Saudi Cultural Missions Theses & Dissertations
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Item Restricted What Does MicroRNA Expression Say about Human Preimplantation Blastocysts: A Descriptive Analytical Study(University College London, 2024) Almutlaq, Arwa; SenGupta, SiobanEmbryo quality is critical in in vitro fertilization treatment, significantly influencing the pregnancy success. While preimplantation genetic testing offers a reliable assessment of embryonic chromosomal status, the investigations of the embryo’s molecular characteristics remain less implemented. MiRNAs, known for their post- transcription regulatory functions, have emerged as promising markers for genetic disruptions. These small non-coding RNAs found both inside and outside cells and typically exhibit altered profiles in disorders with genetic abnormalities. In this study, we utilised next-generation sequencing to explore the miRNA expression profile in 122 cryopreserved human blastocysts collected from CRGH, London. The comprehensive miRNA profiling revealed abundant and stable miRNAs expression in blastocysts, with a substantial increase in the levels of miRNAs encoded in key miRNA clusters, such as C19CM and miR-17/92. Functional analysis linked these miRNAs to crucial biological pathways, including protein modification, cell cycle progression, response to low oxygen levels, and apoptosis. A series of differential miRNAs expression analyses were conducted to identify potential associations between miRNA expression and embryo competence. The findings revealed consistent and significant dysregulation in the miRNA profile in blastocysts with various types of aneuploidies compared to euploid ones. Additionally, differences in miRNA levels were observed among blastocysts at different blastulation days (day5 versus day 6) and between those with varying TE morphology grades. The miRNA expression profile was also assessed in relation to parental factors known to influence implantation potential and pregnancy outcomes. The results indicated that advanced reproductive age, both maternal and paternal, high ovarian stimulation dosage and impaired sperm parameters are potentially associated with altered miRNA expression in the examined blastocysts. Notably, one miRNA, hsa- miR-184, was consistently upregulated across these investigations. The dysregulated miRNAs in these analyses were commonly involved in cell cycle dynamics, metabolic processes and signalling pathways. Understanding the molecular differences between good- and poor-quality embryos through miRNA expression could enhance our knowledge of the underlying causes of poor embryonic development and outcomes. Hypothetically, these miRNAs hold promise as biomarkers for evaluating the quality of preimplantation blastocysts, contributing to advancements in reproductive treatment.8 0Item Restricted Regulation of kinetochore function by post-translational modifications in Saccharomyces cerevisiae(The Cathlic University of America, 2024-05-14) Aljasser, Ali A.; Choy, John S.Aneuploidy, characterized by cells with an incorrect number of chromosomes, is linked to genetic disorders such as cancer, Down’s syndrome, and miscarriage. In cancer, studies show that aneuploidy is associated with cancer aggressiveness and chemotherapy resistance. Aneuploidy in yeast is common and can be beneficial. Yeast rely on genetic heterogeneity to survive in fluctuating environments and toxic stressors. It remains unclear what drives chromosome segregation errors that lead to aneuploidy. The fidelity of chromosome segregation relies on proper kinetochore (KT)- microtubule (MT) attachments, and errors in attachments lead to aneuploidy. The Dam1 protein is a component of the outer kinetochore Dam1 complex (Dam1C), and has a critical function in chromosome segregation by maintaining stable binding to microtubules. It is assembled along with nine other subunits, including Duo1, to form Dam1C. Oligomerization of multiple Dam1Cs around MTs result in stable KT-MT attachments. Dam1 is also a major target that is regulated to correct errors in KT-MT attachments, that left uncorrected could result in chromosome missegregation. Phosphorylation of Dam1 serines by the Aurora Kinase B (Ipl1), promotes KT-MT dissociation. The phosphorylation of Dam1 has been postulated to act by decreasing electrostatic interactions between KT and MT. It remains unclear if these phosphorylation events might modulate another aspect of KT-MT binding. The glucose signaling kinase Protein Kinase A also functions in chromosome segregation by phosphorylating Dam1 S31, yet it is not fully understood how this modification impacts the chromosome segregation process and the implications of being linked to carbon utilization or sensing. Additionally, it remains unclear how much phosphorylation is compatible for optimal (normal) chromosome segregation fidelity. Here, we report that serine phosphorylation reduces Dam1 interaction with Duo1. Dam1 S20 and 31 phosphorylation by PKA and Ipi1 have a limited effect on binding to Duo1, but the C-terminal residues S257, 265, and 292 have a significant impact on Duo1 binding. Modeling and genetic assay, demonstrate that a short alpha helix in Dam1 (from S257 to N278) regulates the interaction with Duo1. We also demonstrate that yeast grown on galactose, glycerol, and maltose all display increased chromosome loss rates. Alterations in chromosome segregation fidelity by growing cells on suboptimal carbon sources were associated with greater survival on antifungals. Fluctuation assays show that 50% or more, but less than 100%, of Dam1 S31 phosphorylation, is associated with optimal chromosome segregation. On the other hand, less than 50% or 100% Dam1 S31 phosphorylation is associated with higher chromosome segregation loss rates. Overall, the results highlight how phosphorylation on the C-terminal tail of Dam1 impacts the formation of the alpha helical structure, which influences the interactions between Dam1- Duo1 and is crucial for binding of the Dam1C to MTs. This brings new insight into how phosphorylation functions to modulate parts of the Dam1C structure. This suggests the possibility that phosphorylation may not only reduce electrostatic interactions between KTs and MTs but also modulate Dam1 binding to Duo1 and together alter KT-MT stability. Finally, we provide evidence that acetylation of Dam1 contributes to regulation of outer kinetochore function raising the possibility that this is another pathway to connect kinetochore activity to glucose availability and chromosome segregation fidelity.17 0