Saudi Cultural Missions Theses & Dissertations
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Item Restricted Investigation of PDI Involvement in Leukocyte Adhesion to Endothelial Cell in Inflammatory State Using Gene Editing (CRISPR/Cas9) Technology(Arabin Gulf university, 2025) almashhur, wedad awadh ali; Fathallah, Mohamed DahmaniLeucocyte adhesion to endothelial cells is a critical step in the inflammatory process, influenced by various molecular mediators, including Protein Disulfide Isomerase 1 (PDI1). This study aimed to investigate the broader roles of additional PDI family members in regulating endothelial cell activation and leucocyte adhesion under inflammatory conditions. We isolated endothelial cells with a cobblestone morphology from human peripheral blood and umbilical cords, achieving yields of 1.2 × 10⁵ and 3.5 × 10⁵ cells/mL, respectively. Neutrophils were isolated from blood samples using magnetic bead technology, and their phenotype was confirmed via flow cytometry. To induce inflammation, endothelial cells were stimulated with TNF-α (100 ng/mL). Quantitative real-time PCR was utilized to assess the expression of PDI genes and adhesion molecules, specifically ICAM-1, VCAM-1, and E-selectin, which serve as established markers of endothelial activation. CRISPR/Cas9 technology facilitated the knockout of PDIA2, PDIA3, PDIA4, and PDIA6 in endothelial cells, with validation achieved through sequencing and Western blot analysis. Efficient gene editing was confirmed by the significant reduction in expression levels of the targeted PDIs. The results demonstrated a 62.5% decrease in the expression of ICAM-1 and VCAM-1, while E-selectin expression was reduced by an average of 55% across all knockouts. Functional assays revealed significant reductions in neutrophil adhesion (p < 0.01) to knockout endothelial cells, with specific decreases of 25% in PDIA2, 20% in PDIA3, 35% in PDIA4, and 2% in PDIA6 knockout cells. These findings underscore the essential roles of various PDIs in the activation of endothelial cells and leucocyte adhesion during inflammation. The study illustrates the effectiveness of CRISPR/Cas9 technology in elucidating gene functions and highlights PDIs as potential therapeutic targets for inflammatory vascular diseases. This research provides valuable insights into the molecular mechanisms underlying inflammation and opens avenues for novel therapeutic strategies.2 0Item Restricted Effect of the inflammatory mediator TNF-α on colorectal cancer epithelial cells development and metastasis, role of dietary carcinogens and miRNA(ICL, 2023-05-01) Alotaibi, Aminah Ghazi; Gooderham, Nigel; Li, JiaColorectal cancer (CRC) is the third most common cancer world-wide and second leading cause of mortality. The majority of CRC cancer cases result from epigenetic and genetic alterations that promote development and metastasis of the disease. Exposure to environmental and dietary carcinogens are strongly associated with CRC. Also, inflammatory mediators are known as a major risk factor for CRC, however the underlying mechanisms are still understudied. Upregulation of pro-inflammatory mediators and dysregulation of miRNAs in the tumour microenvironment (TME) have been observed in CRC. Tumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine thought to play numerous roles in tumour progression including epigenetic gene regulation, activation of tumour promoting signalling pathways, thus presence of TNF- in CRC tumour microenvironment may be key to promoting CRC progression. I hypothesise that the presence of TNF- α in the TME could regulate miRNAs and enzyme expression to induce DNA damage caused by dietary carcinogens, thereby stimulating changes that promote CRC development and metastasis. The present study investigated this hypothesis through a mechanistic approach with in vitro cell line culture. Effects of TNF-α on phenotypic changes were observed and the potential involvement of miRNAs were determined. The results showed that TNF-α enhances dietary carcinogen-induced DNA damage through activation of JNK signalling pathway. Also, TNF-α induced metastatic phenotype cell proliferation and migration through miRNA regulation. Moreover TNF-α regulated expression of CYP450 enzymes through miRNA regulation, which can promote chemical carcinogen genotoxicity. Taken together, the data indicated that CRC progression and metastasis may be related to epigenetic and inflammatory mediators active at the tumour site. Understanding these molecular mechanisms could provide better prevention and therapeutic strategies.27 0