolically distinct breast tumorigenic cells. This study used two breast cancer cell lines: OXPHOS-dependent MCF-7 (luminal, hormone receptor–positive) and glycolysis-dependent MDA-MB-231 (basal, triple-negative). The results obtained demonstrate that ND significantly enhanced the antiproliferative effects of PPV in both MCF-7 and MDA-MB-231 cells. Neither PPV nor ND alone significantly inhibited cell proliferation, indicating that ND sensitizes tumorigenic cells to PPV-mediated mitochondrial complex I inhibition. Morphologically, exposure to ND and PPV significantly increased the number of cells exhibiting enlarged morphology and cell protrusions. Furthermore, cell cycle analysis revealed that exposure to ND and PPV did not affect cell cycle progression but did alter reactive oxygen species (ROS) generation, specifically reducing superoxide (O₂⁻) generation and increasing hydrogen peroxide (H₂O₂) production, potentially independent of SOD activity. Assessment of ΔΨM revealed cell line–specific responses to ND and PPV exposure, with significant depolarization observed in MCF-7 cells and hyperpolarization in MDA-MB-231 cells. Moreover, in MDA-MB-231 cells, ND and PPV exposure led to the activation of AMPK and the inhibition of mTORC1. These findings suggest that combining ND and PPV targets critical biochemical pathways and molecular markers in breast tumorigenic cells, offering a potentially cost-effective and accessible therapeutic approach for resource-limited settings while aligning with Sustainable Development Goal 3 to promote global health and well-being. Although the combination of fasting and PPV shows promise, further in vivo preclinical research is required.
Assessing mitochondrial function, oxidative stress, and vitamin D status in dark-skinned patients with keloid disease
(University of Pretoria, 2024) Nortje, Evangeline; Bester, Janette; Phulukdaree, Alisa; granthattinghsa@gmail.com; Hattingh, Grant
Keloid disease is a challenging condition that occurs worldwide and is mainly experienced by dark-skinned population groups. Keloids are characterised by excessive fibrous tissue formation. Unlike normal scars, keloids extend beyond the boundaries of the original wound and continue to grow for months or years. They are firm, shiny, and irregular in shape. Symptoms, such as itching and pain, are associated with keloid disease. Keloids tend to appear in areas such as the chest, shoulders, earlobes, and upper back and have a high recurrence rate after treatment. Recent evidence has demonstrated that mitochondria play a critical role in meeting the substantial energy requirements of wound repair and facilitate healthy wound healing through cytoskeletal remodelling, mediated by mitochondrial morphological alterations and reactive oxygen species (ROS) signalling pathways. Although the exact pathophysiology remains unclear, emerging evidence suggests that mitochondrial dysfunction and oxidative stress play critical roles. Vitamin D (Vit D), known for its antioxidant properties and its influence on mitochondrial health, may also be implicated, especially in populations prone to Vit D deficiency. This study aimed to assess mitochondrial function, oxidative stress, and Vit D status in dark-skinned patients with keloid disease. Specifically, we explored the complex correlation between mitochondrial dysfunction and oxidative stress, as indicated by malondialdehyde (MDA) levels over a range of Vit D concentration levels. Keloid tissue and blood samples were collected from a cohort of patients with dark-skinned keloids. A total of 44 patients were included in the study, of which 27 were male and 17 were female and were between the ages of 18 ‒ 40 years old. Vitamin D status was assessed by measuring plasma Vit D concentrations, and lipid peroxidation was assessed by measuring MDA levels in the blood plasma, which is a biomarker for oxidative stress in cells. Mitochondrial morphology was examined using Transmission Electron Microscopy (TEM), focusing on the mitochondrial size, membrane integrity, and abundance. Correlation analysis was conducted to evaluate the complex relationship between mitochondrial integrity, MDA levels, and Vit D status. The results revealed a significant negative correlation (r = -0.52) between viable mitochondrial DNA (mtDNA) and MDA levels, indicating that higher levels of oxidative stress are associated with greater mitochondrial dysfunction. Patients that had lower concentration levels of Vit D showed elevated MDA levels within their blood plasma and more severe mitochondrial damage, such as no clear double membrane, improper folding of the cisternae, and small mitochondria compared to those with higher concentration levels of Vit D. In contrast, 32 dark-skinned patients had adequate Vit D levels. The TEM analysis highlighted significant mitochondrial abnormalities in keloid cells, including reduced size, damaged membranes, and decreased mitochondrial quantity. These defects were more pronounced in patients with Vit D deficiency. In conclusion, the findings of this study suggest that mitochondrial integrity and oxidative stress are key components of keloid pathogenesis, particularly in dark-skinned patients with low concentration levels of Vit D. The significant association between reduced mitochondrial function and increased oxidative stress highlights the potential role of Vit D in modulating these processes. This study emphasises the need for further research on Vit D supplementation along with calcium as a therapeutic intervention for keloid disease, particularly in dark-skinned individuals prone to low concentration levels of Vit D and keloid formation.
