3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item A polymeric implant for application in post-surgical resection of osteosarcoma(2024) Suleman, AyeshaOsteosarcoma is a malignant neoplasm of which there is osteoid formation by tumour cells. According to the American Cancer Society, osteosarcoma is prevalent in patients between the ages of 10 and 30 and those diagnosed with osteosarcoma over the age of 60, consist of 10% of the population afflicted. Surgery is a critical component in the treatment of osteosarcoma. Wide margin resections are standard in surgical treatment of osteosarcoma, therefore an osteotomy occurs, inducing a severe bone defect. Adjuvant chemotherapy is administered by IV after surgical removal of osteosarcoma to mitigate the risk of tumour micro metastases. The chemotherapeutic drugs are accompanied by severe side effects. To address the issue of a critical bone defect, the use of bone grafts and prosthetic devices (endoprosthesis or megaprosthesis), have been utilised. However, Osteosarcoma is mainly prevalent in adolescents, their growth poses a constant challenge to prosthetics and bone grafts have their own drawbacks. There is a need for customising implants for individual patients. To this end, this study focused on the development of a mouldable implants. The materials of these implants may have the ability to regenerate bone and provide localised drug delivery which may bypass the severe side effects of IV administered chemotherapeutics. Two hydrogel implants (one containing bioglass and the other without bioglass) were synthesised and compared. The polymers used were Alginate and Polyacrylamide. The hydrogel implants displayed great moulding ability when cast into different shapes and characterisation studies such as FTIR, XRD,TGA and DSC was conducted. Scaffolds containing bioglass initially possessed enough strength to match cancellous bone. Biomineralisation studies proved the formation of hydroxyapatite on the scaffold surface. Both hydrogel implants displayed ~20% of Doxorubicin released over 8 weeks. At 6 weeks, ALG-PAAM hydrogel implants degraded to 87,674% ± 5,042 of their original weight, while BG-ALG-PAAM hydrogel implants degraded to 86,528% ± 0,0987 of their original weight. Cell studies were conducted using MG-63 cells and proved that both scaffolds were non-cytotoxic and could facilitate cell adhesion. However, these hydrogel implants lacked the presence of pores from formation. Therefore, the same polymers were used to fabricate implantable scaffolds. These scaffolds were formed by lyophilisation and contained pores from formation. However, mechanical strength of these scaffold implants did not match the strength of bone and only two of the 5 criteria of the diamond concept were met in implantable scaffolds containing bioglass. Overall, it was concluded that BG-ALG-PAAM was better than the double network hydrogel without bioglass and had potential for application in Osteosarcoma as it met three of the five criteria mentioned in the diamond concept. Further research is needed to improve the scaffolds formed by lyophilization to meet at least three of the 5 criteria of the Diamond Concept.Item DNA damage associated lncRNAs, PANDA, ANRIL and DDSR1, are constitutively active in HT29 cells under hypertonic stress(2022) Patel, AaliaOur genomes undergo DNA damage regularly with the most lethal being DNA double-strand breaks (DSBs). Long non-coding RNAs (lncRNAs) have distinct biological functions and play roles in DNA damage response (DDR). We hypothesized that lncRNA expression would be altered in response to NaCl-induced DNA damage and that these effects may be different in cancer and non-cancer cell lines. Exposure of cells to high NaCl concentrations promotes DSBs in DNA regions near the nuclear periphery. These are rapidly repaired after NaCl withdrawal. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays were performed on HEK293 and HT29 cell lines to determine the EC50 for NaCl. For confocal microscopy, cells were treated with NaCl for 6 hrs or 24 hrs followed by 5 min NaCl withdrawal, and Mre11 was detected. Results indicated that meiotic recombination 11 homolog 1 (Mre11) was localized in the cytoplasm in the presence of NaCl and translocated to the nucleus during 5 min NaCl withdrawal in HEK293 cells. Under high NaCl, Mre11 was present in both the nucleus and cytoplasm in HT29 cells. DSB assessment by western blot analysis for gH2AX revealed no significant change in expression in HEK293 and HT29 cells, but slightly lower levels in HT29 suggest DNA repair may occur in the presence of NaCl. PCR and qPCR were performed for lncRNAs p21 associated ncRNA DNA damage activated (PANDA), Antisense non-coding RNA in the INK4 locus (ANRIL), and DNA damage sensitive RNA1 (DDSR1). qPCR data shows that PANDA, ANRIL, and DDSR1 expression is reduced in HEK293 during NaCl treatment, while in HT29s PANDA was upregulated in response to NaCl treatment and NaCl withdrawal, ANRIL was only upregulated after 4 hrs NaCl and in the 6 hr 5 min NaCl withdrawal treatment and DDSR1 expression was significantly higher in the 6 hr treatment only. Whilst hyperosmotic stress was used to induce DNA damage, we have observed that it results in reduced expression of lncRNAs that play roles in DNA repair and cell cycle regulation in a non-cancer cell line, and enhanced expression in a cancer cell line. This suggests that in hypertonic environments the HT29 cells may still be undergoing cell cycle arrest, DNA repair, and proliferation.Item Comparison of activities, electrophoretic patterns and kinetic properties of some enzymes in normal and cancerous liver(1976-11) Hammond, Kathryn D.Glucose present in tissues is metabolised either by means of glycolysis, gluconeogenesis, glycogenesis or glycogenolysis, as shown in Figs. 1 and 2. Glycolysis occurs in almost all tissues, whereas gluconeogenesis is restricted mainly to liver and kidney cortex; the synthesis and degradation of glycogen takes place mainly in liver, skeletal muscle and heart. The patterns of carbohydrate metabolism characteristic of normal tissue are often altered in various diseased states; this is particularly evident in cancer. Tumour tissues, such as hepatoma, appear to lose the functional properties of adult differentiated tissue and their metabolism often seems to resemble that of rapidly-dividing, undifferentiated foetal tissue.Item Novel LRP/LR induced stem-cell-like cells to aid wound healing and regeneration(2019) Chigumba, StephanieThe 37/67kDa Laminin Receptor (LRP/LR) LR is a multifunctional cell surface receptor that maintains several survival processes. It has recently been found that there is a direct relationship between LRP/LR and the stem cell marker telomerase. Studies have shown that overexpression of FLAG tagged LRP increases hTERT levels and telomerase activity. Telomerase is a reverse transcriptase enzyme found in actively dividing cells whose core function involves telomere maintenance and elongation. The rate limiting component of telomerase, hTERT, is also often upregulated in rapidly dividing cells to aid stem cell renewal and cell survival and its ectopic expression can induce epithelial to mesenchymal transition (EMT) resulting in stem cell like characteristics. In adults, the primary role of stem cells is to repair and regenerate tissue. Hence, this study aims to determine whether overexpressing LRP::FLAG and the subsequent increase in hTERT levels induces stem-cell-like characteristics and promotes repair and regeneration in MRC5 lung fibroblasts and HEK293 embryonic kidney cells. Cells were stably transfected with the pCIneo-moLRP-FLAG plasmid in order to induce LRP::FLAG overexpression. Post-transfection, an increase in hTERT and phospho-TERT protein levels was observed in both cell lines which is crucial in maintaining the self-renewal capacity of stem cells. Additionally, an increase in the levels of pluripotency stem cell markers involved in cell reprogramming and alkaline phosphatase activity was also observed for HEK293 after transfection. However, in MRC5 cells there was an insufficient expression of reprogramming factors but, a Cadherin switch indicative of EMT. Moreover, HEK293 cells overexpressing LRP::FLAG showed no significant changes in the protein levels of the pro-inflammatory cytokine NF-κB1 and an increase in the anti-inflammatory cytokine TGFβ1 which modulates wound healing. In turn, it led to an increase in the adhesion and migratory capacity of HEK293 cells. This data suggests that overexpressing LRP::FLAG induces EMT similar to that observed during induced cell reprogramming and could possibly promote wound healing by upregulating TERT and TGFβ1 protein levels resulting in stem-cell-like characteristics.Item Investigating the effect of LRP/LR down-regulation on malignant melanoma cells and assessing the effect of LRP/LR antibodies on metastatic malignant melanoma mouse models(2017) Rebelo, Thalia MartinsOn a cellular level cancer is described as abnormal cellular growth resulting from uncontrolled cellular proliferation and reduced apoptosis. Cancer is seen as a non-discriminative disease, therefore exhibiting high incidence and mortality rates. Additionally, cancer is classified as a global burden in both economically developed and developing countries. The 37kDa/67kDa laminin receptor (LRP/LR) is seen to be over-expressed in tumor cells when compared to their normal cell counterparts. This receptor has been implicated