3. Electronic Theses and Dissertations (ETDs) - All submissions
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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 Targeting LRP/LR for the treatment of metastatic lung and colorectal cancer through impediment of telomerase activity(2018) Baichan, PavanThe 37 kDa/67 kDa laminin receptor (LRP/LR) plays a vital role in the malignancy of various cancer types contributing to invasion, adhesion, apoptosis evasion, proliferation and tumour angiogenesis. In addition, LRP/LR interacts with the catalytic reverse transcriptase subunit, TERT, of the ribonucleoprotein telomerase. Both LRP/LR and telomerase are implicated in cancer progression and knockdown of LRP/LR causes a decrease in telomerase activity in breast cancer cells. In the current study, LRP/LR was downregulated in lung adenocarcinoma (A549) and late-stage colorectal carcinoma (DLD-1) cells in an attempt to impede telomerase activity and ultimately impede cancer progression. Western blotting analysis showed a significant decrease in LRP/LR levels in HEK293 (Human Embryonic Kidney Cells) and A549 cells after siRNA mediated LRP/LR knockdown. To confirm LRP/LR knockdown confocal microscopy was performed; a reduction in LRP/LR protein levels was observed which also resulted in a subsequent decrease in hTERT mRNA levels with a corresponding decrease in hTERT levels in HEK293, A549, and DLD-1 cell lines. Furthermore, siRNA mediated knockdown of LRP/LR significantly decreased telomerase activity in HEK293, A549, and DLD-1 cells. The effect of LRP/LR downregulation on cellular viability was investigated via the MTT assay and a significant decrease in cell viability in A549 and DLD-1 cells was observed. Since downregulation of LRP/LR impedes telomerase activity and decreases cell viability, siRNAs directed against LRP mRNA acts as potential alternative therapeutic tools for treatment of lung adenocarcinoma and late-stage colorectal carcinoma.