Saudi Cultural Missions Theses & Dissertations
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Item Restricted Quantitative Detection of Hepatitis C Virus RNA in Dried Blood Spots(The University of Manchester, 2024-05-16) Ismaeel, Loui; Klapper, PaulAn estimated fifty-eight million people have chronic hepatitis C virus (HCV) infection, with about 1.5 million new infections occurring each year. A sizeable proportion of these new infections occur among injecting drug users. Dried blood spots (DBS) have revolutionised HCV diagnosis in this difficult to reach population and have further advantages when used in healthcare resource-poor regions of the world. Diagnosis of infection may be made serologically but confirmation of chronic infection necessitates the detection of HCV RNA and response to therapy must be monitored by quantitation of HCV RNA in blood (‘viral load’). A current limitation of the use of DBS is that quantitation of HCV RNA is not possible from standard DBS samples as test blood volume cannot be accurately measured. In the present work a volumetric microfluidic device (HemaXis™ DB 10) was evaluated for preparation of DBS samples to allow quantitation of HCV RNA from the DBS. The HemaXis device was used in conjunction with a custom-made Perkin Elmer 226 paper collection card. Cross contamination between samples was avoided using 10mm diameter perforations to allow individual processing of samples. Extraction of RNA used a modified Qiagen extraction kit and the internally controlled (MS2) HCV RNA quantitative PCR had a limit of detection in DBS specimens of 8.46 IU/mL comparing well with the limit of detection of the reference assay (Cobas® AmpliPrep TaqMan® HCV Test, v2.0; Roche Diagnostics) in EDTA plasma which is 8.5 IU/mL when using a sample size of 500µL. Previously tested HCV RNA positive (n = 125) and HCV RNA and HCV antibody negative (n = 138) serum and plasma samples were used to prepare mock DBS samples by mixing with packed cells from an HCV RNA and HCV antibody negative donor blood. The HemaXis device was used to deliver 10µL samples for each spot. QPCR of mock DBS samples prepared from the 125 HCV RNA positive whole blood samples gave 104 positive HCV RNA results from the DBS. This gave a qualitative test sensitivity of 85.6% and specificity of 100%. A Bland-Altman plot of the differences between the two tests showed that on average the DBS system determines a log 3.37 (2,344 IU/mL) lower value than testing of plasma. The reduction was independent of viral load. Review of the results of internal control testing suggested many samples had evidence of inhibition suggestive of sample deterioration between the time of initial testing of whole blood specimens and the preparation of the mock DBS specimens. Contemporaneous testing of DBS and plasma would be needed to confirm this suggestion. The DBS prepared using the HemaXis were also evaluated for use in genotyping and sequencing of the NS5B and core regions of HCV. Determination of genotype with discrimination of subtypes and of mixed infections was shown to be possible from the DBS samples. The consistent (albeit lower) viral load determination made when using HemaXis prepared DBS shows that this technology has promise as a method for quantitation of HCV RNA load from DBS. Together with the proven ability to genotype HCV using the same sample, the utility of DBS in the diagnosis and management of HCV infection will be further extended.17 0Item Restricted PROTEOMICS ANALYSIS OF RNA BINDING PROTEINS IN PROSTATE CANCER CELLS(Clark Atlanta University, 2024-03-26) Dwaed, Abdulrahman; Cinar, BekirRNA-binding proteins (RBPs) govern various RNA-related functions, and dysregulation of RBPs is linked to numerous human diseases, including cancer. RBPs regulate the intricate facets of RNA biogenesis, impacting its metabolism and functional kinetics. This study investigates the biochemical and functional interactions between the RNA-binding protein NPM1 and the Hippo pathway effector of the transcriptional coregulatory protein YAP1. This study also examined the system-wide changes in RBP patterns in prostate cancer cell lines with differential responses to androgen hormone signaling. This study revealed that androgen hormone signaling regulates the protein-protein interaction of YAP1 with NPM1. GST-pulldown assay combined with western blot showed that the proline-rich region and WW/SH3 domain of YAP1 mediated the interaction with NPM1. The proximity ligation assay combined with co-immunoprecipitation further verified the interaction between YAP1 and NPM1 in the androgen-dependent (AD) and androgen-independent (AI) cell lines. Disruption of NPM1 activity by genetic and pharmacological approaches reduced cell growth and cell migration in cultures, likely mediated by the inhibition of YAP1. Moreover, treatment of AD and AI cells with or without androgen resulted in distinct RBP patterns, as demonstrated by enhanced RNA interactome capture assay combined with quantitative mass spectroscopy-based proteomics. Furthermore, the analysis of prostate cancer tissues showed an elevated NPM1 expression that correlated with prostate tumor progression. These findings suggest that altered RBP patterns downstream of modified androgen hormone signaling contribute to tumor progression, and the YAP1-NPM1 axis may constitute a viable drug target in cancer.17 0Item Restricted Investigating the function of RBMXL2 in spermatogenesis in humans and using mouse models(Saudi Digital Library, 2023-11-20) Aldalaqan, Saad; Elliott, DavidRBMX RBMXL2 and RBMY belong to a family of RNA-binding proteins. How important these proteins are in whole animal biology, what their global RNA targets and mechanisms of action are, and to what extent these proteins overlap in function has been poorly understood. Using histology I found that Rbmxl2 knockout phenotypes differed between mouse strains (C57BL/6 and Sv/129). Both strains, however, were infertile, with no sperm found, which indicates the importance of RBMXL2 in spermatogenesis. I performed an iCLIP analysis to investigate RBMXL2-RNA binding, which revealed that RBMXL2 directly binds to exons and introns of protein-coding genes. The strongest RBMXL2 binding was observed in genes associated with spermatogenesis and reproduction, specifically meiosis, such as Meioc and Esco1. Binding was also enriched within ultra-long exons to prevent the selection of cryptic splice sites. I attempted minigene experiments to try and model RBMXL2 function in transfected cells, but these experiments did not replicate RBMXL2 function in vivo. As an alternative I developed a stable cell line “rescue” approach to over-express RBMXL2 and test if this could replace endogenous RBMX. Using this approach, I demonstrated that RBMXL2 is likely to replace RBMX's function in meiosis when the X and Y are inactive. RBMXL2's ability to restore splicing control is dependent on its disordered domain not the RRM. RBMY, which is distantly related to RBMX, also replaces RBMX function in somatic cells.18 0