Electronic Theses and Dissertations (Masters)
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Item Characterizing Luminal A breast cancer heterogeneity and in vitro response to hormone therapy(University of the Witwatersrand, Johannesburg, 2024) Gallant, SimoneBreast cancer is the most prevalent form of cancer diagnosed amongst women worldwide, responsible for a mortality rate of 6.9% and responsible for 684,996 deaths. Breast cancer is the most heterogeneous disease characterised by variations in genomic, epigenomic transcriptomic and proteomic profiles. The limited research on intratumoural heterogeneity in breast cancer and hormone therapy is the motivation for our study to further aid in understanding stemness markers influencing luminal A breast cancer and the effects hormone therapy has on biomarkers associated with breast cancer. In our study, we optimised modified essential 8 media to culture sorted cell populations in optimal conditions without differentiation ensuring stemness markers are maintained. Magnetic cell sorting was used to separate cells based on stemness markers CD133 and CD44. To verify these sorted markers flow cytometry was performed. The evaluation of the effects hormone therapy had on biomarkers was performed via immunocytochemistry and analysed using cell profiler. Our study revealed significant differences between subpopulations in MCF7 and T47D cell lines. It emphasizes the importance of CD44 and CD133’s role in tumour progression and its possible influence in hormone therapy. Our findings show that in populations with both stemness markers present in T47D cell line there is a reduction in progesterone receptor expression when treated with Tamoxifen. We also noticed the difference between population and hormone therapy impact on these changes. Thus, stemness markers are vital in tumour progression and the interaction of biomarkers and hormone therapy. However future research in the biological process and pathway activation is needed to further understand the intricacies of CD44 and CD133 mechanism of action as well as its association to biomarkers common pathwaysItem Effects of binge alcohol consumption on the development of the femur of adolescent Sprague Dawley rats(University of the Witwatersrand, Johannesburg, 2024) Mngoma, Ndabenzinhle Ronald; Bhika, AkaashniExcessive alcohol consumption adversely affects bone metabolism, thus resulting in reduced bone length, density, and strength. While excessive alcohol consumption is an established risk factor for osteoporotic fractures, there remains a dearth of information in literature regarding bone effects of binge alcohol consumption in adolescents. Therefore, our study aimed to examine the effects of binge alcohol consumption in an acute and chronic binge model, on the development and growth of the adolescent femur. Forty-eight Sprague Dawley rats (24 male and 24 female) aged 7 weeks were randomly allocated to one of the 4 treatment groups (n= 12/group) receiving binge alcohol (3g/kg of 20% alcohol) or caloric equivalent of maltose dextrin (pair-fed control), via oral gavage. The treatment groups were; A1, receiving alcohol on 3 alternating days for one week, C1, receiving the caloric equivalent of maltose dextrin in the same manner as A1 (acute), A4 and C4 received treatments in the same manner as A1 and C1 for four consecutive weeks (chronic). Trabecular morphometry in both the proximal and distal epiphysis, and cortical dimensions were assessed by using three-dimensional Micro- Focus X-ray Computed Tomography (3D-μCT) and Volume Graphics Studio® software. The morphology of the epiphyseal growth plate was examined by Haematoxylin and Eosin staining, whereas Ki-67 immunostaining was employed to quantify the proliferation of chondrocytes in the proliferative zone of the growth plate. A three-point bending test was employed to examine the effects of alcohol on bone strength. Results showed that binge alcohol consumption causes thinner trabeculae that are more widely spaced and with a smaller bone to volume ratio (BV/TV). However, the tensile strength was similar in the alcohol exposed rats and paired fed groups in male rats, whereas it appeared improved in female rats exposed to alcohol. A binge model also affected the number of chondrocytes in the proliferative zone negatively. All the adverse changes observed in the osseous tissue in the current study were shown in the male rats. Our study found alcohol to have no adverse effects on female rats, which could be due to hormonal differences.”Item Impact of Cannabidiol and Tamoxifen