in several tumourigenic processes such as cell migration and adhesion but importantly for the present study, the maintenance of cellular viability and the evasion of apoptosis. The aim of the present study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry revealed that both malignant melanoma cell lines exhibit high cell surface LRP/LR levels and with further analysis using median fluorescence intensity, it was observed that A375SM cells contain approximately 86% more cell-surface LRP/LR than A375 cells. In addition, western blotting and densitometric analysis suggested that A375SM cells contain 60% more total LRP/LR levels than A375 cells. Furthermore, western blot analysis revealed that targeting the mRNA of the 37kDa LRP using a LRP-specific siRNA (Dharmacon ON-TARGET Human RPSA) in A375 and A375SM cells led to significant down-regulation of 77% and 72% in LRP expression, respectively. Consequently, MTT assays showed that LRP down-regulation led to significant reductions of 47% and 61% in the viability of A375 and A375SM cells, respectively. An alternative LRP-specific siRNA (Mission esiRNA-RPSA) was used in order to confirm specificity and to exclude any off-target effects of Dharmacon ONTARGET Human RPSA for LRP. Confocal microscopy with the addition of the Airyscan processing tool indicated nuclear morphological changes suggestive of apoptotic induction in the form of cell death occurring in both malignant melanoma cell lines post LRP down-regulation. Annexin-V/PI assays confirmed this observation, by revealing that A375 and A375SM cells underwent apoptotic induction post LRP down-regulation in comparison to the untreated cells. Additionally, caspase-3 activity assays revealed that both cell lines experienced apoptotic induction after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP down-regulation; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells are thought to undergo apoptosis via the intrinsic pathway. According to Munien et al, 2017 [137], application of the anti-LRP/LR specific antibody IgG1 iS18 has led to a significant reduction in metastatic potential of A375 and A375SM malignant melanoma cells in vitro. Therefore the aim of the present in vivo study was to further investigate the significance of blocking LRP/LR with the IgG1-iS18 antibody and how this will be effective in the treatment of malignant melanoma in vivo using a MF-1 nude mice model. The mice were divided into three groups; with three mice each. This was followed by treatment of group 1 with 0.885mg/ml of the IgG1-iS18 antibody intraperitoneally, twice a week, group 2 was administered with the Phosphate Buffered Saline (PBS) vehicle control solution and the last group remained untreated. The results of this pilot study indicated that three of the nine mice developed external tumour formation; one of the mice being from the untreated group with a tumour volume larger than the other two mice which were from the IgG1-iS18 treatment group. When comparing the tumour formation between the untreated and treated group, there was a large reduction in tumour weight and volume. To conclude, LRP/LR plays a critical role in the maintenance of tumor cellular viability and metastasis, recommending this receptor as a promising therapeutic target and proposing the potential use of siRNA technology as well as the IgG1-iSi8 antibody for the treatment of malignant melanoma.Item Registered nurse knowledge of the early warning signs of childhood cancer(2014) Raymond, NaseerahThe global burden of cancer has more than doubled in the past 30 years. It is estimated that in 2008, 7 million people worldwide died of cancer. Although the majority of deaths were in adults, 90 000 deaths related to childhood cancers. Childhood cancer comprises all cancers arising in children under the age of 15 years. Cancer in children is fairly rare and globally it is estimated that 160 000 children will be diagnosed with cancer each year (World Health Organization, 2008). Early detection is a fundamental goal in oncology nursing as it provides for early treatment of cancers. The onset in children’s cancers is generally short and if not detected early can grow fast and aggressively. Children’s tumours tend to be more invasive but have a better response to treatment in comparison to adults’ cancer. The purpose of this study is to explore the knowledge of registered nurses practicing at primary health clinics situated in the Johannesburg metropolis regarding the early warning signs of childhood cancer. An exploratory research strategy would be used and a contextual study will be done. The context of the study will be Johannesburg and specifically the primary health clinics in the metropolis. A quantitative survey will be conducted. The