Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item Cell Population Shifts and Clinical Heterogeneity in Sjögren's Disease(2024) Pranzatelli, Thomas J; Johnson, Philip L.F.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sjögren's disease (SjD) is a systemic autoimmune disease that causes loss of function of the salivary and lacrimal glands. Those with the disease, overwhelmingly female with an onset of disease in the fourth or fifth decade of life, commonly suffer from dry mouth, cavities and damage to the eyes. Patients present with a wide variety of clinical phenotypes, with variation in degree of immune infiltration and glandular damage as well as positivity for autoantibodies. This thesis uncovers the changes in cell population and gene expression in the gland that underpin diversity in disease severity. SjD patients lose the majority of a specific epithelial population in their labial salivary glands and, as the number of immune infiltrates grows the surviving members of this population can be found colocalizing with invading GZMK+ T cells and expressing markers of increased proliferation. Standard differential gene expression analysis highlighted gene markers of cell types changing in proportion with disease; an unenlightening result when the cell population changes are well-characterized. To avoid this pitfall an ensemble of random forests was trained to find genes predictive of patient subtypes without being correlated with diagnosis. Genes with high importance for autoantibody positivity were enriched for GO terms related to antigen processing and presentation. A master regulator of salivary gland identity, ZBTB7B, was identified from chromatin accessibility data. Mice with this transcription factor knocked out lose salivary flow and develop pockets of tissue in their glands that resemble other glands, eg., labial gland epithelium inside of parotid glands. This work supports a clinical presentation-specific approach to therapy and paves the path for reengineering the glands to correct the effects of disease.Item STUDIES ON VARIABILITY IN CANCER GENE EXPRESSION: FROM SINGLE PROTEINS TO POPULATIONS(2023) Crawford, David Robert; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this dissertation I describe four projects investigating different aspects of the variability of gene expression in human cancers. In the first chapter, we analyze epidemiological incidence rates for autoimmune diseases and cancers across numerous populations and find that sex biases in incidence rates are positively correlated between autoimmune diseases and cancers arising from the same tissue. We find that across these tissues the expression of protein-codingmitochondrial genes is positively correlated with both autoimmune disease and cancer incidence rate sex biases, suggesting a possible direction for further investigation. In the second chapter, I construct a computational pipeline to conduct unbiased searches in large databases for possible events accounting for cancer neopeptides predicted by mass spectrometry. I identify several ribosomal frameshift-derived neopeptides from HLA-peptidomics data and discuss future approaches for further improving the accuracy and flexibility of our approach. In the third chapter, I compare the power of different multivariate Cox proportional hazards survival models based on gene- and below-gene-level expression measures to predict genes whose expression in tumor samples at diagnosis affects subsequent survival of cancer patients. I find that models based on both gene-level expression and isoform-level expression (whether transcript abundance or relative transcript abundance) identify the greatest number of statistically significant genes of interest. Finally, in the fourth chapter I briefly explore how heteroformity and entropy measures can be used to examine differences in mRNA splicing diversity at numerous levels of comparison. I propose some simple visualizations that harness these measures to display patterns in splicing diversity.Item Investigation of progerin expression in non-Hutchinson-Gilford Progeria Syndrome individuals(2023) Yu, Reynold; Cao, Kan; Mount, Steve; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hutchinson-Gilford Progerin Syndrome (HGPS) is a premature aging disease caused by a point mutation in the LMNA gene, which encodes A-type lamins. This mutation activates a cryptic splice donor in exon 11 and leads to the production of a toxic lamin variant called progerin. Interestingly, small amounts of progerin have also been found in cells and tissues of normal individuals. Here we examine the expression of progerin in publicly available RNA-seq data from normal individuals of the GTEx project. Among the 30 available tissues, progerin expression in normal individuals is highest in sun-exposed skin samples, and its expression in different tissues of the same donor is correlated. In addition, telomere shortening is significantly correlated with progerin expression. Transcriptome-wide correlation analyses suggest that the level of progerin expression is highly correlated with switches in gene isoform expression patterns, perhaps