School of Anatomical Sciences (ETDs)
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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 Assessment of disability resulting from degenerative joint disease in a southern African skeletal population(University of the Witwatersrand, Johannesburg, 2023) Gavin, Jessica Robyn Brinkworth; Carrasco, Lorena Nunez; Meyer, Anja; Keyes, CraigAge-related skeletal changes, like degenerative joint disease (DJD), are often used to estimate age in forensic settings, yet these changes also reflect the living experience of individuals as they progress through life. This study set out to assess the impact of DJD in a sample of southern African deceased individuals and the subsequent effects on these individuals’ Activities of Daily Living (ADLs). A novel scoring system was created, translating DJD frequency and severity into how the individual was potentially physically disabled. A sample of 150 southern African individuals between the ages of 35 and 90 years were assessed for signs of DJD in each of the major joints, both for the peripheral (TMJ, shoulder, elbow, wrist, finger, hip, knee, ankle and toe joints) and axial (cervical, thoracic and lumbar vertebral joints) regions. The severity of DJD was then translated into various ADLs (transferring, eating, talking, object manipulation, walking and posture changes: head rotation; twisting and bending) that may have been affected. Socio-economic Status (SES) was included as this would have a significant impact on the types of DJD and subsequent ADL impairment seen, as well as how these individuals may have been impacted within their respective contexts. Results for DJD severity and frequencies indicated that the shoulder most often presented with the most severe DJD score with 42.67% of the pooled sample presenting with a score of 3. It was also interesting to note that most of the upper peripheral joints were affected, with the entire sample presenting with some form of DJD in the elbow, specifically. For the axial skeleton, severity scores were much more variable across different vertebral regions, with the highest severity percentage per region was seen in the thoracic vertebra (5%). In general, females presented with higher severity scores for DJD across all joints, with the TMJ (47%) and toe (22%) joints being statistically higher in females. When controlling for population affinity and sex the same trend was observed in the white sample, specifically the white females (TMJ = 22%). Black males; however, presented with higher severity frequency of DJD in the axial skeleton, specifically the lumbar vertebrae (32%). The white cohort showed very little correlation with age in relation to the TMJ, whereas this was true for the elbow and ankle in the southern African black individuals. This study indicated moderate impacts in transferring (53%), walking (51%), and eating (41%) activities for all individuals. Females presented with significantly higher ADLs for all activities except posture changes whereas problems with eating and talking were more often seen in southern African white individuals. Differences between different socio-economic status groups were also noticed with the lower SES group showing increased levels of impairment across most of the ADLs which may relate to activity differences when age is controlled for. The focus of this research was to provide deeper information into impairment and disability caused by DJD. To conduct research on individuals were their lives and stories were limited and underexplored. This research highlights the need to continue studies on skeletal remains of individuals impaired by DJD, with focus on trends on joint and activity limitations within past and present contextsItem 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 Mathematical representation and analysis of articular surfaces: application to the functional anatomy and palaeo-anthropology of the ankle joint(University of the Witwatersrand, Johannesburg, 1990) Webb, Christie Peter; Tobias, Phillip VallentineThis thesis is a study of quantifiable variation in the geometric shape of the superior articular surface of the talus of higher primates, with special reference to fossil tali of Plio-Pleistocene hominids. (Abbreviation abstract).Item The effect of all-trans retinoic acid on the migration of avian neural crest cells in vitro an in vivo(University of the Witwatersrand, Johannesburg, 2007-02-15T11:43:45Z) Tshabalala, Vincent Abie ThabisoRetinoic acid, the active metabolite of Vitamin A is known to play a major role in embryonic growth and differentiation during development. It has been shown that either excess or deficiency of retinoic acid during embryogenesis can be teratogenic. In order to study the teratogenic effects of retinoic acid, the aim of the present study was therefore to investigate the effect of all-trans retinoic acid on the migration and fate of neural crest cells in vitro and in vivo. In addition, the study investigated the effect of retinoic acid on the cytoskeletal elements of neural crest cells and on Rac and Rho, two members of the Rho family of GTPases. The neural tubes containing neural crest cells of quail embryos were removed at cranial levels and cultured on fibronectin as a substrate. The neural tubes were cultured in either Dulbecco’s minimal essential medium (DMEM) or in DMEM+Dimethylsulphoxide (DMSO) as controls. In order to test the effect of retinoic acid, the neural tubes were cultured in 10⁻⁵M all-trans retinoic acid (RA) which was reconstituted in DMSO. The distance of migration of the cultured quail neural crest cells was measured and compared between the controls and the experimentals. To study the effect of RA on the cell actin cytoskeleton in vitro, cultured neural crest cells were stained with rhodamine phalloidin. In addition, following 24 hours of culture, the quail neural crest cells were brought into suspension and micro-injected into 36 hour-old chick hosts. While the migration of neural crest cells was extensive in the control cultures in vitro, migration was inhibited in the retinoic acid-treated neural crest cells. In addition, retinoic-acid treated neural crest cells showed pigmentation and neuronal processes earlier than did the control neural crest cells. Retinoic acid-treated neural crest cells showed a disarray of the cytoskeletal elements as they were devoid of stress fibres and focal adhesions. In addition, retinoic acid appears to decrease the expression of Rac and Rho of cultured quail neural crest cells. Following micro-injection of cultured control and RA-treated quail neural crest into the cranial region of chick hosts, the control cells populated the beak area, whereas the retinoic acid-treated quail neural crest cells migrated to the retina of the eye, a region they normally do not populate. These results suggest that retinoic acid disturbs the migration of neural crest cells. It appears to do this by affecting the cytoskeletal elements of neural crest cells and the genes that are involved in forming these elements.