Saudi Cultural Missions Theses & Dissertations
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Item Restricted The role of Tet2 and Tet3 in the molecular programming of blood cell differentiation during zebrafish development(University of Nottingham, 2025) Alayed, Amal Saeed; Gering, Martin; Wilkinson, RobDNA methylation is an epigenetic mark that regulates gene expression during cellular differentiation. Ten-eleven translocation (Tet) dioxygenase enzymes are involved in active demethylation and are required for hematopoietic stem cell (HSC) differentiation. HSCs arise from haemogenic endothelial cells (HECs) in arterial vessels, which depend on Notch signalling for their specification. Initially, the loss of HSC formation in tet2/tet3 double mutants was attributed to a failure in HEC formation caused by defective Notch signalling, despite normal vascular patterning and arterial specification. This study aimed to reinvestigate Notch signalling and HEC development in the ventral wall of the dorsal aorta (vDA) of tet2/tet3 double mutants to resolve these inconsistencies. Here, the Notch reporter transgene csl:mCherry was expressed in the endothelium of the vDA in the absence of Tet2 and Tet3. Confocal analysis of the gene trap line qmc551, which exhibits GFP expression in primitive red blood cells (prRBCs) and HECs of the vDA, demonstrated normal GFP expression in HECs of the vDA at 2 days post fertilisation (dpf). It also revealed a few GFP+ hematopoietic stem and progenitor cells (HSPCs) in the mesenchyme below the DA in tet2/tet3 double mutants. Whole-mount in situ hybridisation (WISH) experiments using the HEC marker growth factor independence 1aa (gfi1aa) revealed that, at 40 hours post fertilisation (hpf), gfi1aa expression persisted in the HECs of the vDA in tet2/tet3 double mutant embryos, contrasting with its typical downregulation in wild-type (WT) embryos at this stage. These findings suggest a defect in the endothelial- to-hematopoietic transition (EHT), likely resulting from disrupted epigenetic programming of HECs in the absence of the two Tet proteins. In tet2/tet3 double mutant embryos, circulation appeared normal without evident shunts; however, blood flow velocity was reduced. To investigate the molecular programming underlying these defects, single-cell transcriptome analysis was performed on a cell population enriched for qmc551:GFP and Gata1a:dsRed single and double- positive cells isolated from 2dpf WT and tet2/tet3 double mutant embryos. Transcriptomic analyses revealed significant downregulation of key endothelial genes, such as apelin, plvapb, casz1, and pecam1. These data suggest a critical role for Tet2 and Tet3 in endothelial programming and angiogenesis. The analysis also showed significant downregulation of erythroid genes in prRBCs. WISH experiments revealed increased gata1a expression in circulating prRBCs at 2 and 3dpf, indicating a late maturation defect in tet2/tet3 double mutant embryos. Additionally, reduced slc4a1a mRNA expression in mutants at 20hpf, prior to the onset of circulation, suggests delayed early differentiation of prRBCs. These findings indicate that the absence of both Tet2 and Tet3 leads to delayed erythroid development. In summary, this study elucidates the multifaceted roles of Tet2 and Tet3 proteins in regulating both primitive and definitive hematopoietic waves during zebrafish embryogenesis.31 0Item Restricted Novel Strategy to Unlock Transgenerational Stress Memory in Clonal Plants(warwick university, 2024) Alotaibi, Hidayah; Gutierrez-Marcos, JoséBeing sessile organisms, plants are exposed to a wide range of environmental stress conditions. Recent studies have shown that plants can store information about environmental stresses and access this associated memory to mount a primed response that offers protection from subsequent stress events. This ‘stress memory’ is thought to be mediated by epigenetic modifications, which in turn modulate gene expression, phenotype, and metabolism. However, these environmentally directed epigenetic changes although they are integrated into somatic cells, they are short-lived and/or actively reset during sexual reproduction. Notably, using Arabidopsis as a model system we have recently found that clonal plants generated using zygotic transcription factors display epigenetic and transcriptional features present in the founder