reflecting widespread isoform shifts in these samples. Differential expression analyses show that progerin expression is correlated with significant changes in the level of transcripts from genes involved in splicing regulation and a significant reduction of mitochondrial transcripts. Interestingly, 5’ splice sites whose use is correlated (either positively or negatively) with progerin expression have significantly altered frequencies of consensus trinucleotides within the core 5’ splice site. Furthermore, introns whose alternative splicing is correlated with progerin have reduced GC content. Together, our study suggests that progerin expression in normal individuals is part of a global shift in splicing patterns and provides insight into the mechanism behind these changes.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 IDENTIFICATION AND ANALYSIS OF GENETIC VARIANTS PERMITTING DISSEMINATED COCCIDIOIDOMYCOSIS(2022) Hsu, Amy Pepper; Mosser, David M; Holland, Steven M; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the Southwestern United States and Mexico. While the majority of those infected have minor symptoms or remain asymptomatic, illness requiring medical attention occurs in approximately 30%, with <1% developing extrapulmonary dissemination. To address why some individuals allow dissemination, we performed whole-exome sequencing on an exploratory cohort of 67 DCM patients. Using standard genetic analysis for identification of novel or rare Mendelian mutations only two patients were identified, both with STAT3 premature termination codons causing haploinsufficiency. Since Coccidioides are geographically isolated, I explored the possibility that dissemination could be a combination of more common genetic variants plus exposure. Defects in sensing and response to -glucan, the major component of Coccidioides cell wall, were seen in 34/67 (50.7%) cases. Damaging variants in CLEC7A, encoding DECTIN-1, (n=14) and PLCG2 (n=11) were associated with impaired production of -glucan-stimulated TNF from peripheral blood mononuclear cells compared to healthy controls (P<0.005). Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were over-represented in DCM (P=0.0206, P=0.015, respectively) including CLEC7A Y238* (P=0.0105) and PLCG2 R268W (P=0.0025). A validation cohort of 111 DCM patients confirmed over-representation of the specific variants, PLCG2 R268W (P=0.0276), CLEC7A I223S (P=0.044), and CLEC7A Y238* (P=0.0656). Lastly, I identified a novel pathway of pulmonary-epithelial fungal recognition by DECTIN-1 leading to activation of the NADPH oxidase complex, DUOX1/DUOXA1. Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived H2O2 in transfected cells. Heterozygous DUOX1 or DUOXA1 variants which impaired H2O2 production were overrepresented in discovery and validation cohorts. Together these studies highlight the importance of fungal recognition and response for control of infections. Patients with DCM have impaired -glucan sensing or response affecting TNF and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.Item MECHANISM OF DREAM COMPONENT TSO1 IN PLANT STEM CELL REGULATION(2022) Wang, Fuxi; Liu, Zhongchi ZL; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plants are important for human survival and the environment. They provide oxygen, food, medicine and fuel. Understanding the development of plants has been a fundamental research question. Among all the plant tissues, the most important ones are the meristems. Sitting at the tip of the shoot and the root are the shoot apical meristem (SAM) and the root apical meristem (RAM). The shoot apical meristem gives rise to the above-ground organs like leaves and flowers while the root apical meristem produces all the root tissues that help to anchor the plants and transport water and nutrients. As the meristem is capable of producing new organs throughout the lifespan of a plant, the study of meristem maintenance and development provides the key to the understanding of plant development.Arabidopsis transcription factor TSO1 plays an essential role for the proper development of shoot apical meristem and root apical meristem. TSO1 encodes a protein with a cysteine-rich repeats domain and TSO1 is a potential component of a cell cycle regulating complex, the DREAM complex. The tso1-1 mutant has fasciated SAM due to shoot meristem cell over-proliferation and complete sterility due to lack of differentiated female and male floral organs. Interestingly, the tso1-1 mutant also produces shorter root than the wild type, presumably caused by early differentiation of the cells in the RAM. A prior mutagenesis screen identified two major suppressors of tso1-1. Characterization of these tso1-1 suppressor mutations provides important insights to the understanding of TSO1-regulatory pathways. My dissertation project focuses on analyzing one of these suppressors that was shown to be a mutated type-A cyclin gene named CYCA3;4. Mutations in CYCA3;4 suppress the shoot phenotype but not the root phenotype of tso1-1. The suppressed plants can produce normal floral organs and become partially fertile. Using transgenic method, I showed