The effect of aspirin and lipopolysaccharide binding protein on hypercoagulability induced by lipopolysaccharide
(University of Pretoria, 2019) Pretorius, Elna; Bester, Janette; u26170147@tuks.co.za; Sibanda, Sthembile Valencia
Inflammation is a well-known underlying cause in many diseases. Chronic inflammation has a role in a host of common and often deadly diseases, including Alzheimer’s disease, rheumatoid arthritis, cancer, type 2 diabetes, heart disease, and even conditions like depression. A highly potent inflammagen might be one of the drivers of systemic inflammation, when present in circulation and it is the bacterial component called Lipopolysaccharide. It is known that lipopolysaccharide is located on the outer membrane of gram-negative bacteria. Recently, research has shown that this bacterial (and other) membrane components, when in circulation, might have a direct effect on blood hypercoagulability, which is a prominent hallmark of chronic, systemic inflammation. In this study it is shown that Lipopolysaccharide caused marked changes in the nature of the fibrin network formed. Furthermore, irregular red blood cells’ membrane, platelets activation and formation of microparticles were seen when examining with scanning electron microscope and confocal microscope. Addition of Lipopolysaccharide caused significant hypercoagulation and changes to viscoelasticity of whole blood, as noted using Thromboelastograph. Airyscan confocal microscopy was used to study abnormal fibrin(ogen) protein folding in the presence of Lipopolysaccharide, using a well-known amyloid protein marker of anomalous blood protein clotting, called Thioflavin T. Previously, it was shown that this anomalous clotting of fibrin(ogen) fibres is amyloid in nature. Mopping agents are needed to reverse the hypercoagulation induced by Lipopolysaccharide. Biochemical agents (mopping agents) that will prevent anomalous blood clotting are therefore needed to reverse the hypercoagulation induced by Lipopolysaccharide. Such agents could, in future be used to prevent anomalous clotting in inflammatory diseases, where increased Lipopolysaccharide levels are known to be present. The first Lipopolysaccharide mopping agent of choice is Lipopolysaccharide binding protein. The Lipopolysaccharide binding protein is a serum molecule that arbitrates cellular activation in reaction to endotoxin by ensuring the delivery of Lipopolysaccharide to either soluble or membrane bound forms of CD14 also known as mCD14. The second Lipopolysaccharide mopping agent in this study, is aspirin. Aspirin is an anticoagulant and commonly used anti-inflammatory agent. In literature aspirin has been found to increase fibrin clot porosity and susceptibility to lysis. Lipopolysaccharide binding protein, and the combination with aspirin, and the effects on anomalous blood clotting, is novel, and has not previously been studied. The research questions that this thesis thus aims to address is, at what minimal concentration can the mopping agent Lipopolysaccharide binding protein be used to effectively “mop up” Lipopolysaccharide, and can Lipopolysaccharide binding protein be used in combination with Aspirin, an anticoagulant agent, to reduce the signs of hypercoagulation produced by Lipopolysaccharide ex vivo? Which techniques can be used to visually see the difference in structure and elastic function of fibrin(ogen) as well as cells in the coagulation system, namely red blood cells and platelets? The results shown here reveal that Lipopolysaccharide binding protein can reduce the effect of Lipopolysaccharides on fibrin(ogen) and cells of the coagulation system at an exposure concentration of 2ng.L-1. The coefficient of variation from Airyscan confocal between control and treatments indicated a decrease in the intensity of samples treated with Lipopolysaccharide binding protein mixed with Lipopolysaccharide and a decrease in intensity from whole blood treated with aspirin compared to control (p=0.0106). The coefficient of variation calculated from Scanning electron microscopy, showed a distinct alteration in the clots from samples treated with Lipopolysaccharide binding protein and aspirin alone and mixed together gave (more variation of light coming from fibres, neatly branched forming three layers) as significant difference, when aspirin was mixed with Lipopolysaccharide showed less variation light from fibres fussed into one layer with the contact surface and the same with aspirin mixed with Lipopolysaccharide (less light variation) compared to the control (untreated PPP) (p<0.0001). From the Thromboelastography® results there was a statistically significant differences in clot strength between whole blood and whole blood mixed with Lipopolysaccharide binding protein and Lipopolysaccharide, (p=0.0412). This shows a decrease in platelet and/or fibrin(ogen) interaction resulting in a less dense clot that is less rigid in fibrinogen or platelets. The time from clot initiation to maximum clot formation decreased when whole blood was mixed with aspirin together with Lipopolysaccharide binding protein and Lipopolysaccharide as compared with naïve whole blood, (p=0.0071). This usually occurs in hypercoagulation seen by decreased time from clot initiation to maximum clot formation. The amount of thrombus total resistance decreased significantly when whole blood was mixed with aspirin and Lipopolysaccharide verses whole blood alone, (p=0.0078). This shows that the addition of aspirin to Lipopolysaccharide can lead up to hypocoagulable state were the clot strength decreases. When measuring fibre thickness it was found that Lipopolysaccharide binding protein alone and in combination with aspirin effectively reduced the large fibres produced by Lipopolysaccharide, almost back to normal size which is +-110nm. The difference was statistically significant (p<0.05). Lipopolysaccharide binding protein and aspirin showed impeccable results reducing the signs of Lipopolysaccharide-induced hypercoagulation, both in plasma and whole blood. Many non-communicable diseases have been shown to have a bacterial component. The amyloid protein created with Lipopolysaccharide, can be reversed in the blood model with the study’s novel combination of Lipopolysaccharide binding protein/aspirin could be implemented in applications for treatment of conditions where there is presence of Lipopolysaccharide particularly sepsis.
The effects of whole body electromyostimulation exercise training on physical fitness in middle aged sedentary females
(University of Pretoria, 2019) Wood, Paola Silvia; Clark, James R.; burbidgemichael8@gmail.com; Burbidge, Michael
The purpose of this study was to determine the effects of whole-body electromyostimulation (WB-EMS) augmented calisthenic training on physique, maximum strength, balance and aerobic endurance and compare said effects to an active control only performing calisthenic exercise. Electromyostimulation (EMS) also known as neuromuscular electrostimulation (NMES) produces a continuous muscle contraction via an electrode placed over a target muscle without any effort from the individual. EMS can be applied locally to a single muscle or in a WB-EMS fashion which allows for the stimulation of 16 different muscular regions simultaneously. Up to 2800 cm2 of muscle mass. EMS has been observed to significantly increase maximum isometric and isokinetic strength. The effects of EMS on body composition and power parameters has varied between different research studies and styles of application. EMS training does not appear to positively affect aerobic fitness in healthy populations though it has in patient populations. The effects of EMS on balance have so far not been explored. In the current study 39 sedentary females aged 35-55 years were allocated to either a control group performing 10-weeks of calisthenic training or an experimental group performing 10-weeks of WB-EMS augmented calisthenic training. Both groups completed a pre-testing (one week prior to the start of the 10-week training) and post-testing (one week following the conclusion of the 10-week training). The main outcomes were physique, determined by evaluating mass (kg), stature (cm) which were used to calculate Body Mass Index (kg/m2), waist and hip girth (cm) which were used to calculate the waist-hip ratio. Strength evaluated by measuring maximum handgrip and leg-and-back strength. Balance assessed by the functional reach (FR) test and aerobic endurance determined by measuring the distance covered with the Cooper-12-minute run/walk test and calculating maximal oxygen uptake (VO2 max) with the use of a regression equation. Following ten-weeks of exercise training significant increases were observed in mass and BMI (kg/m2) in the WB-EMS group and high between-group effect sizes were observed. Significant decreases in waist girth and waist hip ratio were observed in the control with high between-group effect sizes observed for both. Handgrip strength did not change significantly in either group. While leg-and-back strength increased significantly in the WB-EMS group and did not change in the control group. A significant large between-group difference and effect size was also observed in leg-and-back strength. The reach distance achieved in the FR test increased significantly in the WB-EMS group and did not change in the control, however no between-group differences, but a large between-group effect size was observed. Distance covered in the Cooper-12-minute run/walk test and VO2 max did not change significantly in the WB-EMS group but did significantly increase in the control group with medium effect sizes observed. No between group differences were observed with medium between group effect sizes. Findings support that WB-EMS significantly increases strength. However, it is important to carefully place electrodes to stimulate target muscles and any strength increases will not translate to improvements in dynamic strength unless dynamic movements of the same kind are applied during the exercise training sessions. This can be observed in the significant increases in leg-and-back strength as many of the exercise completed during the training session simulated the test action. While neither right nor left handgrip strength changed significantly and none of the exercises performed simulated the test action. WB-EMS does not affect aerobic endurance as shown by the lack of increase in the distance covered and VO2 max. WB-EMS also appears to significantly increase forward reach and dynamic balance, this is curious as no dynamic movements related to the test movement were applied during the exercise sessions. It may be that the primary muscles involved in the test movement were strengthened during the training and this in turn improved the test distance achieved. As there is currently very little information on WB-EMS effect on balance this area should be researched more in the future and the precise mechanisms involved should be investigated.