cells used for regeneration. Moreover, these molecular signatures are stably transmitted over multiple generations of sexual reproduction, creating distinct phenotypic variants. Therefore, we hypothesised that cloning from somatic cells exposed to distinct environmental stimuli could be used to engineer specific primed responses in plants. To test this hypothesis, we have generated clonal lines from tissues exposed to a short abiotic stress pulses that were propagated sexually over three generations in stress free conditions. Our data shows clonal plants derived from primed tissues display a transgenerational stress memory response, which is associated with specific transcriptional states, and enhanced tolerance under stress. Our study also aiming to identify the molecular mechanisms implicated in the integration, storage and retrieval of the acquired stress memory with the aim of engineering specific primed responses in plants. Our data shows that the transcriptional activity of DREB2A genes is necessary and partly sufficient for a salt-induced transgenerational salt memory in clonal plants46 0Item Restricted THE IMPACT OF O-GLCNAC SIGNALING ON DNA HYDROXYMETHYLATION IN TEMPORAL LOBE EPILEPSY(the University of Alabama at Birmingham, 2024) Bahabry, Rudhab; Lubin, FarahTemporal Lobe Epilepsy (TLE) is the most prevalent form of focal epilepsy and the most treatment-resistant type. This condition is characterized by recurrent, unprovoked seizures typically originating in the hippocampus. Epigenetic modifications such as DNAme changes have been implicated in brain functions such as synaptic plasticity, learning and, memory, and cognition. The oxidation of DNA 5-methylcytosine (5-mC) and its oxidized form, 5-hydroxymethylcytosine (5-hmC) catalyzed by Ten- Eleven Translocation (TET) family of dioxygenases and linked to epilepsy's hyperexcitable state. Post‐translational modifications (PTMs) of proteins, such as O- GlcNAcylation, facilitate cells' immediate responses to intracellular or extracellular environmental stimuli by modifying the functions of targeted proteins. Recent evidence suggests a significant interaction between O-GlcNAc transferase (OGT) and TET enzymes, affecting TET activity and chromatin structure, thus influencing gene expression. This dissertation hypothesized that decreased TET1 O-GlcNAcylation in the epileptic hippocampus contributes to pathological hyperexcitability via decreased 5-hmC levels in a TLE rat model. We found a significant reduction in 5-hmC levels in the hippocampi of both human TLE patients and kainic acid-induced TLE rats, without affecting 5-mC levels. hydroxyMethylated DNA immunoprecipitation sequencing (hMeDIP-Seq) analysis indicated a notable loss of 5-hmC in intergenic regions of the epileptic hippocampus, with identified pathways related to GABA signaling and ion transport. In-vivo manipulating hippocampal Tet1/5-hmC levels showed direct implications on seizure susceptibility and resilience. Furthermore, a co-immunoprecipitation (co-IP) assay was utilized to investigate the interactions between TET1 and OGT within the hippocampus of epileptic tissues, revealing a decrease in their interaction compared to the controls. Additionally, we further identified the presence of a TET1-OGT complex by Western blot assays, emphasizing the presence of physical interaction between these proteins. Moreover, we aimed at detecting protein O-GlcNAcylation levels through the use of sWGA assay demonstrating a significant reduction in the O-GlcNAcylation of TET1 in epileptic animals, to the point where it was nearly undetectable when compared to control animals. Finally, using Thiamet-G treatment, we were able to increase O-GlcNAcylation and global 5-hydroxymethylcytosine in our epileptic animals, restoring the levels to levels similar to the control. These results elucidate the significant role of TET1 levels and O-GlcNAcylation in epilepsy, establishing a foundational link between PTMs and epigenetic regulation in the disorder and suggesting the potential of targeting O-GlcNAcylation pathways as a novel therapeutic strategy for epilepsy.6 0Item Restricted Investigating the Regulation of Early Development of the Amphipod Crustacean Parhyale hawaiensis(Saudi Digital Library, 2023-10-13) Rawas, Salha; Aboobaker, AzizThe amphipod crustacean Parhyale hawaiensis is an ideal model organism for studying