Saudi Cultural Missions Theses & Dissertations
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Item Restricted EPIGENETIC AND TRANSCRIPTIONAL DYNAMIC IN PERIODONTAL DISEASE(University of North Carolina at Chapel Hill, 2017-03-19) Hefni, Eman; Barros, SilvanaObjectives: Several studies have shown the involvement of epigenetics with periodontal disease. Since functional dissociation of Paracellular permeability is expected during bacterial infection, we hypothesize that the methylation of host oral epithelial DNA represents an important element in the disruption of barrier function and pathogenesis of periodontal diseases. With this In vitro study, we aimed to assess whether there is altered epithelial permeability measuring trans epithelial resistance after Porphyromonas gingivalis (P. gingivalis), Campylobacter rectus (C. rectus) and Fusobacterium nucleatum (F. nucleatum) infection. Plakophilin2 (PKP2) methylation status and expression levels were also investigated. In addition, investigate the potential effects of DNA methyltransferase (DNMT) inhibitors on epithelial barrier function in response to infection with periodontal pathogen in human gingival epithelial cells. Methods: Primary human gingival epithelial cells (HGEPs) were stimulated with P. gingivalis, strain, C. rectus and F. nucleatum (MOI 50) either in the presence or absence of DNMT inhibitors (10 μM of RG108 or EGCG). CellTiter-Blue® Cell Viability Assay (Promega) was used to determine an optimum cell density and maximum inhibitor concentration at which cell viability is maintained. Transepithelial electrical resistance (TER) at various time points were performed using an EVOM® electrical resistance system. DNA methylation was quantified by PCR using EpiTect Methyl II PCR Primer Assays for PKP2. Immunofluorescence analysis was performed using PKP2 antibody and analysis performed using Zeiss710 confocal microscope. Results: Exposure of HGEPs to P. gingivalis resulted in decreased TER (P=<0.001) associated with increased cell permeability. Methylation assays showed increased methylation levels of the PKP2 in comparison to non-infected controls (P=<0.001) and an associated PKP2 down-regulation (P=<0.005). For infected cells treated with DNMT inhibitors, PKP2 mRNA expression was increased (P=<0.001) and TER values similar to non-infected cells. Comparatively, immunofluorescent staining of the PKP2 protein showed reduced protein expression in infected cells not treated with DNMT inhibitors. Conclusion: DNA methylation levels of PKP2 can affect epithelial barrier function potentially conferring increased susceptibility to infection. DNMT inhibitors can affect cell adhesion dissociation in response to infection minimizing the disturbance to the barrier function.12 0Item Restricted Potential epigenetic biomarkers of circulating tumour DNA to improve detection of endocrine therapy resistance in breast cancer(Saudi Digital Library, 2023-03-07) Alahmari, Areej; Guttery, DavidBackground and aims: Endocrine therapy resistance is a major clinical problem and leading cause of metastatic breast cancer (MBC) death. Epigenetic changes via aberrant DNA methylation play an important role in therapy resistance. This thesis aimed to investigate aberrant methylation in oestrogen responsive elements (EREs) as a biomarker of hormone therapy resistance using MCF7 BC cell lines resistant to tamoxifen (TAMR-MCF7) and fulvestrant (FULVR-MCF7), with a view to utilising these signatures for early detection of hormone therapy resistance through circulating-tumour DNA (ctDNA). Methods: The four methylation conversion kits were compared for DNA recovery of to select the most efficient method for ctDNA methylation analysis. Aberrant DNA methylation was analysed in cell lines by shallow-depth whole genome bisulfite analysis (WGBS), and correlated with RNA-Seq data. Lastly, sets of primers were designed and validated the analysis of aberrant ctDNA methylation to apply to longitudinal plasma from patients with MBC. Results: The Premium bisulfite kit from Diagenode was the optimal kit for methylation conversion. EREs were hypermethylated and oestrogen target genes significantly downregulated in hormone therapy resistant cell lines. The hypermethylation phenotype existed more in ERE enhancers than promoters. EREs were not the dominant responsive elements in the aberrant DNA methylation analysis. FOX and AP-1 responsive elements were the top hits for hypermethylation in both resistant cell lines, while TEAD and MYC responsive elements were hypomethylated in FULVR and TAMR, respectively. The designed methylated-specific assays for OSMR promoter, and the enhancer of CBX4, TFF1, TRAF7 TERT, and RASA3 validated the enriched methylation level of these regions at resistant cell line. Conclusions: Results generated in this thesis has identified potential candidate regions that can be applied to longitudinal ctDNA samples from MBC patients to determine whether aberrant ctDNA methylation in EREs can be analysed as a marker of endocrine resistance.12 0