treatment on cell death and cell survival in breast cancer in vitro(University of the Witwatersrand, Johannesburg, 2024) Mahasha, Mahlatse Fortunate; Augustine, TanyaThe main non-psychotropic component of Cannabis sativa, Cannabidiol (CBD), alleviates breast cancer treatment-associated side effects but its effects with standard therapy remain unclear. In breast cancer, CBD has been shown to exhibit anti-cancer properties by inducing apoptosis and pro-death autophagy. This study aimed to investigate the effects of combined CBD and Tamoxifen treatment on metabolism, cell death, and cell survival mechanisms in luminal-A breast cancer cell lines MCF7 and T47D. The CBD concentration relative to IC50 was established by testing a range of CBD concentrations: 5 μM, 7 μM, and 10 μM, at 24 h, 48 h, and 72 h using the neutral red cell viability assay. The scratch assay was used to determine the effects of the concentrations on migratory capacity. Two models of treatment were used, single-dose treatment (model 1) and daily-replacement treatment (model 2), and appropriate controls were included. Treatment with 2 μM Tamoxifen and 5 μM CBD for 48 h was determined to be the optimal treatment condition. The MTT assay was performed, and the absorbance ratio indicative of cell proliferation was calculated. The ability of the cells to metabolize the drug components was examined through an assessment of CYP450 reductase (CPR) enzyme activity. The mRNA and protein expression levels of three autophagic markers; BECN1, LC3B, and p62, were investigated using qPCR and immunocytochemistry, respectively. Friedman’s Anova (p<0.05) and Kruskal Wallis (p<0.05) post hoc tests were used to statistically analyse the data. Combined CBD and Tamoxifen treatment showed the greatest decrease in the proliferation of MCF7 cells and T47D cells compared with all other treatments across both treatment models, with the daily- replacement treatment model (model 2) showing more efficacy thus suggesting that combined treatment may inhibit cell proliferation. CYP450 enzyme reductase activity was higher in T47D cells compared with MCF7 cells in both treatment models suggesting increased metabolic activity and susceptibility to combined treatment. However, in the daily replacement model, MCF7 cell CPR activity could not be ascertained, suggesting either prodrug availability or reduced CPR activity. Further analysis is required in this regard. For immunolocalization, optimization was conducted in late 2021 and all three antibodies showed clear and expected immunolocalization but when the experiments were repeated early 2022, immunofluorescence was reduced (P62 and BECN1), with LC3B not detectable. P62 and BECN1 were expressed in both MCF7 and T47D cells across both treatment models although BECN1 expression was not sufficient to be quantified and assessed statistically. LC3B protein levels could not be accurately quantified irrespective of the treatment model used. Low amounts of target mRNA in MCF7 cells resulted in undetermined Cq values of LC3B, P62 and BECN1 genes across both treatment models. In T47D cells, Cq values of target genes were determined across both treatment models and the fold change in gene expression indicated that combined CBD and Tamoxifen treatment effectively upregulates target genes albeit not significantly (LC3B, P62 and BECN1) with the single-dose treatment model (model 1) compared with the daily replacement model. Both the immunofluorescence and qPCR experiments would be required to be repeated to ensure conclusive results. The findings of this study nevertheless indicate that combined CBD and Tamoxifen treatment may inhibit tumour growth, but tumour cells may be able to evade cell death pathways resulting in tumour cell survivalItem The effects of ibogaine on myelination in Sprague Dawley rats(University of the Witwatersrand, Johannesburg, 2024) Govender, Demi NatishaIntroduction: The growing opioid epidemic is a worldwide issue which is prevalent in South Africa with the use of opioid cocktails such as nyaope. A possible solution to this problem is the use of psychedelic assisted psychotherapy. Ibogaine is a psychedelic that has been shown to curb addiction cravings and have neuroplastic effects in the brain. Ibogaine is extracted from the root bark of a West African plant and has shown to have neuroplastic effects in the brain. We investigated whether these antiaddictive properties are due to remyelination of the brain’s white matter. Methods: This study uses qPCR and western blotting to determine how myelin specific proteins and genes