that the expression of CYCA3;4 was increased in the tso1-1 SAM, and overexpression of CYCA3;4 in the tso1-3 mutant enhanced the fertility defect, suggesting that overexpression of CYCA3;4 partially mediates the tso1-1 shoot phenotype. In addition, I provided evidence supporting that TSO1 likely represses CYCA3;4 gene expression indirectly through MYB3R1, whose mutations also suppress tso1-1 mutants. My dissertation provides an important link between TSO1, a potential cell cycle regulatory complex component and meristem regulator, and cyclin A, a protein directly involved in cell cycle regulation. This link provides an important mechanistic insight into how plant meristems maintain their identity by limiting their cell division activity. To further investigate the mechanism of TSO1 action in the root, I collaborated with two other scientists to profile the gene expression in the tso1-1 root at single cell level. I compared the single cell RNA sequencing data of tso1-1 and wild type roots and identified molecular defects in the tso1-1 root vasculature. Correspondingly, the known regulators of vasculature development, the HD-ZIP III genes, are ectopically expressed in some of the vascular cells in the tso1-1 root. It suggests that the defects of root vasculature may be attributed to mis-expressed HD-ZIP III genes in the tso1-1 mutant. The HD-ZIPIII function was previously linked to their regulation of cytokinin biosynthesis genes, which were ectopically expressed in tso1-1 roots as revealed by our scRNA-seq data. Together, our data suggest that the over-production of cytokinin might be the cause of tso1-1 short root phenotype. In summary, my dissertation research revealed previously unknown links between TSO1 and cell cycle regulation in the shoot and root meristems as well as the molecular mechanisms of TSO1 function in the root vascular development at single cell level. These findings have furthered our understanding of how cell cycle regulation is integrated with plant development.Item Intercellular transport of RNA can limit heritable epigenetic changes(2021) Shugarts, Nathan Maxwell; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNAs in circulation carry sequence-specific regulatory information between cells in animal, plant, and host-pathogen systems. The accumulation of specific RNA species in circulation during human disease states therefore implicates such RNAs in disease-related gene regulation. However, mechanisms of RNA secretion, accumulation and import into cells are not well understood and yet are directly taken advantage of in the delivery of recently approved RNA-based therapeutics. In the tractable animal Caenorhabditis elegans, double-stranded RNA (dsRNA) can be delivered into circulation, accumulate within the germline and reach progeny, raising the potential for intergenerational effects from endogenous RNAs released into parental circulation. Here we provide evidence for spatial, temporal, and substrate specificity in the transport of dsRNA in C. elegans from parental circulation to progeny. Temporary loss of dsRNA transport resulted in the persistent accumulation of mRNA from a germline gene. The expression of this gene varied among siblings and even between gonad arms within one animal. Perturbing RNA regulation of the gene created new epigenetic states that lasted for many generations. Thus, one role for the transport of dsRNA into the C. elegans germline in every generation is to limit heritable changes in gene expression. We speculate that transport of extracellular RNA into germ cells in other systems could similarly buffer against heritable change across generations.Item Chromatin Control of Papillomavirus Infection(2020) Porter, Samuel Stephen; McBride, Alison A; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genomes of papillomaviruses are packaged into chromatin throughout the entire viral lifecycle. A peculiar feature of papillomaviruses genome organization is that the viral DNA is associated with host histones even inside the virion particle. However, little is known about the nature of the epigenome within papillomavirions, or its biological impact on early infection. Here, we use three approaches to study the epigenome of papillomavirions. Papillomaviruses can be assembled in packaging cells by expression of the capsid proteins in the presence of the viral genome. We have optimized and manipulated this process to generate viruses with replicated and genetically modified virion DNA and have used these “quasivirions” to evaluate early infection of primary human keratinocytes. We have also profiled the histone modifications on chromatin extracted from native virions isolated from human and bovine warts. We find that, compared to host cells, the viral chromatin is enriched in histone modifications associated with transcriptionally active chromatin (including histone acetylation), and depleted in those associated with transcriptional repression. To examine the biological role of histone acetylation in the early virus lifecycle, we produced HPV quasivirions with highly acetylated chromatin by assembling the virions in cells treated with