population targeted is all registered nurses practicing in the 35 primary health clinics in the Johannesburg Metropolis Region B, D, E, F and D. A census will be done and a self- administered questionnaire will be used to gather self-report data. The data will be analyzed by means of descriptive statistics.Item PI3K in human oesophageal squamous carcinoma cells : a critical modulator in the PKB signalling pathway(2013-03-05) Shaw, NicoleneThe phosphotidylinositide-3-kinase (PI3K) pro-survival signalling pathway is critical in the development of cancer. Major contributors to the proliferative and/or anti-proliferative signalling in human oesophageal squamous cell carcinoma (HOSCC) are currently unknown. Based on the Ser473 phosphorylation state of PKB (pPKB), this study dissects the overall activation status of the PI3K/PKB pathway. Despite the prevalent membrane expression of PI3K determined through western blotting and immunofluorescence, pPKB levels were shown to be surprisingly low. Activation of EGFR did not produce a hyperactivation of the PI3K/PKB pathway. Neither PI3K nor PKB sequence isolated from the 5 HOSCC cell lines possessed any of the ―hotspot‖ mutations described previously for other tumours. Inhibiting phosphatase protein 2A (PP2A), an integral antagonist of PKB, indicated that its activity in respect of PKB is diminished in HOSCC cells. Despite the low concentration of pPKB, the reciprocal relationship with PTEN expression was not evident in the WHCO and SNO HOSCC series. Moreover, reversible oxidization and inhibition of PTEN served to augment the activation of the PI3K/PKB pathway. Since oxidation of PTEN is imperative for effective signal propagation from activated EGFR and PI3K, these data reveal an aberrant EGFR-PI3K-H2O2 mediated PTEN inhibition in HOSCC. Allied to this discovery, was the finding that HOSCC cells are highly susceptible to oxidative stress induced by H2O2. This was suggested to play an essential part in maintaining the low PI3K/PKB activation status. Although the decrease in PTEN activity was required for the induction of pPKB, PTEN may not be the only limiting component for the activation of the PI3K/PKB pathway in HOSCC. In addition to its overexpressed EGFR status, the WHCO and SNO HOSCC series have the propensity to appropriate nuclear β-catenin. Interruption of the PI3K/PKB signalling pathway caused a small, yet significant, depression in the nuclear localization of β-catenin in 3 of the HOSCC cell lines. Together, this work greatly expands our understanding of the major influences behind the proliferative and/or anti-proliferative signalling in HOSCC, primarily that, the EGFR overexpression status does not propagate these transforming capabilities via activation of the PI3K/PKB pathway, and that this may be a reflection of its transformation potential. The findings derived from this study are likely to have a profound impact on future therapeutic targets for this disease.Item Molecular analysis of the domain with no name (DWNN)/RBBP6 in human cancers(2012-10-08) Mbita, ZukileRetinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. It is a multi-domain protein containing a Zinc finger, a RING finger, an Rb binding domain, a p53 binding domain and a novel N-terminal protein domain, the so called, Domain With No Name or DWNN. The RBBP6 gene encodes three isoforms of this particular protein. A common feature of all three isoforms of RBBP6 is the presence of the N-terminal DWNN domain. RBBP6 isoform 3 is comprised of the DWNN domain only. The DWNN itself has a ubiquitin-like fold, sharing 22% similarity with ubiquitin. It is likely that DWNN regulates intracellular levels of the two tumour suppressors, Rb and p53 through the ubiquitin-proteasome pathway and as such, DWNN may therefore play a role in the deregulation of cell cycle control in cancer cells. A mouse homologue, P2P-R of the gene has been implicated in mitotic apoptosis. The expression of DWNN, RBBP6 and their roles in the cell cycle, apoptosis and human cancer were investigated. RT-PCR and real-time PCR were used to determine the gene expression of DWNN and RBBP6 variants in human cancer cells. An anti-human DWNN antibody was characterized using both Western Blotting analysis and MALDI-TOF mass spectroscopy to determine whether the antibody specifically recognizes DWNN and RBBP6 isoforms, or if it recognizes other proteins. Western blotting was also used to determine the nature of the DWNN in human cell lines. A DWNN probe and the characterized anti-human antibody were used to localize DWNN and RBBP6 gene products at the mRNA and protein levels using ISH/FISH and Immunohistochemistry respectively. Cell labelling was also performed using this antibody to localize RBBP6 products