development, evolution, and regeneration. Its ease of rearing in the lab, accessibility of embryos at all developmental stages, large broods produced year-round, and the variety of functional experiments that can be conducted on animals and embryos make it a valuable resource for investigating many biological questions. As an out group to insects, Parhyale offers a platform for studying biological diversity through comparative studies. Recent research has identified a full repertoire of single-copy genes encoding the machinery associated with DNA methylation in the Parhyale genome. This discovery provides an opportunity to explore the role of DNA methylation in early embryogenesis using an invertebrate model system. In this thesis, we present Parhyale hawaiensis as a tractable model to study DNA methylation dynamics during embryonic development. Our aim was to investigate the regulation during early embryo development, describe the maternal-to-zygotic transition (MZT) and zygotic-genome-activation (ZGA) in Parhyale, and explore the potential function of DNA methylation during embryogenesis while examining its regulatory role in gene expression. Parhyale possesses a large genome size of approximately 3.6 Gb, which was initially sequenced by Kao et al. in 2016. An update using Dovetail technology enhanced the assembly by generating large scaffolds butleft significant gaps. Therefore, our objective was to further improve the existing assembly by integrating PacBio data to close the gaps and correct assembly errors. This effort proved successful, as over 70% of the gaps in the assembly were closed, suggesting further coverage would give further improvement. Subsequently, we performed an expression-driven annotation using a wide range of RNA-seq data from various embryonic stages and different adult conditions. The well-annotated genome served as the foundation for analyzing transcriptomic data from early embryonic stages to detect de novo zygotic transcription using intronic RNA signals. We described the transcriptome of early Parhyale embryos and proposed a model for the maternal-to-zygotic-transition (MZT) and zygotic-genome-activation (ZGA) timelines. Our findings demonstrated that zygotic transcription begins as early as 11 hours post-fertilization and occurs in two waves. The minor wave of ZGA in Parhyale commences at the 32-cell stage, while the major wave takes place at the start of the blastodisc formation stage. We discovered that the earliest transcribed genes in Parhyale are typically short, intron-less or intron-poor, and newly evolved. Furthermore, we validated the presence of DNA methylation mediator genes, with a focus on DNMT1, DNMT3, and MBD2/3. We analyzed the expression pattern of DNA methylation machinery genes during the early stages of Parhyale embryogenesis and found that they are provided maternally. To explore the relationship between DNA methylation and gene expression, we correlated our gene expression datasets with methylseq datasets. Our results revealed a positive correlation between gene-body methylation and gene expression levels. Lastly, we conducted functional experiments targeting DNMT1, DNMT3, and MBD2/3 to understand their roles during embryonic development. We utilized CRISPR/Cas9 to generate knockout animals for each of the three genes, revealing that the loss of any of these genes is lethal to embryos. Additionally, we performed RNA interference (RNAi) knockdown on MBD2/3, which also resulted in an early embryonic lethality, confirming its essential role in embryogenesis. Profiling the transcriptome of knockdown embryos revealed that the knockdown of MBD2/3 altered the expression of many genes, including developmental transcription factors with low levels of gene-body methylation.The work presented in this thesis offers a comprehensive understanding of the early embryonic development of Parhyale and emphasizes the crucial role of DNA methylation during embryogenesis. In future, we aim to investigate whether MBD2/3 regulates gene expression through its association with the NuRD complex, and if this occurs in a DNA methylation-dependent, independent, or both manners. Furthermore, the improved assembly and annotation presented in this thesis will greatly facilitate more precise analyses, enabling us to address intriguing questions regarding the promising model organism Parhyale hawaiensis.40 0