Theses and Dissertations from UMD
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Item TRANSLATION, REPLICATION AND TRANSCRIPTOMICS OF THE SIMPLEST PLUS-STRAND RNA PLANT VIRUSES(2024) Johnson, Philip Zhao; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plus (+)-strand RNA viruses are among the most common pathogens of plants and animals. Furthermore, they present model systems for the study of basic biological processes, including protein translation and RNA replication, and shed light on the versatile roles that RNA structures play in these processes. After cell entry, the next step in the (+)-strand RNA viral life cycle is translation of the viral genome to produce the viral RNA-dependent RNA polymerase (RdRp) and associated replication proteins necessary for viral replication to occur. For many (+)-strand RNA viruses lacking a 5´cap and 3´ poly(A) tail, translation depends upon RNA structural elements within their genomes capable of hijacking the host translation machinery, which for plant viruses are commonly located in their 3´ proximal regions and are termed 3´ cap-independent translation enhancer (CITE) elements. In Chapter 2, I report upon my work characterizing a new subclass of panicum mosaic virus-like translation enhancer (PTE) elements, which bind and co-opt for viral use the host translation initiation factor 4E (eIF4E) – the translation initiation factor normally responsible for binding and recognition of mRNA 5´caps during canonical eukaryotic translation initiation. Thus, PTE 3´CITEs present a novel mechanism for co-opting the critical host factor eIF4E. My work characterizing a new subclass of PTE 3´CITEs further revealed characteristics common among all PTE 3´CITEs pertaining to their mechanism of binding eIF4E.After translation of the necessary viral replication proteins, replication of the viral RNA occurs, which again is in large part mediated by RNA structural elements within the viral genome that can bind to the viral RdRp and/or host factors involved in viral replication. Indeed, RNA structural elements often serve dual roles in viral translation and replication and/or are located proximal to RNA structural elements involved in the alternate function. In Chapter 3, I discuss my work characterizing novel replication elements in the 3´ terminal regions of umbraviruses (family Tombusviridae). The uncovered replication elements appear to be specific to umbraviruses and are located immediately upstream of replication/translation elements that are common throughout the Tombusviridae, lending greater complexity to the already complex 3´ proximal structures of umbraviruses. While the study of (+)-strand RNA viruses has historically focused on their protein-coding transcripts, (+)-strand RNA viruses also commonly produce additional non-coding transcripts, including recombinant defective RNAs, typically containing 5´ and 3´ co-terminal viral genome segments, and (+/-)-foldback RNAs, composed of complementary (+)- and (-)-strand viral sequences joined together. Long non-coding RNAs that accumulate to high levels have also been reported for plant and animal (+)-strand RNA viruses in recent years, and truncations of viral transcripts also commonly arise due to host nuclease activity and/or premature termination of replication elongation by the viral RdRp. The rise of long-read high-throughput sequencing technologies such as nanopore sequencing presents an opportunity to fully map the complexity of (+)-strand RNA viral transcriptomes. In Chapter 4, I present my work performing this analysis, employing direct RNA nanopore sequencing, in which the transcripts present in an RNA sample of interest are directly sequenced. This analysis revealed for the umbra-like virus citrus yellow vein-associated virus (CY1): (i) three novel 5´ co-terminal long non-coding RNAs; (ii) D-RNA population dynamics; (iii) a common 3´ terminal truncation of 61 nt among (+)-strand viral transcripts; (iv) missing 3´ terminal CCC-OH motif in virtually all (-)-strand reads; (v) major timepoint- and tissue-specific differences; and (vi) an abundance of (+/-)-foldback RNAs at later infection timepoints in leaf tissues. This work also sheds light on the current shortcomings of direct RNA nanopore sequencing as a technique. Finally, the importance of RNA structural biology in the study of (+)-strand RNA viruses presents the need for specialized RNA structure drawing software with functionality to easily control the layout of nucleobases, edit base-pairs, and annotate/color the nucleobases and bonds in a drawing. It is through the visual exploration of RNA structures that RNA biologists routinely improve upon the outputs of RNA structure prediction programs and perform crucial phylogenetic analyses among related RNA structures. Large RNA structures, such as whole viral genomes thousands of nucleotides long, can only be studied in their entirety with the aid of RNA structure visualization tools. To this end, I have developed over the course of my doctoral education the 2D RNA structure drawing application RNAcanvas, which is available as a web app and has grown popular among the RNA biology community. RNAcanvas emphasizes graphical mouse-based interaction with RNA structure drawings and has special functionality well suited for the drawing and exploration