Saudi Cultural Missions Theses & Dissertations
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Item Restricted Transcriptomic Data Interpretation of Stickleback Embryos response to: Cd, Ibuprofen, and DB(University of Birmingham, 2024-08-27) Alarfaj, Manal; Williams, TimothyThis study aimed to biologically interpret the impacts of three common chemicals found in aquatic environments: Cadmium, Ibuprofen, and Dibutyl Phthalate (DBP). We analysed data from previous experiment conducted on three-spined stickleback (Gasterosteus aculeatus) embryos, utilizing microarray technology to identify upregulated genes. Using DAVID bioinformatics, we focused on clusters with high enrichment scores and significant fold changes, specifically examining the effects of these chemicals on small heat shock proteins (sHSPs), muscle tissue, coagulation processes, and eye lens proteins. By investigating the potential mechanisms behind these upregulated genes and referencing relevant literature, we hypothesize that these chemicals impact muscle tissues, the eye lens, and possibly neurons. However, it remains uncertain whether these effects result primarily from oxidative stress or other toxic mechanisms inherent to the chemicals. Additionally, based on existing research, we anticipate that these chemicals may cause defects in embryos, including cardiac issues, impaired swimming, and eye problems. Further research is needed to fully understand these mechanisms, validate our findings, and explore the broader effects of these chemicals on aquatic organisms.10 0Item Restricted STUDIES ON THE ROLE OF TRANSMEMBRANE PROTEINS IN HEMOSTASIS(University of North Texas, 2024-09) Deebani, Afnan; Jagadeeswaran, PudurTransmembrane proteins (TMEMs) are a unique group of cell membrane-embedded proteins with largely undefined functions. Since we found 89 tmems in zebrafish thrombocytes by our RNAseq analysis, we hypothesized that they may play a role in thrombocyte biology and hemostasis. To verify this hypothesis, we screened these tmem genes by knockdown in zebrafish and assaying for gill bleeding. We found tmem242 and tmem183a knockdowns led to greater bleeding suggesting their roles in hemostasis. Therefore, we investigated Tmem242 and Tmem183a roles by knocking them down in zebrafish followed by assessing thrombocyte production, thrombocyte aggregation, fibrin formation, in vivo thrombus formation, and coagulation factors levels. We found that tmem242 knockdown did not affect thrombocyte production and aggregation, but it affected coagulation pathways observed by delaying fibrin formation and in vivo thrombusformation, which indicate bleeding phenotype. However, tmem242 knockdown led to increased mRNA levels of coagulation factors, especially f9a. Further investigations into Tmem242 role in hemostasis and F9a were done by conducting various assays including, western blot, microthrombi detection, ROS detection, and qRT-PCR to detect mRNA levels of hepatocyte transcription factors, such as sirt6 and nrf2. We found that tmem242 knockdown increased ROS production which signals to increase sirt6 transcription. This subsequently raises nrf2 expression, which in turn elevates f9a transcripts, leading to DIC-like conditions. On the other hand, tmem183a knockdown in zebrafish led to reduced thrombocyte counts, diminished aggregation, delayed fibrin generation, and prolonged in vivo thrombus formation. These findings were explained by the observed reduction in mRNA levels of thrombocyte producing genes, thpo and fli1 and by the decreased mRNA levels for several coagulation factors after tmem183a knockdown. Taken together, the knockdown of tmem183a in zebrafish resulted in bleeding phenotype linked to the downregulation of key genes involved in thrombopoiesis and decreased mRNA levels of coagulation factors. In summary, these studies highlight the crucial roles of Tmem242 and Tmem183a in maintaining normal hemostasis.12 0Item Restricted STUDIES ON ZEBRAFISH THROMBOCYTES(University of North Texas, 2024) Fallatah, Weam; Jagadeeswaran, PudurZebrafish thrombocytes exhibit characteristics of human platelets and megakaryocytes, making them valuable for studying megakaryopoiesis and thrombopoiesis. Using single-cell RNA sequencing, we analyzed gene expression in young and mature zebrafish thrombocytes. We identified 394 protein-coding genes unique to young thrombocytes, many corresponding with human orthologs, suggesting shared regulatory mechanisms in zebrafish and humans. We hypothesized knocking down these 394 genes should identify the novel regulatory genes that control thrombocyte maturation. To address this, we used the piggyback knockdown method to knock down these genes to study their biological functions in zebrafish thrombopoiesis. We first found the knockdown of nfe2, nfe2l1a, and nfe2l3 reduced both young and mature thrombocyte counts, confirming their role in thrombopoiesis. A comprehensive knockdown screening of the uniquely expressed genes in young thrombocytes identified 7 candidate genes associated with thrombopoiesis. We selected the spi1b gene for further mutant characterization, which revealed its critical role in young thrombocyte development, with homozygous mutations leading to embryonic lethality. Considering megakaryocyte properties in thrombocytes, we studied the potential for polyploidization in zebrafish thrombocytes. The inhibition of AURKA led to the development of polyploid thrombocytes resembling mammalian megakaryocytes, suggesting the retention of genetic programs for megakaryocyte development in zebrafish thrombocytes and providing insights into the evolutionary basis of thrombopoiesis. Thus, our study reveals critical gene expression patterns and regulatory factors in zebrafish thrombocyte development, offering insights into conserved mechanisms relevant to developmental biology and research in thrombosis and hemostasis disorder.21 0