such as CNPase (CNP), Myelin Basic Protein (MBP) and Proteolipid Protein (PLP) are affected by morphine (opioids) and ibogaine. The experimental rat groups included a saline, morphine and ibogaine only controls, a combination morphine and ibogaine and a second combination morphine and ibogaine which included a 3 day withdrawal after ibogaine injection. Results: CNP protein was increased in the second morphine ibogaine group (p<0,0001) and the CNP mRNA fold expression was increased in the first morphine ibogaine group compared to the second morphine ibogaine group (p=0,0343). The 18,5 kDa isoform of MBP had increased expression in the ibogaine control (p=0,0384) and second morphine ibogaine group (p=0,0037). PLP shows increased protein expression in the second morphine ibogaine group when compared to the first group (p=0,0464). There is decreased PLP mRNA expression in the ibogaine control group when compared to morphine control (p=0,0033), first morphine ibogaine (p<0,0001) and second morphine ibogaine groups (p=0.003) Conclusion: Ibogaine may cause remyelination following demyelination by morphine. A consistent trend in the data shows that the myelin proteins were increased after the 3 days after administration of ibogaine following chronic morphine administration compared to 1 day after administration of ibogaine. This suggests that remyelination takes between 24-72 hours before it begins to produce new myelin around the axons due to ibogaine. These results also shows that CNP and MBP increase in expression earlier than PLP and are good markers for early remyelination. This is consistent with increase in CNP mRNA expression for CNP seen in the first morphine ibogaine but not the second group revealing an immediate effect on mRNA but a delay in protein expressionItem The role of human papilloma virus (HPV) in oropharyngeal squamous cell carcinoma: a clinicopathologic and molecular analysis(2024) Disenyane, DineoContext: Human papillomavirus (HPV) is an established aetiology in a subset of oropharyngeal squamous cell carcinoma (OPSCC). However, literature on the association between HPV and OPSCC in the African context is lacking. Objective: To determine the association of HPV in OPSCC and to correlate it with the clinicopathologic and molecular analysis. Design: A total of 60 OPSCCs were evaluated with HPV in-situ hybridisation (HPV ISH) and immunohistochemistry (IHC) for p16, p53 and Ki67. This was correlated with the site of occurrence of the tumours, the patients' smoking and alcohol usage and microscopic features of tumours (NKSCC;n=30 and KSCC;n=30). Results: HPV DNA was found in 24/60 (40%) cases. HPV16, was positive in 18/60 (30%) cases and HPV6 in 8/60 (13.3%) tumours. Two of the 60 (3.3%) cases were positive for both HPV16 and HPV6. HPV positive (+ve) tumours occurred more in males (n=22; 73%) than females (n=7; 23%); median age: 62.5 years. The primary tumour sites were the tonsil (n=7; 11.6%) and posterior tongue (n=6; 10%). Of the NKSCCs, 11 (36.7%) were HPV16+ve and 8 (26.7%) HPV6+ve. Seven (23.3%) KSCC were HPV16+ve and none (0%) HPV6+ve. Of the 24 HPV+ve tumours, 13 (54.1%) were p16+ve and 14 (58.3%) were p53+ve. Three (12.5%) tumours exhibited the molecular phenotype: p16+ve in > 70% of tumour cells, a low (25%) proliferation index. Conclusion: HPV positive OPSCC is seen more in older male patients. The distinct molecular phenotype, (p16 positive, low p53, high KI67) highlights tumours that are of true viral aetiology. In this study, the low p16 IHC stain sensitivity towards HPV ISH positive tumours infers that p16 is less reliable when used alone as a surrogate marker for HPV associated OPSCC. Therefore, in OPSCC with a high suspicion for HPV and which display a basaloid and non-keratinising morphology, a negative p16 IHC stain should be followed by other molecular techniques such as HPV ISH.Item The epidemiology of paediatric burn injuries in 6 Johannesburg, South Africa(2024) Banga, Agatha TafadzwaBackground Children remain the most common victim of burns in Sub-Saharan Africa. We investigated the epidemiology of burn injuries of the paediatric patients admitted to the Paediatric Burns Unit (PBU) at the Chris Hani Baragwanath Academic Hospital (CHBAH). Objectives To describe the epidemiology of paediatric burn injury among those admitted to CHBAH. Methods This was a hospital based cross-sectional study, with data collected at the time of admission via an accompanying caregiver questionnaire and via clinical examination of the patient by the admitting doctor. Results A total of 509 patients were admitted to the unit over a 12-month period, with 482 patients included for baseline analysis. 