Diagnostic measures to inform training prescription to alter countermovement jump strategy and improve performance
(University of Pretoria, 2024-07-31) Bayne, Helen; Cronin, John; dsangari1@gmail.com; Sangari, Darius B.
Jumping is a fundamental demonstration of lower-body power across various sports, where the ability to generate maximal force quickly is crucial. Central to optimising vertical jumping performance is the stretch-shorten cycle (SSC), which involves the coupling of eccentric and concentric muscle actions with a rapid transition period. This cycle allows for the storage and release of elastic energy, enhancing force production in activities like the countermovement jump (CMJ). However, traditional measures of CMJ performance, such as jump height, often overlook the intricacies of how effective an athlete is at utilising the SSC mechanism. Recent research emphasises the importance of analysing jump strategy – how an athlete moves their centre of mass (COM) during the CMJ – metrics derived from ground reaction force (GRF) data to understand and improve SSC utilisation. Despite this, challenges persist in assessing the variability and influence of these metrics on CMJ performance. Moreover, while lower-body strength is known to enhance CMJ performance, its relationship with jump strategy is less understood. This thesis aims to evaluate jump strategy metrics, investigate the influence of lower-body physical characteristics, and test the effects of a training intervention on jump strategy and CMJ performance. The first study explores the variability and consistency of CMJ performance and jump strategy metrics, revealing the need for careful metric selection and interpretation. The second study examines the relationship between eccentric phase biomechanical parameters and CMJ performance, highlighting the importance and influence jump strategy has on optimising measures of CMJ performance. The third study investigates the influence of timing of peak GRF during the CMJ and maximal strength on jump strategy and CMJ performance, showing that stronger athletes achieve better performance outcomes and that the combination of increased strength and an optimised jump strategy produces the best CMJ performance outcomes. The final study of this thesis includes a six-week training intervention, which assess the impact of assisted CMJ exercises on jump strategy and performance. Despite changes in jump strategy metrics, no significant performance improvements were observed, suggesting that increased strength may be necessary to benefit from altered jump strategies. This research provides insights into the complex interactions between jump strategy, SSC utilisation and lower-body strength, offering practical implications for optimising CMJ performance. The findings underscore the importance of holistic metric analysis and strength development in enhancing jump performance outcomes, paving the way for further research and refined training methodologies.
The in vitro effects of a polyphenol epigallocatechin gallate on the cell viability, adhesion, and morphology of melanoma and endothelioma cells
(University of Pretoria, 2024-07-22) Hlophe, Yvette Nkondo; Serem, June Cheptoo; Bipath, Priyesh; u17070466@tuks.co.za; Adigun, Aminat Opeyemi Iyanuoluwa
Adhesion molecules play a crucial role in the process of cancer cells adhering to and metastasising to secondary locations within the body. Adhesion molecules such as E-cadherin, play a crucial role in the adhesion and metastasis of cancers. A polyphenol compound known as epigallocatechin gallate (EGCG) is predominantly found in green tea and has been demonstrated to possess health benefits, including antioxidant and anti-inflammatory properties. This study focused on investigating the impact of EGCG on the adhesion of tumour cells across four distinct cell lines. These included B16F10 melanoma cells, sEnd.2 endothelioma cells, and two control cell lines, namely RAW 264.7 macrophage cells and EA.hy926 endothelial cells. The study determined the half maximal inhibitory concentrations (IC50) values for four cell lines using a crystal violet assay and examined their morphological changes post EGCG treatment using haematoxylin and eosin (H&E) staining and polarisation optical differential interference contrast (PlasDIC) imaging. The study also used flow cytometry to study intracellular proteins and conducted an adhesion assay to evaluate the adhesion of the cell lines, pre-treated with fibronectin or collagen IV, after EGCG exposure. Crystal violet assays revealed that EGCG induced cell death in the cancerous cell lines, namely B16F10 and sEnd.2, while its impact on the non-cancerous cell lines, RAW 264.7 and EA.hy926, was less pronounced. Morphological results revealed that both cancerous cell lines exhibited characteristic features of cell death (membrane blebbing, compromised cell membrane, nuclear condensation and cell swelling) following treatment with EGCG. Flow cytometry analysis provided evidence that EGCG had halted the two types of cancerous cells (B16F10 and sEnd.2) and non-cancerous cells (RAW 264.7) cell cycle in the G1 phase. Furthermore, EGCG had enhanced the expression of E-cadherin in the B16F10 and RAW 264.7 cell lines, but E-cadherin expression in the sEnd.2 cell line was not as substantial. EGCG exposure on B16F10 cells pre-treated with fibronectin did not have the ability to reduce the adhesive ability on the B16F10 cells. However, EGCG exposure on B-16F10 cells pre-treated with collagen IV significantly reduced the adhesive ability of the B-16F10 cells. In contrast, sEnd.2 cells exposed to EGCG coated with either fibronectin or collagen IV, significantly reduced adhesion ability of the sEnd.2 cells. RAW 264.7 cells, responded differently: cells pre-treated with fibronectin showed a decrease in adhesion at the higher EGCG concentration compared to the control group. While cells pre-treated with collagen IV showed a general decrease in adhesion, irrespective of EGCG concentration. Page 11 of 125 EGCG demonstrated the ability to induce cell death in cancerous cells. However, the exact biological pathways and mechanisms that EGCG utilises to induce cell death are not yet fully understood and may vary between different types of cancerous cells. Therefore, further understanding of these mechanisms could have significant implications for cancer research.