in human cell lines. RNA interference and over-expression of DWNN and RBBP6 gene products was carried out to further investigate the role of RBBP6 products in the cell cycle, apoptosis and carcinogenesis. Cloned RT-PCR products of RBBP6 binding domains, the RING finger domain, pRb-binding and p53-binding domains in human cancers cell lines (Hek 293T, MCF7, HeLa and HepG2 cells) showed no mutations, but MCF-7 cells showed the lowest expression of the RBBP6. Real-time PCR and Western blotting analysis confirmed that MCF-7 cells express very little DWNN (RBBP6 isoform 3) and RBBP6 gene products when compared to Hek 293T, HeLa and HepG2 cells. It was also shown that the anti-human DWNN antibody recognizes the DWNN domain (RBBP6 isoform 3) and the larger RBBP6 isoforms. Using 2D gel electrophoresis and MALDI-TOF spectrometry, it was also found that DWNN is associated with other proteins namely, Recoverin and a hypothetical protein XP_002342450. This result suggested that DWNN may be a ubiquitin-like protein, which may be specific to these proteins in human cells. FISH and IHC demonstrated that the DWNN domain and its relatives are down-regulated in human cancers at both mRNA and protein levels, respectively. In contrast, however, cell staining showed that the expression of the DWNN gene products was high during the G2/Mitosis transition. Knocking-down the DWNN domain or over-expressing it did not sensitise the Hek 293T cells to Camptothecin (CPT)-induced apoptosis but rather slowed down cell growth. These results strongly suggest that the DWNN gene is likely to be involved in cell cycle control. Up-regulation in mitotic cells and down-regulation in cancers also implies that RBBP6 gene products may additionally be involved in cell cycle arrest. Moreover, down-regulation in human cancers particularly indicates that the loss of its function which correlates with loss of cell cycle control in this disease may be involved in the pathogenesis of cancer. This was confirmed by up-regulation of the DWNN in arsenic trioxide induced cell cycle arrested cells specifically at G2/M phase where a p53-dependent cell cycle arrest ensued. It is thus proposed that the DWNN may be implicated both as a p53 stabilizer and additionally as a G2/M progression regulator.Item Characterization of 1-ACBP B-ACBP and PBR in oesophageal cancer(2006-10-27T07:44:47Z) McCabe, Michelle LynnBackground: Cancer of the oesophagus ranks as the ninth most common malignancy in the world, and recent evidence shows that its incidence is increasing. Apoptosis is a process of programmed cell death, which is as essential as cell growth, for the maintenance of homeostasis. When these processes lose integration, such as cancer, then uncontrolled cell growth occurs. There are at least five ACBP subgroups and the two being focused on in this study is B-ACBP (brain specific) and 1-ACBP (found in nearly all tissues). ACBPs act as intracellular carrier-proteins for medium to long chain acyl-coA, mediating fatty acid transport to the mitochondrion for ß-oxidation. ACBPs are also believed to be putative ligands of PBR (Peripheral Benzodiazepine Receptor), and bound to this receptor facilitates mitochondrial membrane permeabilization giving the notion that it favours apoptosis. Aim: To establish the expression patterns of 1-ACBP, B-ACBP, and PBR in oesophageal cancer, and to characterize their roles in this disease. Methodology: Paraffin-embedded sections of normal and malignant oesophageal tissues were utilized for localization studies. RNA probes was synthesized and labelled using Digoxigenin for colorimetric and fluorescent detection during the in situ hybridization (ISH) technique for localization. Real time quantitative RT-PCR was performed to determine the expression levels of the three genes in oesophageal cancer RNA using the Roche Lightcylcer .Results: All three genes showed substantial upregulation within the malignant tissue sections compared to normal oesophageal sections, all three transcripts localized specifically to plasma cells and lymphocytes in diseased and normal tissue section. In the diseased tissue B-ACBP and 1-ACBP mRNA localized to endothelial cells of blood vessels in the submucosa. B-ACBP also localized to the nucleus of squamous epithelium cells. PBR localization occurred in tumour islands in invasive tissue sections. Quantitative RT-PCR also illustrated PBR expression level was the highest compared to the ACBP genes expression in tumours. Conclusion: These results show that 1-ACBP, B-ACBP and PBR play a role in the pathogenesis of oesophageal cancer as well as immunology. Further experiments are still required to determine the function of these genes and the role they play in apoptosis and oesophageal cancer.