Saudi Cultural Missions Theses & Dissertations
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Item Restricted Investigating the Impact of Inherited Genetic Variations on Antidepressant Drug Response Using ARMS Genotyping(University of Glasgow, 2024) Alward, Dana; Marks, LeahMajor Depressive Disorder (MDD) is a severe mental health condition affecting millions of people worldwide that can impact daily functioning and is caused by the regulation of serotonin. Selective serotonin reuptake inhibitors (SSRIs) are the primary treatment for MDD. CYP2D6 encodes an enzyme involved in drug metabolism of SSRIs antidepressants. This study aimed to evaluate pharmacogenetics' ability to improve SSRI treatment outcomes by genotyping CYP2D6 SNPs associated with poor SSRI metabolism. This was done using the Amplification Refractory Mutation System (ARMS), where primers were designed to produce specific amplicons for different alleles of the targeted SNPs. Three SNPs were examined with random HRC-2 DNA samples. The results showed that the primers for SNP rs35742686 and SNP rs5030655 worked well after optimization, while SNP rs3892097 primers were deemed unreliable for further use. A tri-ARMS protocol with three primers for each SNP was successfully combined for SNPs rs35742686 and rs5030655. However, multiplexing both SNPs in one ARMS reaction proved difficult to interpret due to close fragment sizes. Genotyping CYP2D6 SNPs provided insights into the metabolism of SSRIs. In conclusion, the findings from the CYP2D6 gene analysis have significant implications for personalised medicine and drug therapy. As research in pharmacogenomics continues to advance, it is essential to address the complexities of genetic diversity and the translation of genomic data into clinical practice. Further studies with larger sample sizes and diverse populations will be essential for refining our understanding of drug metabolism variability and improving therapeutic outcomes.28 0Item 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 0