Saudi Cultural Missions Theses & Dissertations
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Item Restricted Bioengineering of transcriptional elements driving MMP13 gene in skeletal development(University of Liverpool, 2024-08) AlSalhi, Sara Ibrahim; Bou-Gharios, GeorgeMmp13 is a primary catabolic factor involved in cartilage degradation through its ability to cleave collagen type II and other cellular components. In addition to being necessary for the formation of various cells, organs, and tissues, Mmp13 expression is regulated transcriptionally by two main elements: the proximal promoter and distal enhancers. This study aims to identify the transcriptional elements that regulate Mmp13 gene expression. Identification of novel Mmp13 enhancers was conducted in silico using the Encyclopaedia of DNA Elements (ENCODE), based on histone modifications (Limb H3K4me1 and Limb H3K27Ac), fibroblast coverage, chondrocyte, and embryonic limb regulatory elements from public ChIP-Seq data, and evolutionarily conserved sequences, in addition to transcription factor profiles of Runx2 and vitamin D. All prospective Mmp13 enhancer sequences were analysed using three different software tools: CIIIDER, TRANSFAC, and JASPAR, for the prediction of transcription factor binding sites. Each enhancer sequence was cloned upstream of the Hsp68 silenced promoter and lacZ gene to create a β-galactosidase reporter construct, which was then used to generate transgenic mice. Embryos were collected at E15.5 and tested for lacZ gene expression and tissue expression analysis. Constructs were also transfected in vitro into pre-osteoblasts (MC3T3- E1), NIH3T3 mouse embryo fibroblasts, human chondrocytes (SW1353), and primary chondrocytes extracted from OA patients. Among the seven tested Mmp13 enhancer regions, strong skeletal element expression was detected in the region from -21.9 to -21.1 kb, which overlaps with Runx2 and Sox9 binding sites. Other enhancers revealed some skeletal element activity but were not as prominent. Expression in various tissues and organs, including skin, was observed in multiple regions. In contrast, sequences aligning with the highest peaks of Runx2 at -29 kb and -32.5 kb did not show significant expression. In vitro, Mmp13-transfected enhancer sequences demonstrated enzyme activity, with the highest responses observed in chondrocyte and human cells at -10 kb and -29 kb regions, along with -21.4 kb that indicate a potential regulatory influence. Comparisons of potential enhancers in mouse embryos highlighted the sequences in the intronic 5', -10 kb, -19.2 kb, and -21.4 kb regions as significant Mmp13 enhancers regulating gene expression but not -29 and -32.5 kb regions. Identifying these specific enhancers could lead to targeted therapeutic strategies to modulate MMP13 activity, potentially slowing or preventing cartilage degradation in degenerative diseases.18 0Item Restricted Repurposing Approved-Chemotherapeutics for Head and Neck Squamous Cell Carcinoma(Saudi Digital Library, 2022) Alkurdi, Khlood; Tappuni, Anwar; Muy, Teck- TehThe advancement in head and neck squamous cell carcinoma (HNSCC) therapy necessitates the acquisition of a superior understanding of cancer molecular biology and the capability to use this knowledge to adapt the therapy to achieve best patient outcome. HNSCC is heterogeneous in its basis, mechanism, and behaviour, and its biomarkers are considered greatly important in identifying the tumorigenesis mechanism that is involved in therapy resistance to increase patient survival. Resistance to chemotherapy is a major cause of treatment failure in HNSCC patients; the long-term assessment of a patient’s compliance with chemotherapy is key to overcoming treatment resistance and controlling cancer. FOXM1 is a known oncogene and plays an important role in conferring chemoresistance. The objective of the current study was to investigate candidate approved-chemotherapeutic drugs to inhibit FOXM1 in HNSCC cells in the aim to counteract chemoresistance. Methods: Dose-response assay was performed to investigate the potency of 16 candidate drugs using crystal violet cell viability assay in 11 human cell lines consisting of normal oral keratinocytes, oral fibroblasts, normal skin keratinocytes, premalignant oral buccal mucosa and 7 HNSCC cell lines. The most potent drugs with least toxicity towards normal control cells were selected to investigate their effect on FOXM1 gene expression using reverse transcription quantitative PCR. Results: Vincristine, lanatoside A and topotecan were found to be the most potent drugs with least toxicity across the 11 cell lines. Lanatoside A was the only effective drug for a paclitaxel resistant cell line (CaLH2-PTX). All three drugs significantly suppressed FOXM1 gene expression levels in all 7 HNSCC cell lines. Conclusion: This study identified 3 existing candidate approved chemotherapeutic drugs demonstrating potent anti-proliferation effects against a panel of HNSCC cell lines and with low toxicity towards normal cells. Further investigations are needed to understand their mechanisms and their effects on FOXM1 in HNSCC cells.7 0