50% of admitted patients were between 15 and 47 months with a median age of 25 months. 205 58% of participants were male. 53% of all admissions had burns above 10% total body surface area and were considered severe burns. The predominant mechanism of injury was scalding (84%), with most overall in the winter season (32%). The most common site of burn was upper limb (75%). 63% of all admissions received 208 first aid. Among those who received first aid,an appropriate first aid method was provided in 74% of the cases. 226 out of 482 participants (47%) provided sociodemographic information. Access to basic amenities such as electricity was high, with most admissions coming from households with access to electricity (91%) and using electricity for cooking (83%). Basic level education was shown by 90% of caregivers holding at least a high school leaving certificate. Migrant caregivers made 19% of the caregivers, which was four times the proportion of foreign nationals counted in the national census. Over three quarters of admissions (79%) were 214 referrals from other centres. Severe burns were associated with thermal mechanism of injury (p<0.01), multiple burn sites (p <0.01), and receiving first aid prior to admission (p=0.01). No sociodemographic factors were associated with increased burn severity. Conclusion Children under two years of age and minority groups are at greatest risk for burn injury and should therefore be targeted for burn injury prevention. Education on appropriate first aid is essential to educate the community. Future research should investigate caregivers of children with burn injuryItem A comparison of malignant histopathological diagnoses on uterine curettings and hysterectomy specimens(2024) Ismail, AbdullahBackground. Endometrial carcinoma (EC) is a common gynaecological malignancy in postmenopausal females. Diagnosis is made on endometrial biopsy, where histological subtype and tumour grade are used to predict disease progression and to plan surgical management. Objectives. We aimed to determine the accuracy of preoperative biopsies compared to the final diagnosis on hysterectomy specimens in our department. Methods. This was a retrospective, cross sectional study in which 126 biopsies and corresponding hysterectomy specimens, over a 3-year period, were reviewed. Patient demographics and histological features were recorded and statistically analysed. Results. The most prevalent tumours were endometrioid endometrial carcinoma (EEC) (48.5%), serous carcinomas (25.4%) and carcinosarcomas (16.7%). The majority (66.7%) of tumours were high-grade tumours on biopsy and hysterectomy specimens (58.7%). EECs had a poor sensitivity level (65.12%) compared to other subtypes but had a high specificity rate 90%. There was moderate agreement between biopsy and excision specimen diagnoses. High-grade tumours had a high sensitivity (94.29%) level. Conclusions. Our study showed moderate agreement between histopathological diagnoses on biopsy, and excision specimens. EEC was the most prevalent tumour subtype. There was a high sensitivity (94.29%) level for biopsies of high-grade tumours, concordant with other studies. The sensitivity of low-grade EECs (42-46%) was lower than international studies, likely due to the comparatively low prevalence of EECs in our population. Accurate preoperative tumour subtyping and grading are needed to guide surgical management. It is envisaged that use of a combined histological and molecular tumour classification will better guide patient treatment and allow for reproducible results.Item Evaluation of histomorphometric changes in the small intestinal epithelium and paneth cells of male Sprague Dawley rats exposed to alcohol and/or combination anti-retroviral drug (Atripla) therapy (cART)(2024) Maseko, Zekhethelo LeticiaSignificant number of individuals who have HIV/AIDS are also chronic alcohol consumers (Pandrea et al., 2010). Alcohol intake has been found to disrupts ARV drug bioconversion and innate immunity of the gut (Bishehsari et al., 2017), but the exact effects of the combined use of alcohol and/or cARV on small intestinal epithelium and Paneth cells remains unclear. Paneth cells are the main regulator of innate immunity of the gut (Salzman et al., 2010). This study evaluated the histomorphologic appearances of Paneth cells and crypt-villous morphology in small intestine of rats exposed to alcohol and/or combination anti-retroviral drug therapy (cART). The study utilized an