Saudi Cultural Missions Theses & Dissertations
Permanent URI for this communityhttps://drepo.sdl.edu.sa/handle/20.500.14154/10
Browse
3 results
Search Results
Item Restricted Epigenetic Gene Regulation by the Type I Restriction Modification Systems(University of Leicester, 2024-04) Althari, Yasmeen; Oggioni, MarcoEpigenetic modifications mediated by type I restriction modification systems in prokaryotes have been linked to gene regulation. In Streptococcus pneumoniae, differential expression of alternative specificity subunits within the SpnIII type I restriction modification system had been shown to selectively modulate bacterial virulence. Given the presence of this system in the core genome of pneumococci, but not of the related Streptococcus mitis, SpnIII could represent a conserved phase-variable regulatory mechanism operating on a global scale. However, this phenomenon had yet to be examined at the single-gene level, and the underlying molecular mechanism remained unexplored. In this study, I utilised pneumococcal strains locked for alternative specificity subunits (no phase-variation at the locus) to validate methylation-dependent differential gene expression of several model genes using various transcriptomic and translation reporter assays. Additionally, I delved into the complexity of recombination within the spnIII locus, which is partially governed by a site-specific tyrosine recombinase whose mechanism of controlling the rate of recombination remains elusive. To investigate the regulation of this recombinase, I explored the possibility of its control by a hairpin structure in the 5-prime UTR of the gene representing a potential RNA thermosensor, considering the recognised temperature sensitivity of recombination. To provide evidence to support my hypothesis on epigenetic gene regulation, I analysed the non-phase variable prototype type I EcoKI system in Escherichia coli which revealed a similar methylation-dependent differential expression following deletion of the EcoKI methyltransferase. This discovery underscores the epigenetic impact of type I RMSs, suggesting a widespread occurrence and possibly a global relevance of this phenomenon across bacterial genera. Overall, my findings propose that methylation influences both local gene topology and global genome architecture, thus playing a crucial role in methylation-mediated regulation. This mechanism involves the interplay between methylation and DNA-binding proteins, which collectively shape the overall genome architecture and transcriptional landscape.12 0Item Restricted Biomedical and Public Health Studies on Susceptibility to Post-Traumatic Stress Disorder(Saudi Digital Library, 2023-05) Al Jowf, Ghazi Ibrahim Abdurahman; Rutten, BartTraumatic stress exposure can induce the development of mental disorders such as post-traumatic stress disorder (PTSD), resulting in a decreased quality of life and increased disease burden. This thesis investigated and addressed the gaps of knowledge in the understanding of the association between epigenetic mechanisms, gene activity, and differential susceptibility to PTSD. It also identified the public health measures that can be effectuated for the prevention of PTSD and how biomarkers may support these measures. Accordingly, it discusses the possible ways that can aid in the early identification and detection of PTSD resulting from traumatic stress, and the possibility of developing biomarkers of increased susceptibility. It also discusses how this early detection can help with early intervention, and if early enough, prevention. Application of such models, with findings from future research to establish cost-effective measures, can greatly enhance the patient’s quality of life and decrease the disease burden.8 0Item Restricted HISTONE H3K27ME3 IN PREIMPLANTATION MOUSE EMBRYO DEVELOPMENT(Saudi Digital Library, 2023-06) Harasani, Ahlam; Pennings, Sari; Taylor, JaneDuring the developmental stages of the mouse preimplantation embryo, totipotent blastomeres generate the first three cell lineages of the embryo: trophectoderm (TE), epiblast (EPI) and primitive endoderm (PrE). The first cell fate decision consists of the separation of cells that will comprise the TE and the ICM (inner cell mass). Then, further segregation of ICM cells takes place into EPI-fated cells and PrE-fated cells. It is thought that this cell lineage specification is mediated in part by epigenetic processes, in which genes are regulated both temporally and spatially without any changes in the genome. Certain transcription factors such as Oct4, Sox2, Cdx2, Gata6, Nanog, and Fgf4 coordinate pluripotency and early cell fates during development. They are part of a network of pluripotency and associated genes that control each other’s expression as well as that of other major transcription factors through epigenetic mechanisms and simultaneous signalling pathways. Epigenetic mechanisms such as DNA methylation and post-translational histone modifications play a critical role in regulating gene expression in preimplantation embryos. During the embryonic development process, methylation of DNA is required for overall development. Similarly, trimethylation of lysine 27 on histone 3 (H3K27me3) is required to regulate developmental genes. This histone modification is facilitated by the polycomb repressive complex 2 (PRC2) subunit named enhancer of zeste homolog 1/2 (EZH1/2), an enzyme that epigenetically modulates chromatin structure and gene expression through H3K27me3 and may be involved in recruitment of DNA methyltransferases for gene silencing. The SET domain of EZH2 is thought to serve in the mechanism for transferring methyl groups from S-adenosylmethionine (SAM) to H3K27. EZH2 has a pivotal role to play in cancer progression and malignancy, but it also plays a vital role in the renewal, maintenance and differentiation of stem cells into specific cell lineages. Several recent studies have shown that blocking PRC2-EZH2 alone is not enough to suppress cancer-related genes. The dual inhibitors of both PRC2-EZH2 and PRC2-EZH1 were found to suppress H3K27me3 completely in cells after treatment. It is also suggested that Ezh1 may be involved in enhancing the maintenance of ES cell identity when EZH2 is not present. In this study, two small molecules, GSK-126 and EPZ-6438, have been used to inhibit the catalytic subunit of EZH2 in preimplantation mouse embryos in vitro to study the contribution of this enzyme through its inhibition in the embryos' developmental process. The objectives of this study are to determine the role of PRC2 complex methyltransferase EZH2 and the H3K27me3 epigenetic mark in the development of pluripotent ICM cells and other early cell fates in the mouse preimplantation embryo, based on morphology, epigenetic and cell fate markers and gene expression. This study was carried out using cultured preimplantation embryos from superovulated B6CBA F1 mice. Fertilised zygotes were collected at embryonic day 0.5 (E0.5). Embryos were cultured from age E0.5 (1-cell stage), to E1.5 (2-cell stage), E2.5 (8-cell stage) or until E4.5 (blastocyst stage) in the presence of either GSK-126 (1μM, 3μM), EPZ-6438 (0.5μM, 0.75μM, 1μM) or dimethyl sulfoxide (DMSO) as a vehicle. The effect of EZH2 inhibition was assessed based on morphology and using two techniques: reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for determining gene expression and immunocytochemistry (ICC) for measuring protein abundance. Quantitative analyses of the gene expression data of the relevant genes were conducted using the housekeeping genes GAPDH and H2Afz as endogenous controls. Indirect immunofluorescence staining was used in embryos, images were captured using confocal microscopy, and analyses were performed using MATLAB, IMARIS, and ImageJ software programs. The findings showed that inhibition of EZH2 reduced H3K27me3 levels in blastocysts. Also, the number of blastomeres was lower in the treated blastocysts than in control. EZH2 appears to influence developmental delay in the preimplantation mouse embryo. Furthermore, ICC results showed a decrease in the number of blastocyst cells expressing CDX2, the trophectoderm marker, compared with the control group. In conclusion, the results of this study will contribute to our understanding of the role H3K27me3 plays in early mammalian embryos during the specification and maintenance of cell fates.24 0