A description of the profile of paediatric brain tumours in a tertiary neurosurgery service
(University of Pretoria, 2024-05-07) Padayachy, Llewellyn; Grobbelaar, Craig; debbiemags@gmail.com; de Beer, Debbie
Background Central nervous system (CNS) tumours are the most common form of solid tumours in children, leading to significant mortality and morbidity. In developing countries, survival rates for children with CNS tumours are lower than in developed nations. In South Africa, brain tumours account for 13% of paediatric cancers, yet local epidemiological data is scarce. No regional data exists for Steve Biko Academic Hospital in South Africa. Aim and Objectives This study aimed to describe the profile of paediatric brain tumours at Steve Biko Academic Hospital by examining patient demographics, histopathology, and imaging data. Methods This retrospective study reviewed medical records from the neurosurgery department at Steve Biko Academic Hospital. Data on patient demographics, tumour histopathology, and imaging from January 2019 to June 2023 were analysed to compile a comprehensive tumour profile. Results The study included 52 patients, with a male-to-female ratio of 1.74:1 and a mean age of 6.8 years. The highest tumour prevalence was in the 3–8-year age group. Infratentorial tumours were the most common. In descending order, the most prevalent tumour types were mixed glioma, medulloblastoma, astrocytoma, and ependymoma. Discussion The male predominance aligns with existing studies, potentially due to sex differences in brain development. The high incidence of infratentorial tumours may be linked to genetic susceptibility and rapid cell proliferation in this region. The common tumour types in this study all originate from highly prolific cells, contributing to an elevated risk of tumorigenesis. Conclusion The findings in this study, such as mean age, tumour location, and tumour prevalence, corroborate similar studies from Westernised countries. Understanding the epidemiology of paediatric brain tumours is vital for improving diagnosis, treatment, and healthcare policies. Regional data is essential for enhancing medical knowledge and improving patient outcomes. Keywords: Paediatric brain tumours, malignancy, cancer, supratentorial, infratentorial, posterior fossa glioma, medulloblastoma.
Investigating the use of neurorehabilitation scales in paediatric neurosurgical patients at a tertiary academic hospital in Gauteng
(University of Pretoria, 2024-03-30) Padayachy, Prof. L.C; Grobbelaar, Dr. C.W; Padayachy, DR. V; u17001014@tuks.co.za; Smit, Nicoleen
Introduction: Children with central nervous system disorders frequently display altered synaptic processes, discordant brain activity, delayed development, and reduced neuroplasticity. A major hallmark of neuroplasticity is the ability of the brain to reorganize and heal after injury, and may be enhanced through neurorehabilitation. Early rehabilitation, particularly for conditions such as traumatic brain injury, brain tumours, spina bifida, and hydrocephalus, can dramatically improve patients’ functional outcomes. This study sought to examine the criteria used for identifying paediatric neurosurgery patients’ eligibility for neurorehabilitation, evaluate the clinical outcomes assessed by particular neurorehabilitation scales, and examine the time between surgery and rehabilitation, as well as the length of the overall inpatient stay. Materials and Methods: This retrospective study was conducted at Steve Biko Academic Hospital (SBAH) and Tshwane Rehabilitation Hospital (TRH), using patient records. The study evaluated the length of hospital stay, neurorehabilitation eligibility criteria, and the specific scales used in neurorehabilitation. Results and Discussion: A total of 51 patients were included in the study. The study revealed that, despite the absence of defined criteria, clinical judgment based on physical, cognitive, developmental, and social factors was the primary determinant for transferring patients to the rehabilitation facility. Interestingly, 21.5% of the study population demonstrated cognitive and physical improvement based on the documented Glasgow Outcome Scale, Glasgow Coma Scale, and Waterlow Scales. The data revealed that patients with brain tumours spent the longest time at the tertiary academic hospital (approximately 47 days), and had the longest overall length of stay (119 days) with the least number of transit-affecting factors. Patients with hydrocephalus had the longest hospitalisation stay at the neurorehabilitation facility (approximately 70 days). The study highlighted disparities in hospitalisation periods based on neurological conditions, with notable impacts on hydrocephalus patients. Neurorehabilitation scale usage exhibited inconsistencies, and a fraction of patients demonstrated improvement in functional outcomes. Furthermore, other reasons for requiring transfer to a rehabilitation facility such as nutritional and psychosocial support were also considered. Rehabilitation interventions, including occupational therapy, physiotherapy, and speech therapy, were tailored to specific needs. Conclusion: The study's results highlight the complex nature of neurorehabilitation in children, marked by extended hospitalisation and resource-intensive care demands, underscoring the vital necessity for tailored support. Furthermore, these findings emphasize the ongoing imperative of enhancing rehabilitation strategies, particularly for patients with limited cognitive and physical progress, stressing the continued dedication to advancing rehabilitative approaches to benefit a broader range of patients and enhance their overall well-being. Therefore, the inclusion of the Bayley Scales of Infant and Toddler Development may serve to improve the comprehensiveness of the assessment, particularly for younger patients, highlighting a potential area for improvement in the rehabilitation protocol.
