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 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 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 settingItem Evaluation of the neuroprotective effects of simvastatin against alcohol-induced damage to the sciatic nerve and the somatosensory barrels in adolescent C57BL/6J mice(2024) Efuntayo, Alice AdetokunboAlcohol is a commonly used and abused drug among adolescents which has an adverse effect on the body’s overall health, especially on the developing brain. It causes neurodevelopmental, neurobehavioral, neurocognitive, and social problems because alcohol exerts its neurodegenerative effects by up-regulating oxidative stress which is responsible for neuronal death. The rising prevalence of alcohol-related diseases and disabilities and the cost to the government necessitates investigation into interventions that could protect the neurons against the damaging effects of alcohol. One drug with antioxidant properties is Simvastatin, a U.S. Food and Drug Administration (FDA) approved drug for lowering blood cholesterol levels. The neuroprotective effects of Simvastatin against alcohol neurotoxicity were evaluated on the sciatic nerves and the somatosensory barrel cortices of adolescent mice. 40 four–week old C57BL/6J male and female mice were administered 20% alcohol (i.p.), 5 or 10 mg/kg Simvastatin orally followed by 20% alcohol (i.p.) or the controls (i.e. 5 mg/kg Simvastatin only or non-treated) consecutively for 28 days. The axonal density, myelin thickness and g-ratio of the sciatic nerves were assessed as well as the sizes of the Posteromedial barrel subfield (PMBSF) barrels. The results confirmed alcohol neurotoxicity on the axonal density and myelination in both sexes. At the same time, Simvastatin was effective against the onset of alcohol nerve damage. For the somatosensory barrels, alcohol did not significantly reduce the mean areas of (I) the PMBSF barrels, (II) the enclosure, or (III) the septal portion in both sexes. However, the barrel-to-barrel comparison revealed alcohol toxicity on specific barrels in specific rows and arcs of the PMBSF barrels. Both concentrations of Simvastatin were also effective against alcohol–induced damage on those specific barrels. These may explain the reasons for the sensory-motor delays that are often seen in alcoholics due to possible delays in the relaying of sensory input and the processing and interpreting of information from the somatosensory cortex. Simvastatin seems to have the ability to protect against the damaging effect of alcohol on the peripheral nerves and the somatosensory cortex and this may be beneficial in reducing the prevalence of alcohol-related diseases or disabilities, especially in adolescents that are prone to abusing alcohol.