experimental study design of 32 adult male Sprague-Dawley rats which were divided into 4 groups and treated with normal saline, alcohol, cART or a combination of alcohol and cART. The animals were sacrificed after 90 days. Segments of small intestine were collected and studied to evaluate the morphometric changes of crypts and villi dimensions in the jejunum and ileum, determine the location of the Paneth cells along the axis of the intestinal crypts of the jejunum and ileum. Furthermore, to examine the histomorphological appearance of Paneth cells including their morphology and amounts of secretory granules, in the jejunum and ileum and examine the histomorphological appearance of stem cells in the crypts of the jejunum and ileum using H&E,special stains and immunohistochemistry. Histomorphometric measurements were done using ImageJ software. Analysis of data was done using STATA SE 15 statistical software. Morphometry and morphological analysis showed significant (p < 0.05) reduction in villous height, villous width, crypt’s depth, intestinal stem cells, increased villous stripping, increased crypt’s width, increased muscular wall thickness, increased number of Paneth cells and staining intensity of Paneth cell granules in alcohol + cART treated group. The increase in number of villi was in all experimental groups and highest following treatment with cART alone or in combination with alcohol. The shortest villi, shallowest crypts and the least number of crypts were seen in ileum of animals that had cART alone. The alcohol alone group had the least number of villi but showed increased collagen content. Paneth cells were noted in the proliferation zone of intestines of animals that had combined treatment (alcohol +cART). Concomitant use of alcohol and cART led to thickening of small intestinal wall, shortening and/or stripping the villi, reduction of crypt depth, appearance of Paneth cells in proliferation zone and a decrease in intestinal stem cells. The structural changes in the small intestine and Paneth cells may adversely affect the regulation of gut innate immunity. These findings are clinically invaluable in the management of HIV patients considering the critical significance of innate immunity amongst HIV patients.Item Testing the use of three-dimensional surface rendering and mesh-to-mesh comparisons as a method of pair-matching commingled human skeletal remains(2024) Pillay, KaminiWhen dealing with mass graves, natural disasters, or mass fatalities, forensic anthropology faces a complex scenario that necessitates specific methods for sorting, matching, and identification. The main aim of dealing with commingled human remains is to sort the remains to identify the minimum number of individuals (MNI). Traditional sorting methods, such as visual-pair-matching, articulation, process of elimination, and taphonomy, are welldocumented but subjective and rely heavily on the experience and knowledge of the forensic anthropologist. The study aimed to evaluate the effectiveness three-dimensional surface renderings of bone and the subsequent comparison of mesh-to-mesh values as a more objective and repeatable method for pair-matching commingled human remains. The Structure-from-Motion (SfM) technique was used to create and render 171 three-dimensional mesh model samples using Agisoft Metashape. These models were used to test the effectiveness of a new digital pairmatching method, the mesh-to-mesh value comparison (MVC) method against a South African populated sample. To generate the mesh-to-mesh values, different softwares were used: Viewbox 4.1, which is proprietary and uses a Trimmed Iterative Closest Point (ICP) algorithm to run the program, and Meshlab 2022.12, an open-source software that does not require a license and uses a general ICP algorithm to run the program. Each software was programmed to generate a root mean square value, which was used as the mesh-to-mesh value required for comparison. Analysis was performed using two techniques: LCV (Lowest Common Value) mesh-to-mesh comparison using Microsoft Excel and ROC curve analysis performed using MedCalc. The LCV mesh-to-mesh comparison requires the user to filter, sort, and eliminate values based on observation using specific commands on Microsoft Excel until the lowest agreed upon value is found across left-right and right-left. The ROC curve analysis on MedCalc follows the principle stated by Delong et al. (1988), which works with a scoring system. The closer the threshold value is to the top-left corner of the graph, the higher the accuracy of the test