An ex vivo study on the hypercoagulability of brain cancer patients at an academic hospital by studying the morphological and viscoelastic properties of platelet-poor plasma
(University of Pretoria, 2024-07-01) Bester, Janette; Mahlangu, Thandi; Padayachy, Llewellyn; u17014222@tuks.co.za; Rademeyer, Lilu
Cancer and its associated outcomes rank among the primary contributors to global mortality. Tumour cells are not only able to alter their cellular physiology to promote their own growth but can also disrupt the coagulation system by interfering with natural signals and pathways in the body, particularly the inflammatory and coagulation pathways. As a result, about 20% of brain cancer patients suffer complications associated with coagulopathies. Extensive research is required to understand the coagulation potential in brain cancer patients and identify the factors that trigger hypercoagulation in these patients. This study aimed to investigate the potential hypercoagulable state in brain cancer patients at an academic hospital, comparing them to healthy individuals by studying the morphological and viscoelastic properties of platelet-poor plasma (PPP), specifically focusing on fibrin formation. This study measured the viscoelastic properties of PPP during clot formation using Thromboelastography®. Additionally, scanning electron microscopy was employed to analyse and compare the clot ultrastructural morphology of fibrin networks between patients and healthy individuals. The fibrin fibre thicknesses of both groups were then measured and compared using the ImageJ software. Branching of the fibrin fibres was measured by determining the fractal dimensions from the scanning electron microscope images with Fractalyse software. Clinical tests—including the international normalised ratio (INR), C-reactive protein (CRP), and procalcitonin tests (PCT)—were obtained from patient records and used to create a clinical profile of the patient population. These values were compared to healthy reference ranges to identify any potential abnormalities in the patient group. The INR was used to determine the time it took for the patients’ blood to clot, whereas the CRP and PCT were used to evaluate their inflammatory status. The clinical tests showed normal INR and PCT values but elevated CRP values when compared to normal ranges. This indicated that the brain cancer patients in this study exhibited normal clotting times and no signs of bacterial infection. The elevated CRP values could be indicative of elevated inflammation caused by the brain cancer. The viscoelastic and ultrastructural results showed that there were no significant differences in any of the analyses between the healthy individuals and those with brain cancer, except with regards to the fibre thickness. Specifically, this study found that brain cancer patients have thinner fibrin fibres than healthy individuals. Thinner fibres exhibit a reduced rate of dissolution compared to thicker fibres, resulting in the persistence of clots and, consequently, increasing the patient's susceptibility to thrombotic events. The results from this study open avenues to further study the impact of brain cancer on the formation of fibrin fibres during clot formation. While the tests used in this study might not have been sensitive enough to identify subclinical changes, future tests measuring fibrinogen levels, coagulation factors, and clot lysis could provide valuable insights into how coagulation is affected in these patients. Such insights might reveal potential targets for more effective patient management.
An investigation of somatosensory evoked potential responses during brain tumour surgery
(University of Pretoria, 2024-07-02) Padayachy, Llewellyn; Grobbelaar, Craig; u17044792@tuks.co.za; Rasool, Muhammed Yusuf
Introduction: Intra-operative neurophysiological monitoring (IONM) is the use of electrophysiological tools to evaluate and monitor the functional status of the nervous system during surgery. The main aim of IONM is to mitigate the risk of damage to nervous tissue during neurological surgery, such as brain tumour resection surgery, and to reduce the incidence of postoperative neurological complications. The IONM techniques commonly employed include somatosensory evoked potentials (SSEPs). The main use of SSEPs is the indirect warning of possible sensory nervous pathway injury. Intraoperative SSEP monitoring requires adroit coordination by healthcare professionals. Despite progression in this field, there is rather limited research comparing responses in the cortical, sub-cortical contralateral, and ipsilateral SSEP responses. Aim: This study aimed to evaluate the use of continuous SSEP monitoring during resection of intracranial brain tumours to provide an ongoing functional assessment of the somatosensory pathway. Methods: This retrospective study was conducted using data from patients who underwent continuous somatosensory evoked potential (SSEP)monitoring during brain tumour reresection surgery between January 2019 and December 2021 at Steve Biko Academic Hospital (SBAH). The data was compiled electronically and then subjected to statistical analysis as per the study the objectives. Results: Contralateral latencies showed consistently higher values than ipsilateral readings across all the cortical measurements. In addition, the cortical latencies consistently exceeded the subcortical latencies. Particularly, the latencies prior to brain tumour resection tended to exhibit greater values than those recorded during and after the resection process. Conclusion: The data suggests that latency tends to decrease over the course of surgery, reflecting improvements in sensory pathways following tumour removal. This pattern suggests a dynamic relationship between the timing of the surgical intervention and the somatosensory evoked potential latencies.
The in vitro effects of diallyl trisulfide on osteoclast formation and function in RAW 264.7 murine macrophages
(University of Pretoria, 2024-03) Kasonga, Abe; Visagie, Michelle; u15001327@tuks.co.za; Mukozi, Maria Goretti Burungi Agnes
Osteoclasts are large multinucleated cells that resorb or degrade bone. Over-active osteoclasts result in fragile bones that fracture easily, causing bone degenerative disorders such as osteoporosis. Inhibiting osteoclast activity represents a viable strategy to combat bone degeneration. When the receptor activator nuclear factor-kappa B ligand (RANKL) interacts with the receptor activator nuclear factor-kappa B (RANK), found on osteoclast precursors, precursors of osteoclasts develop into mature osteoclasts. Reactive oxygen species (ROS), nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinase (MAPK) pathways are crucial for osteoclast differentiation and activation in a process known as osteoclastogenesis. Diallyl trisulfide (DATS) is an organosulfur compound produced in garlic which has shown powerful anti-cancer effects in vitro including decreased expression of MAPK proteins. However, the effects of DATS on osteoclast differentiation, stimulation, and function are unknown. As a result, this study aimed to investigate how DATS influences osteoclast differentiation and function by utilising RAW 264.7 murine macrophages. In this study, various methods were carried out to establish the effects of DATS on osteoclast formation and function in RAW 264.7 murine macrophages. Using a resazurin assay, 1-5 µM of DATS was shown not to affect the viability of the RAW 264.7 murine macrophages. RAW 264.7 murine macrophages were then differentiated into osteoclasts using RANKL and stained for tartrate-resistant acid phosphatase (TRAP). It was found that DATS significantly decreased the number of TRAP-positive osteoclasts formed after exposure to a DATS concentration of 2 µM. The effects of DATS on MAPK expression were then investigated through western blotting. It was shown that DATS significantly decreased the expression of crucial MAPKs, c-Jun N-terminal kinase (JNK) at 2 minutes of RANKL exposure, potentially inhibiting c-Jun phosphorylation and impeding nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expression crucial for osteoclastogenesis. While ERK protein expression remained unchanged, the significant reduction in P38 protein expression at 5 minutes of RANKL exposure could diminish NFATc1 levels, thereby reducing osteoclast formation. The effects of DATS on the expression of oxidative stress markers were investigated in RAW 264.7 murine macrophages through western blotting. It was shown that DATS exhibited a significant increase in the expression of the antioxidative protein, nuclear factor erythroid 2-related factor 2 (NRF2) while significantly decreasing the expression of oxidative stress marker nicotinamide adenine dinucleotide phosphate oxidase-1 (NOX1), suggesting a potential reduction in ROS-mediated MAPK activation and mature osteoclast formation. This study represents a significant milestone in elucidating the impact of DATS on osteoclastogenesis using RAW 267.4 murine macrophages. The findings underscore the novel role of DATS in influencing osteoclast formation and key proteins essential for osteoclastogenesis. Notably, DATS was shown to be a potent inhibitor of osteoclastogenesis by decreasing the number of TRAP-positive osteoclasts, targeting MAPK signalling and ROS pathways. The comprehensive investigation conducted in this study provides valuable insights into the intricate interplay between DATS and osteoclastogenesis, offering a promising avenue for future research and therapeutic interventions in the realm of bone degenerative diseases.
