Theses and Dissertations from UMD
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Item AN INTERSECTING NETWORK OF REGULATORS IS REQUIRED FOR RNA SILENCING AND NUCLEAR INTEGRITY IN C. ELEGANS(2024) Knudsen-Palmer, Daphne R; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Regulation of gene expression is required for an organism to develop, maintain homeostasis, and respond to environmental stimuli. While each cell in a multicellular organism contains the same genetic information, the epigenetic control of the expression of genes at different times is crucial for processes such as cell differentiation, division, and for allowing for cells to carry out different functions from one another. One type of such epigenetic regulation is mediated by small non-coding RNAs. Introduction of double-stranded RNA (dsRNA) and subsequent production of small interfering RNAs can result in sequence-specific mRNA silencing, creating the potential for highly specific therapeutics and pesticides. However, some targets are more easily silenced than others, and the mechanisms of silencing are not fully understood. Here we investigate regulators of small RNA-mediated silencing in the nematode C. elegans and find that they function in an intersecting network, allowing the potential for regulators to contribute to the silencing of any target. Quantitative modeling suggests that the production and turnover rates of a target at steady-state can affect the ease with which a target can be knocked down, and experimentally we found that changing the cis-regulatory sequences of a target can make it more susceptible to silencing. We found restricted production of RNA silencing intermediates, allowing for the recovery of a target in response to dsRNA, which we observed experimentally in non-dividing cells. In addition to roles in response to dsRNA, we report that disruption of small RNA-based regulation can result in germline nuclear defects. In the absence of the intrinsically disordered and perinuclear granule-forming protein MUT-16, some of the nuclei in the syncytial germline appear enlarged, suggesting that small RNA-based regulation may be playing an active role in maintaining nuclear size. Taken together, these findings suggest that (1) regulators of small RNA silencing can contribute to the silencing of all targets as part of an intersecting network, as opposed to operating in specialized pathways and (2) small RNA-based regulation is required for nuclear integrity, providing a paradigm for studying control of nuclear size, where enlarged nuclei can be compared with wildtype nuclei in a shared syncytium. We speculate that these findings will improve understanding of RNA silencing across species and provide insight into understanding how nuclear size is controlled, a fundamental ability of all eukaryotes.Item Mobile RNA reveal differential requirements for gene silencing in C. elegans at single-cell resolution(2018) Ravikumar, Snusha; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the worm C. elegans, such organism-wide silencing relies on the transport of dsRNA to most cells and requires amplification mechanisms for effective gene silencing. Amplification of silencing signals is accomplished by two tissue-specific RNA-dependent RNA polymerases - EGO-1 in the germline and RRF-1 in somatic cells. Here, we reveal instances of RRF-1-independent silencing in somatic cells, which are dictated by three variables. First, when the same intestinal target gene was silenced using ingested, intestinal, or neuronal dsRNA, only silencing by mobile RNAs derived from neuronal dsRNA was independent of RRF-1. Second, when the same source of mobile RNA was used to silence a target sequence in different genomic contexts, the requirement for RRF-1 could change. Third, measurement of silencing by mobile RNAs at single-cell resolution revealed cell-to-cell and animal-to-animal variation in the requirement for RRF-1. Therefore, the requirements for gene silencing can vary based on the source of dsRNA, the target context, and even the particular cell examined, suggesting that each C. elegans animal is a functional mosaic with respect to RNA interferenceItem HEMIPTERAN INSECTS AS MODELS FOR UNDERSTANDING SEGMENTATION(2018) Chen, Mengyao; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although segmentation is highly conserved in arthropods, diverse mechanisms underlie segmentation. Pair-rule genes (PRGs) are a group of genes controlling segmentation in Drosophila melanogaster, a holometabolous insect. While Drosophila are long-germ insects, most insects add segments sequentially. Studying the role of PRGs in sequentially-segmenting species will provide a deeper understanding in terms of developmental biology. Here, I studied two such insects: Halyomorpha halys and Oncopeltus fasciatus, hemimetabolous insects in a sister order to Holometabola. I annotated segmentation genes in the Halyomorpha genome and tested its response to RNA interference which I showed to be effective in this species for the first time. I further showed that three orthologs of Drosophila PRGs are present in the Oncopeltus genome and are expressed during stages at which segments are specified. Surprisingly, only one of these orthologs is expressed in a PR-pattern, indicating that PRG expression and function have changed during insect evolution.Item RNA Interference Mediated Suppression of Tn-Caspase-1 as a means of investigating apoptosis and improving recombinant protein production in Trichoplusia ni cells(2008-11-17) Hebert, Colin G; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The baculovirus expression system has proven to be a robust and versatile system for recombinant protein production in insect cells. A wide range of promoters is available for the facile expression of transgenes, and yields of up to 50% of total protein have been reported. However, in many cases production is decreased as a result of proteases and host cell apoptosis. To combat this problem, RNA interference (RNAi) has been used as a metabolic engineering tool to knockdown host genes responsible for decreasing the yield of recombinant protein. A novel caspase (Tn caspase-1) derived from Trichoplusia ni cells has been identified and characterized. Through modulation of caspase levels via either RNAi or through interaction with baculovirus protein p35, the overall level of apoptosis present in cell culture has been decreased. In addition, the use of in vitro RNAi targeted against Tn caspase-1 has increased the production of recombinant green fluorescent protein. To further study the effect of suppressing Tn caspase-1, a stable cell line producing in vivo RNAi was developed, resulting in a nearly 90% decrease in caspase enzymatic activity. This suppression was able to improve culture viability under adverse conditions and increase recombinant protein production levels up to two-fold that of standard cells.Item RNA packaging and gene delivery using Tobacco mosaic virus pseudo virions(2008-04-28) Hung, Chi-Wei; Bentley, William; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA interference (RNAi) has emerged as a powerful tool for the study of gene function and post-transcriptional regulation. However, the lack of a proper delivery system for RNAi is a major problem for its application as a therapeutic agent. In this study, Tobacco mosaic virus (TMV) is utilized as an RNAi carrier for gene delivery into mammalian and insect cells. The self assembly and disassembly of TMV is investigated to create chimeric viruses for gene delivery. The origin of assembly sequence (OAS) within the TMV RNA initiates its association with coat protein through a unique hairpin structure. Studies in this dissertation show that by incorporating TMV OAS into an RNA of interest, the RNA can assemble into "pseudo-virions" by the virus coat protein. The length of the pseudo-virions changed in proportion with the size of the RNA. To deliver the RNA to the targeted cells, virions are further surface-modified with synthetic cell-penetrating peptides to facilitate cell endocytosis. Two genes were selected as targets: 1) EGFP as a visual marker and 2) Cyclin E for control of cell cycle. EGFP is expressed in a transient expression experiment using a plasmid vector, pEFGP-N1. Cyclin E is regulated endogenously in High FiveTM cells, and its translation is targeted using the pseudo virions. Pseudo-virions targeting EGFP RNA (antisense EGFP) are able to suppress transient EGFP production by 61% whereas pseudo virions targeting cyclin E (antisense cycE) are capable of arresting cells at G1 phase. This RNA packaging system protects packaged RNA and provides a means of delivering RNAi constructs into various host cells.