histone deacetylase inhibitors. We show that acetylation of viral chromatin results in a reduction of early viral transcription in primary keratinocytes indicating that the histone modifications on virion chromatin do influence the early stages of infection. Collectively, these studies demonstrate that histone modifications on virion chromatin are important for the HPV infectious cycle.Item SYSTEMIC AND TRANSGENERATIONAL REGULATION OF GENE EXPRESSION BY SMALL RNAS IN C. ELEGANS(2019) Raman, Pravrutha; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Development of an organism requires information contained minimally within a single cell. This information is inherited in two forms- the genome sequence and regulatory molecules. Little is understood about the types of the regulatory molecules inherited or the impact of parental experiences on them. However, environmental stimuli can alter gene expression without changing DNA sequence and these changes can be inherited suggesting heritable regulatory molecules are influenced by parental experience. Such changes could require communication of regulatory information between cells within an animal (systemic regulation) and across generations via germ cells (transgenerational regulation). Double- stranded RNA (dsRNA) introduced to an animal can silence a gene of matching sequence within that animal and this silencing can persist in progeny suggesting that RNA has the potential to transfer gene-specific regulatory information. Using RNA silencing in C. elegans, we identify conditions that facilitate systemic and transgenerational regulation of gene expression. Previous work suggested that two forms of dsRNA, short and long, could move between somatic cells to cause systemic silencing. However, we show that the movement of short dsRNA is not an obligatory feature of systemic silencing and that long dsRNA introduced by feeding likely enters every cell to cause silencing. Silencing by dsRNA can also be communicated to the germ cells, however this does not guarantee persistence of silencing in descendants. Even the same target sequence expressed from different genetic contexts shows varying susceptibility to transgenerational silencing. Most tested genes recover from silencing in a few generations suggestive of mechanisms that repair changes induced in ancestors. We characterize a unique gene that is exceptionally susceptible to transgenerational silencing that lasts for >200 generations and find that non-genomic signals mediate its expression pattern in every generation. A forward genetic screen to isolate mutants exhibiting re-activation of gene expression (Rage) after many generations of silencing revealed additional defects indicative of endogenous processes that utilize transgenerational silencing mechanisms. We speculate that homeostatic mechanisms that prevent or preserve induced changes maintain form and function across generations in living systems.Item THE IMPACT OF TRANSLATIONAL FIDELITY ON HUMAN HEALTH(2018) Marques dos Santos Vieira, Carolina; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ribosomopathies are a class of diseases resulting from mutations in genes encoding ribosomal proteins and ribosome biogenesis factors. Pleiotropic clinical presentations of different ribosomopathies has been taken as evidence of specialized ribosomes. Alternatively, gene dosage effects have been proposed to account for the observed differences. A yeast genetics approach was used to address this issue. Due to a historical gene duplication event, S. cerevisiae cells harbor two ohnologs for most ribosomal proteins. Deletion of one yeast ribosomal protein ohnolog was used to mimic haploinsufficiency in diploid cells (i.e. pseudo-haploinsufficient yeast). Further, insertion of a second copy of the undeleted ohnolog into the locus of the deleted ohnolog enabled separation of effects due to gene dosage from those due to ribosomal protein ohnolog identity. We found that significant changes in translational fidelity in the ribosomal protein ohnolog deletion strains were corrected by ohnolog duplication. Changes in gene dosage, particularly as they may affect the abundance of an enzyme as central as the ribosome, can impart stress through far reaching effects on cellular metabolism. Thus, as an orthogonal approach, we also examined the stress profiles of cells harboring the cbf5-D95A allele (model of X-linked Dyskeratosis Congenita) and the rps23a-R69K allele (model of MacInnes Syndrome). RNA-seq analysis revealed increased expression of proteins involved in response to oxidative stress in cbf5-D95A cells. Growth curve analysis revealed a longer plateau of the cbf5-D95A cells upon reaching stationary phase, suggestive of a pre-adaptive stress response. Decreased ROS abundance, tunicamycin resistance and increased basal levels of HAC1 mRNA in the mutant cells support this hypothesis. Similar results were observed with regard to the ohnolog deletion strains. Taken as a whole, these data support the gene dosage model as opposed to the specialized ribosome hypothesis with the caveat that this conclusion is limited to yeast cells growing in rich medium.