Investigating hypercoagulability in brain cancer patients by studying the viscoelastic and ultrastructural properties of whole blood
(University of Pretoria, 2024-04-02) Bester, Janette; Padayachy, Llewellyn; Mhlanga, Thandi; zenobiaseyfert@gmail.com; Seyfert, Zenobia Jacomine
Brain cancer is a condition with a relatively high rate of loss of life, regardless of patient demographics. The location and malignancy of the tumour are both determinants of this mortality rate, however there are complications associated with the disease that also contribute to this mortality rate. One of these complications are coagulopathies which may lead to thrombotic events. Thrombosis is a reality for many brain cancer patients which may contribute to a poor prognosis. This study analysed the contribution of different components that may contribute to the overactivation of the coagulation pathway in this patient group. Brain cancer in South Africa has an incidence rate of about 1.5/100000. There is also a strong association of coagulopathy in cancer patients that may be attributed to morphological changes in red blood cells, inflammation, as well as the influence of inflammation on the release of certain procoagulants. This study aimed to investigate the morphological and viscoelastic changes during coagulation in patients with brain cancer by studying the components involved in coagulation and their contribution to hypercoagulability in these patients. This was done using ultrastructural and viscoelastic techniques. Light microscopy was used to determine the deformability of red blood cells by calculating the axial ratios. Scanning electron microscopy was used to study the ultrastructural properties of clots as well as red blood cells, and platelets. Lastly, the viscoelastic properties of whole blood were quantitatively analysed using thromboelastography®. This provided insight into the contributing factors to coagulopathy in brain cancer patients. When these factors are analysed and understood, insights into the clot formation in brain cancer patients may contribute to understanding the thrombotic risk in these patients and possible interventions based on the effects of the contributing factors. From the results it was established that the red blood cell deformability, ultrastructural properties of fibrin fibres, and viscoelastic profiles during clot formation of this patient group are changed to develop denser and faster forming clot types. This research therefore contributed to the field by providing information that can guide understanding about the relationship between primary brain cancer and coagulation of whole blood. By using this research alongside current knowledge, targeted monitoring of the contributing coagulation factors, such as changes in fibrin formation, and subsequent intervention can be applied to treat brain cancer-associated thrombotic risk.
Biomechanical investigations of coordination during initial acceleration in highly trained to world class sprinters
(University of Pretoria, 2023) Bayne, Helen; Bezodis, Neil; u05038601@tuks.co.za; Donaldson, Byron John
Initial sprint acceleration is a complex and dynamic skill, requiring the application of large forces to propel the body forwards. Effective force application is achieved through the use of joint and segment rotations in an organised and inter-related manner. While many of the isolated angular kinematic features associated with effective external force profiles are established, little is currently known about the relationships that exist between the key segments during the first steps of acceleration, i.e., the coordination of movement between functionally related elements. Through a series of three studies, this thesis explores inter- and intra-limb coordination during initial acceleration in sprinters ranging from highly trained to world class level, to enhance the understanding of sprint acceleration technique and performance. The first study provided a detailed description and quantification of inter-limb thigh-thigh, intra-limb shank-foot, and trunk-shank coordination during the first four steps of acceleration, and investigated changes in coordination between steps. Specific coordination features were identified and between-individual variation in coordination patterns in preparation for, or response to, the major transitions in the step cycle, i.e., touchdown and toe-off, were observed. Additionally, step-to-step changes in coordination and angular kinematics were identified, showing clearly differentiated coordination in step 1 compared to later steps. The second study utilised a novel application of hierarchical cluster analysis to vector coding data in order to identify and characterise sub-groups of sprinters with similar thigh-thigh and shank-foot coordination patterns, and subsequently explored discrete kinematic and performance differences between sub-groups. Three sub-groups were identified in step 1 and two sub-groups over steps 2-4. Sub-groups tended to be differentiated by differences in thigh-thigh coordination at the beginning and end of the step, and shank-foot coordination during flight as well as during ankle dorsiflexion in early stance. Combining sub-groups from step 1 and steps 2-4 to describe entire initial acceleration strategies, cluster combinations identified coordination approaches more likely to be associated with higher level sprinters and better performance. In the final investigation, relationships between coordination and lower body strength were evaluated in the context of dynamical systems theory, and the interaction of these two factors with regard to acceleration performance was explored. Several correlations existed between measures of lower body strength and features of thigh-thigh and shank-foot coordination, while multiple regression analysis suggested the presence of interaction effects between coordination and tests associated with lower body power in relation to performance. Thus, lower body power appeared to influence the relationships between coordination features and performance, such that the effectiveness of particular coordination patterns varied depending the lower body power of the athlete. The work included in this thesis provides a basis for understanding coordination during initial sprint acceleration, and includes several novel and exploratory approaches to investigating these questions which provides relevant information for practitioners and coaches interested in exploring the organisation of the body and coordination of segments during initial acceleration. Moreover, this work facilitate the generation of new hypotheses and encourages new directions in future research.
A comparison of the risk profile for developing illness-related medical encounters in half- and ultra-marathon runners
(University of Pretoria, 2023) Wood, Paola Silvia; Camacho, Tanya; Schwellnus, Martin Peter; dean.chris.baker@gmail.com; Baker, Dean
Research comparing risk profiles for medical encounters in race entrants at distance running events is limited. The aim of this study was to determine and compare the risk profile for developing illness-related medical encounters in half-marathon compared to ultramarathon runners. Online pre-race medical screening questionnaire data from 76654 consenting race entrants (71.8% of all entrants) over four years of Two Ocean Marathon (2012-2015) were analysed, using a prospective cross-sectional observational study design. Study participants were classified in four risk categories (‘very high risk’, ‘high risk’, ‘intermediate risk’ and ‘low risk’) based on history of the following: existing cardiovascular disease (CVD), history of any symptoms of CVD, or any risk factor for CVD, disease in other organ systems, medication use and history of collapse in half-marathon and ultramarathon. We report the prevalence (%; 95%CI) in each risk category for half- and ultra-marathon entrants. When comparing the OR between the ultramarathon participants compared to the halfmarathon participants the OR was 43% (OR 1.43; CI 1.19-1.73) more for an ultramarathon participant to be categorised in the “very high” risk category compared to a half-marathon participant. The OR for an ultramarathon participant to be categorised in the “high” risk category was 28% (OR 1.28; CI 1.14-1.43) more compared to the half-marathon participant, whilst the OR for an ultramarathon participant to be categorised in the “intermediate” risk category was 83% (OR 1.83; CI 1.71-1.96) more compared to the half-marathon participants. This finding indicates a higher number of participants in the ultramarathon will compete with either/or existing CVD, symptoms of CVD, risk factors for CVD, diseases in other organ systems, prescription medication use, use of NSAIDs before/during a race, history of collapse compared to the half-marathon participants. This may suggest that the ultramarathon participants have a higher risk for sustaining a medical encounter during a race compared to the half-marathon participants during a half-marathon race.