being performed. For the complete cadaveric humeri and femora analysis, both elements performed moderately well with the LCV mesh-to-mesh comparison method, while the ROC curve analysis produced considerably higher results, comparatively. For the complete cadaveric bones, the femur performed better with both softwares when compared to the humerus. Mesh-to-mesh values iv obtained through Meshlab and analysed with the ROC curve method generated significantly higher results and were more adept at correctly identifying and distinguishing differences between matches and non-matches. Differences in mesh-to-mesh values between sexes and different population groups were observed. In the simulated fragmented remains, the distal mesh fragment performed the best with 100% sensitivity for Viewbox, and the shaft performed the best using Meshlab with sensitivity results greater than 79%. For the fragmented remains, the distal mesh fragment and the shaft mesh fragments generated significantly high results when using the values obtained through Viewbox and statistically analysed with the ROC curve method. The mesh-to-mesh value comparison method was applied to a real-world forensic commingled assemblage with high fragmentation and taphonomically altered bones. The Structure-fromMotion technique was effective in capturing distinct details on bones, aiding in 3D modelling. However, the method failed to identify true positive matches in the humeri bone sample, as there were no individuals matched with visual pair matching in the original case. In conclusion, the mesh-to-mesh value comparison method has the potential to improve the accuracy and objectivity of forensic anthropology in handling commingled human remains. By utilizing advanced technologies like SfM, ROC curve analysis, and real-world forensic commingled assemblages, this method can be applied to enhance the accuracy and reliability of forensic anthropological research.Item An assessment of developmental anomalies in the thoraco-lumbosacral region of South Africans(2024) Nchabeleng, Elsie KoketsoDevelopmental anomalies, typically caused by epigenetic interactions, are very common in the human vertebral column. Many studies have been conducted to assess their prevalence in different populations. Several studies have shown differences in the prevalence and expression of vertebral anomalies among populations and between the sexes. These differences may be related to different geographical areas and the environmental conditions posed by these, socioeconomic status, diets, lifestyles and/or gender roles, to name a few. Therefore, this study aimed at assessing the prevalence and pattern of six developmental anomalies in the thoracolumbosacral regions and their possible associations with vertebral pathologies in South Africans. The study comprised skeletal remains of 902 individuals. The remains were procured from the Raymond A. Dart Collection of Modern Human Skeletons, the Pretoria Bone Collection and the Kirsten Bone Collection. The sample included South African Blacks (SAB) (n=325), South African Coloureds (SAC) (n=286) and South African Whites (SAW) (n=291). The most common vertebral developmental anomaly observed in this study was sacralisation (5.7%), followed by spina bifida occulta (4.5%), spondylolysis (4.5%), L6 (3.1%), and T13 (2.2%). The lowest prevalence was seen for lumbarisation at 1.9%. Overall, developmental anomalies were most prevalent in the SAB sample compared to the SAC or SAW. These anomalies were generally more prevalent in males of the total sample and within the three populations groups of the study. The high prevalence of most developmental anomalies in the SAB sample may be mostly related to the low socio-economic status associated with Black South Africans under the apartheid era. The presence of multiple anomalies was more prevalent in Blacks (44.4%). Blacks also had a higher prevalence for multiple anomalies accompanied by some form of spinal pathology (50%). Whites, on the other hand, had a higher prevalence of a single developmental anomaly accompanied by some form of spinal pathology (50%). In general, males were more prone to developing multiple anomalies accompanied by some form of spinal pathology. There was substantial variation observed with regards to the presentation of the six developmental anomalies in and between the samples under study. Therefore, inter- and intrapopulation differences, as well as sex differences, should be considered when dealing with the above developmental anomalies in biological anthropological and clinical setting