Hawkins, R DavidOverbey, Eliah Goldsmith2021-07-072021-07-072021-07-072021Overbey_washington_0250E_22643.pdfhttp://hdl.handle.net/1773/47078Thesis (Ph.D.)--University of Washington, 2021Throughout the process of cellular differentiation, cell trajectories are shaped by different epigenomic and epitranscriptomic regulatory mechanisms. These trajectories eventually land cells into their terminal cell state, where they are no longer undergoing differentiation and have a stable gene expression profile. Throughout my thesis, I profiled gene expression of various cell states examining the impact of both natural variation between cell types and the effects of environmental perturbation. Specifically, I examined the natural variation found within chicken tissues and found a variety of alternatively spliced transcripts and differentially expressed genes between different cell and tissue types. To assess the effects of environmental perturbation, I examined the effects of a growing area of interest, the spaceflight environment, on mammalian cephalad cell types. From these experiments, I discovered that the combinatorial effect of spaceflight factors, specifically low-dose radiation and microgravity simulation, produce many more differentially expressed genes in murine brain tissue between the spaceflight simulated group and the control group than either low-dose radiation or microgravity simulation produced in isolation. The results of this experiment demonstrated that there is a compounding effect of the different environmental factors of spaceflight on mammalian brain tissue. The second experiment I analyzed on the spaceflight environment were the gene expression profiles of retinal tissue from mice flown aboard the International Space Station. Compared to the ground control, mice flown in spaceflight had hundreds of differentially expressed genes, a handful of which overlapped with genes associated with the cone-rod degenerative disease, retinitis pigmentosa. After examining differential gene expression, due to both natural variation and the environmental perturbations caused by spaceflight, I examine how gene expression is regulated post-transcriptionally. I did this by performing direct-RNA sequencing using the Oxford Nanopore MinION and employing base-modification prediction software in order to determine the global RNA modification landscape across three distinct states of pluripotent stem cells. Doing this, I uncover that pluripotency-relevant genes are enriched for transcript modifications. I then used the known motif DRACH to specifically locate m6A modifications. From this, I can identify uniquely modified genes in each pluripotent state. Collectively, these studies demonstrate the spectrum of transcriptional variation that can arise from natural and perturbed cell states, as well as the importance of incorporating mRNA modifications into analysis to understand the transitions and uniqueness between cellular states.application/pdfen-USCC BY-NC-NDMolecular biologyGeneticsThesis