Between-session reliability of performance and asymmetry variables during lower limb strength tests and sport-specific tasks in netball players
(University of Pretoria, 2023) Bayne, Helen; Clark, James; charnesctt@gmail.com; Britz, Charne Beatrix
Title: Between-session reliability of performance and asymmetry variables during lower limb strength tests and sport-specific tasks in netball players Inter-limb asymmetry, the ratio that represents the performance comparison between two limbs, has been quantified during lower body plyometric, ballistic and isometric strength assessments. Asymmetries vary across different tasks and performance metrics, making asymmetries very task specific and metric dependent. In addition, inertial measurement units (IMUs) have recently developed into a popular tool to quantify training load and inter-limb differences in a sport-specific environment. The reliability of a measure shows its reproducibility across repeated trials. Any assessment requires a high reliability to ensure low measurement error. Measurement studies assessing inter-limb differences have generally shown acceptable within- and between-session reliability for observed performance measures. However, recent research investigating the within- and between-session reliability of the actual derived asymmetry value found this to be highly variable and unreliable. This measurement study aimed to investigate the within-day and between-day reliability of force metrics and inter-limb force asymmetry during unilateral and bilateral variations of isometric, ballistic and plyometric laboratory-based strength assessments. A second aim was to investigate the between-day reliability of IMU-derived impact load and impact load asymmetry during sport-specific drills. During the netball pre-season, 25 healthy female university netball players (mean ± SD age: 20 ± 1.7, stature: 177.6 ± 7.0 cm, mass: 69.9 ± 8.3 kg) participated in this study. Testing consisted of four days. On Day 1 participants performed three trials of both unilateral and bilateral variations of the drop jump (DJ), countermovement jump (CMJ), and isometric squat (ISQ). On Day 2 participants performed six routine warm-up drills with IMUs attached to the shin to measure the frequency and intensity of ground contacts experienced. Day 1 and Day 2 was repeated on Day 3 and Day 4. Inter-limb asymmetries were quantified for peak force in the drop jump, countermovement jump and isometric squat. For the field-based assessments inter-limb asymmetry was quantified for impact load. The coefficient of variation (CV) and intraclass correlation coefficient (ICC) was determined for each participant for all performance and asymmetry metrics to define the within-and between-day reliability. Good to excellent within- and between-day reliability was seen for all force metrics for both variations of the strength assessments. Relative within- and between-day reliability for force asymmetry variables during the bilateral strength tests ranged from good to excellent (ICC: 0.89 – 0.94). For the unilateral strength assessments relative within- and between-day reliability were poor to moderate (ICC: 016 – 0.67). Absolute within- and between-day reliability for all force asymmetry variables were unacceptable (CV: 26.4 – 645.5%). During the sport-specific drills, moderate relative reliability (ICC: 0.50 – 0.60) and unacceptable agreement (CV: 13 – 19%) were seen for impact load in all the controlled drills. When considering all the drills together, impact load reliability was moderate (ICC: 0.58 – 0.60), with a CV of 11%. In all the sport-specific drills impact load asymmetry was inconsistent between days and showed very poor between-day reliability (CV: 44.3 – 422.6%; ICC: -0.21 – 0.15). Performance variables used to quantify inter-limb asymmetries are reliable within- and between sessions, however, high variability is seen when considering the reliability of asymmetry measures. When describing, comparing, or tracking lower limb asymmetries during unilateral and bilateral strength assessments, as well as during sport specific drills, practitioners should carefully consider test selection, and metric- and asymmetry reliability. Practitioners should not only look at the ecological validity of a specific test to create an asymmetry profile of an athlete, but also consider the reliability and variability of the test, test metrics and asymmetry measures.
Investigating clotting changes in patients from Steve Biko Academic Hospital with intracerebral haemorrhage by studying the viscoelastic and ultrastructural properties of whole blood.
(University of Pretoria, 2023) Bester, Janette; Padayachy, Llewellyn; Alummoottil, Sajee; ferreirashene99@gmail.com; Ferreira, Shene
Patients with intracerebral haemorrhage (ICH) have an increased risk of experiencing clotting changes when compared to healthy individuals, with recent research indicating that a bleeding event may enhance the prothrombotic effects of ICH. Although the effects of inflammation on the properties of whole blood (WB) in these patients have been studied extensively, there is a scarcity of research on the effects of ICH on the haemorheological-, and morphological properties of WB in patients with ICH. Therefore, this study utilised microscopy and viscoelastic techniques to examine clotting in these patients, in order to obtain a better understanding of the changes in clot formation in ICH patients. This may give more insight into thrombotic risk assessment and management. Whole blood from traumatic ICH (TICH) and non-traumatic ICH (NTICH) patients were compared to healthy controls. For a haematological overview of the ICH patients, routine clinical test results were utilised. Light microscopy (LM) was used to quantify the amount of deformed red blood cells (RBCs) present in each patient group. Scanning electron microscopy (SEM) was used to study the ultrastructural changes in blood cells and formed clots. Thromboelastography (TEG®) was used to study the changes in clot kinetics during clot formation. Results from the full blood count and C-reactive protein (CRP), demonstrated a tendency toward inflammation in both patient groups. No significant difference was seen in RBC deformation in both groups compared to the controls, indicating there was no significant RBC deformation in the patient groups. Ultrastructural studies on RBCs using SEM in both patient groups showed fine membrane changes and increased aggregation when compared to healthy controls. Platelets (PLTs) also appeared to be spread and fibrin fiber formation was disorganised. Viscoelastic results showed that clots formed faster in ICH patients, with increased strength and rigidity, thus revealing a hypercoagulable nature during clotting in these patient groups. The results of this study have revealed the marked differences in coagulation and associated blood components in TICH and NTICH patients compared to healthy controls. They provide a greater understanding of clot dynamics that could contribute to an increased risk of thrombotic events, traceable through viscoelastic techniques. This justifies further investigation into the utilisation of these techniques in a clinical, point-of-care setting, in order to enhance the prevention and management of thrombotic events in these patients.
