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 Genome-wide identification and analysis of imprinted genes in strawberry seed development(2022) Joldersma, Dirk; Liu, Zhongchi Anne; Taneyhill, Lisa Anne; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The activation of zygotic gene expression is of fundamental importance to reproductive biology, but its regulation remains poorly understood. Within Angiosperm plants, fertilization occurs simultaneously in two locations, the embryo and its genetic twin, the endosperm, a nutritive tissue that is a defining feature of Angiosperm reproduction. Auxin hormone synthesized in the endosperm is essential to seed and fruit development. In the diploid strawberry Fragaria vesca, that auxin synthesis is regulated by FveAGL62, which is expressed specifically after fertilization in endosperm. How fertilization activates FveAGL62 expression in the endosperm, however, is presently unknown. I investigated the hypothesis that epigenetically regulated maternally- and paternally expressed genes (MEGs and PEGs, and together, “imprinted genes”) regulate the expression of FveAGL62. I hybridized two F. vesca accessions, isolated the endosperm from the F1 seeds, and sequenced the transcriptome of the F1 endosperm—a result facilitated by strawberry’s uniquely accessible seed. To identify imprinted genes within the endosperm, I assembled and annotated the genome of the maternal parent, F. vesca accession “Yellow Wonder” (FvYW5AF7), a model for the commercial strawberry. The paternal parent genome was obtained from a collaborator. 809 PEGs and 825 MEGs were identified from RNA sequencing reads that align uniquely to the maternal or paternal genome. MEGs are enriched in genes catabolizing auxin and hence limit seed growth, while PEGs are enriched in genes involved in histone modification, thereby promoting cell differentiation and seed growth. The distinct roles of MEGs and PEGs supports and can be explained by parental conflict and kinship theories, which predict a maternal genome tends to restrict progeny consumption of maternal resources, while a paternal genome will encourage such consumption. In contrast to findings in other species, I find that the endosperm-specific auxin biosynthetic gene FveYUC10 is maternally expressed, but while its imprinting status has changed, it may still function as a fertilization sensor. The maternally expressed gene FveMYB98 contains a binding domain that targets motifs present in FveAGL62’s promoter and its homolog binds AtAGL62 promoter in Arabidopsis. With collaborators, I showed that overexpression and CRISPR knockout of FveMYB98 changes seed size. Transient expression, yeast one hybrid and quantitative PCR analyses suggest that FveMYB98 represses FveYUC10 expression directly and FveAGL62 expression indirectly. These results suggest that FveMYB98 expression is a vehicle for maternal regulation of the level of auxin in the endosperm and thereby endosperm proliferation and seed size. My dissertation research has produced a new genome assembly of a model strawberry, a transcriptome of strawberry endosperm, and identified imprinted genes at genomic scale. I find FveMYB98 regulates seed size—a function echoed broadly within MEG and PEG classes—providing supporting evidence for the parental conflict theory within the developing progeny. These results improve our understanding of zygotic expression in developing seeds, addressing a fundamental scientific gap and, more tangibly, may enable future production of fertilization-independent seeds and seedless fruits.Item LARGE SYSTEMS OF MANY INTERCONNECTED DYNAMICAL UNITS: GENE NETWORK INFERENCE, EPIGENETIC HERITABILITY, AND EMERGENT BEHAVIOR IN OSCILLATOR SYSTEMS(2014) Ku, Wai Lim; Ott, Edward; Girvan, Michelle; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this thesis, which consists of three parts, we investigate problems related to systems biology and collective behavior in complex systems. The first part studies genetic networks that are inferred using gene expression data. Here we use established transcriptional regulatory interactions (TRIs) in combination with microarray expression data from both Escherichia coli (a prokaryote) and Saccharomyces cerevisiae (a eukaryote) to assess the accuracy of predictions of coregulated gene pairs and TRIs from observations of coexpressed gene pairs. We find that highly coexpressed gene pairs are more likely to be coregulated than to share a TRI for Saccharomyces cerevisiae, while the incidence of TRIs in highly coexpressed gene pairs is higher for Escherichia coli. The data processing inequality (DPI) of information theory has previously been applied for the inference of TRIs. We consider the case where a transcription factor gene is known to regulate two genes (one of which is a transcription factor gene) that are known not to regulate one another. According to the DPI if certain conditions hold, the non-interacting gene pairs should have the smallest mutual information among all pairs in the triplets. While we observe that this is sometimes the case for Escherichia coli, we find that it is almost always not the case for Saccharomyces cerevisiae, thus indicating that the assumed conditions under which the DPI was derived do not hold. The second part of this dissertation is related to the dynamical process of epigentic heritability. Epigenetic modifications to histones may promote either activation or repression of the transcription of nearby genes. Recent experimental studies show that the promoters of many lineage-control genes in stem cells have "bivalent domains" in which the nucleosomes contain both active (H3K4me3) and repressive (H3K27me3) marks. Here we formulate a mathematical model to investigate the dynamic properties of bivalent histone modification patterns, and we predict some interesting and potentially experimental observable features. The third part of this dissertation studies dynamical systems in which a large number $N$ of identical Landau-Stuart oscillators are globally coupled via a mean-field. Previously, it has been observed that this type of system can exhibit a variety of different dynamical behaviors including clumped states (in which each oscillator is in one of a small number of groups for which all oscillators in each group have the same state which is different from group to group), as well as extensive chaos (a situation in which all oscillators have different states and the macroscopic dynamics of the mean field is chaotic). One of our foci is the transition between clumped states and extensive chaos as the system is subjected to slow adiabatic parameter change. We observe and analyze explosive discontinuous transitions between the clumped states and the extensively chaotic states. Also, we study the fractal structures of the extensively chaotic attractors.