Investigating the role of oxidative stress in apoptosis induced by a sulphamoylated estradiol analogue in breast cell lines
(University of Pretoria, 2020) Visagie, M.H. (Michelle Helen); Joubert, Annie M.; tebogo_lebelo@yahoo.com; Lebelo, Maphuti Tebogo
2-Methoxyestradiol (2ME), a 17β-estradiol metabolite, exerts anticancer properties however, the compound was found to possess low bioavailability. This resulted in the in silico-design of 2ME analogues with a sulphamoyl moiety which made them more potent than the parent compound. Sulphamoylated 2ME analogues are suspected to induce the antitumourigenic effects through the induction of reactive oxygen species. However, the exact role of oxidative stress in the activity exerted by these compounds remains elusive. In the current study, 2-ethyl-13-methyl-17-oxo-7,8,9,11,12,13,14,15,16,17- decahydro-6-cyclopenta[a]phenanthrane-3 sulphamate (ESE-one) was chosen as a sulphamoylated estradiol analogue representative to investigate the role of reactive oxygen species (ROS) in the effects exerted by these sulphamoylated compounds on cell proliferation, morphology, cell cycle progression, antioxidant activity and mitochondrial membrane potential in estrogen receptor positive breast epithelial adenocarcinoma (MCF-7) cells and estrogen receptor negative breast epithelial adenocarcinoma (MDA-MB-231) cells. Fluorescent microscopy data revealed that sulphamoylated estradiol analogues induced more ROS production compared to their non-sulphamoylated counterparts in both MCF-7- and MDA-MB-231 cells. Crystal violet staining demonstrated a significant growth inhibition in cells exposed to sulphamoylated estradiol analogues compared to cells exposed to the non-sulphamoylated compounds. ESE-one exposure resulted in a ROS-dependent growth inhibition which was repressed by tiron (superoxide inhibitor), trolox (peroxyl inhibitor) and DMTU (hydrogen peroxide inhibitor). ESE-one exposure to MCF-7- and MDA-MB-231 cells resulted in an accumulation of cells in G2/M phase after 24 hours and sub-G1 phase after 48 hours. The effect induced after 24 hours exposure was inhibited by tiron and trolox, and that induced after 48 hours exposure was inhibited by tiron, trolox and DMTU. Proliferation data was confirmed by morphology studies. Tiron, trolox and DMTU significantly decreased the number of rounded cells, shrunken cells and apoptotic bodies in MCF-7 and MDA-MB-231 cells induced by ESE-one exposure; cell density was recuperated indicating the rescue effects of ROS inhibitors. Antioxidant activity data demonstrated that ESE-one induced cell rounding and antiproliferative effects via ROS evident in the reduced catalase protein concentration in MCF-7 cells which was opposed by tiron and DMTU and in MDAMB- 231 cells, inhibited by tiron and trolox. Reduction in mitochondrial membrane potential was inhibited by tiron in MCF-7 cells and DMTU in MDA-MB-231 cells. This in vitro study suggests that ESE-one induces growth inhibition, cell rounding, cell cycle arrest, catalase inhibition and depolarization of the mitochondrial membrane by production of superoxide anion, peroxyl radical and hydrogen peroxide which culminates in apoptosis. This study contributes to targeted therapy based on ROS-dependent cell death pathways in tumourigenic breast cells.
Investigating lipopolysaccharide-induced inflammation in Sprague-Dawley rats on liver function with specific focus on coagulation factor production
(University of Pretoria, 2023) Bester, Janette; Allummoottil, Sajee; karabor8@gmail.com; Rathebe, Karabo
Neurodegenerative diseases, such as Parkinson’s disease (PD) and Alzheimer’s disease (AD), have long confounded the scientific and medical communities. The extensive research into these diseases, particularly AD has produced several plausible theories on its progression, but this research has so far failed to yield any lasting results in producing an effective treatment of AD. With the increase of the prevalence of AD in an increasingly aging world population, elucidating a model of disease progression that takes into account the neurodegenerative characteristics that are hallmarks of the disease, along with its related metabolic and organ system effects has become a public health priority. Systemic inflammation has become one of the main components of several non-communicable diseases, in particular AD. In this study, the aim was to investigate an emerging theory of AD pathogenesis that may combine the neurological and metabolic aspects that are often seen in these patients, the gut-liver axis theory. This theory states that the liver is the first organ to come into contact with circulating lipopolysaccharide (LPS), which would cause the kind of low-grade and chronic inflammation that would cause significant long-term damage to the liver, thus resulting in the organ system failure secondary to AD. Therefore, a model of LPS-induced inflammation was studied in adult male Sprague-Dawley rats to determine the effect of the systemically -induced LPS inflammation on the liver with particular focus on coagulation factor production and the possible prevalence of liver damage and fibrosis. The model was successfully created over a 10-day period, after which the animals were terminated, and the liver tissuetissues collected and processed for use in light-microscopy and transmission electron microscopy (TEM). The levels of coagulation factors, tissue factor (TF) and fibrinogen (FG), were also determined by use of ELISA assays. Behavioural testing was used as a means to confirm the presence of AD-like symptoms in the model induced by the LPS administration. Due to the low concentration of LPS administered into the animals during the experimental period, the levels of tissue factor and fibrinogen were not significantly higher or changed when compared with the control group. Similarly, the morphological analysis of liver tissue showed that the administration of LPS as designed in this study did not significantly affect the liver tissue. At an organelle level, it was found that the LPS administration produced a mild to moderate negative effect on the liver, an indication of the initial stages of liver damage due to the exposure of LPS. In conclusion, the administration of LPS in Sprague-Dawley rats as modelled in this study did not induce the systemic inflammation that would cause significant liver damage and subsequent fibrosis, only producing the initial stages liver damage seen at sub-microscopic level. This however shows that short-term exposure to low levels of LPS will induce changes on a cellular level and that long-term exposure could accumulate to complications that could lead to clinical presentation. Key words: inflammation, lipopolysaccharide, liver

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