of large RNA structures, such as automatic layout adjustment and maintenance, complementary sequence highlighting, motif finding, and performance optimizations. Large viral structures such as that of the 2.7 kb CY1 genomic RNA could not have been characterized without the aid of RNAcanvas. In Chapter 5, I present my work developing RNAcanvas.Item EXPANDING THE TOOLKIT: STRUCTURE, DYNAMICS, AND DRUG INTERACTIONS OF THE “PRIMING LOOP” FROM HEPATITIS B VIRUS PRE-GENOMIC RNA BY SOLUTION NMR SPECTROSCOPY(2022) Olenginski, Lukasz Tyler; Dayie, Theodore K; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNAs are dynamic macromolecules that function as essential components of biological pathways that result in human disease, making them attractive therapeutic targets. Yet, RNA structural biology lags significantly behind that of proteins, limiting mechanistic understanding of RNA chemical biology. Fortunately, solution NMR spectroscopy can probe the structure, dynamics, and interactions of RNA in solution at atomic resolution, opening the door to their functional understanding. However, NMR analysis of RNA – with only four unique ribonucleotide building blocks – suffers from spectral crowding and broad linewidths, especially as RNAs grow in size. One effective strategy to overcome these challenges is to introduce NMR-active stable isotopes into RNA in an atom- and position-specific manner. Here, we outline the development of labeling technologies, their use in benefiting RNA dynamics measurements, and applications to study the structure, dynamics, and interactions of a conserved regulatory RNA stem-loop from hepatitis B virus that is critical for viral replication.Item Strategies for small RNA loading into extracellular vesicles(2022) Pottash, Alex; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Small RNAs are an exciting class of therapeutics with significant untapped therapeutic potential, due to their ability to affect cell behavior at the RNA level. However, delivery of RNA is a challenge due to its size and labile nature. Extracellular vesicles (EVs) are promising as delivery vehicles due to their natural role as physiological intercellular microRNA transporters, and research has shown that EVs have significant advantages compared to competing technologies such as lipid nanoparticles. Specifically, EVs more readily transport through biological barriers, deliver RNA more efficiently, and are less immunogenic. However, intrinsic microRNA content in EVs is low and thus active small RNA loading strategies are needed to enable therapeutic use. Consequently, a variety of small RNA loading methods for EVs have been developed. These include endogenous and exogenous approaches. Exogenous approaches, in which EVs are loaded directly, have been shown to enable loading of hundreds to thousands of small RNAs per EV, but they are not readily amenable to scalable production processes. Endogenous approaches, in which EVs are loaded by upstream manipulation of the producer cell, are compatible with large scale EV production, but loading by these approaches is inconsistent and has scarcely been quantitatively analyzed. The work in this dissertation is focused on enabling small RNA therapeutics via EV delivery. The lack of an ideal small RNA loading approach for EVs is addressed by tackling important issues of both endogenous and exogenous loading. First, the loading capacity of several common endogenous loading methods was optimized and quantitatively analyzed. Additionally, new approaches to endogenous small RNA loading involving genetic manipulation of the RNA structure and the microRNA cellular processing pathway were developed and evaluated. Finally, exogenous loading via sonication was applied to enable delivery of a novel microRNA combination that was identified via a rational selection process. This combination of miR-146a, miR-155, and miR-223 was found to have potentially synergistic anti-inflammatory activity, and EV-mediated delivery of the combination opens the possibility for therapeutic application in inflammatory diseases and conditions such as sepsis. Overall, this work both improves understanding of current techniques for small RNA loading into EVs and opens new opportunities for advanced strategies, bringing EV-based small RNA therapeutics closer to clinical application.Item DISSECTING THE GENE REGULATORY FUNCTION OF THE MYC ONCOGENE WITH SINGLE-MOLECULE IMAGING(2020) Patange, Simona; Larson, Daniel R; Girvan, Michelle; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The MYC oncogene contributes to an estimated 100,000 cancer-related deaths annually in the United States and is associated with aggressive tumor progression and poor clinical outcome. MYC is a nuclear transcription factor that regulates a myriad of cellular activities and has direct interactions with hundreds of proteins, which has made a unified understanding of its function historically difficult. In recent years, several groups have put forth a new hypothesis that questions the prevailing view of MYC as a gene-specific transcription factor and instead envision it as a global amplifier of gene expression. Instead of being an on/off switch for transcription, MYC is proposed to act as a `volume knob' to amplify and sustain the active gene expression program in a cell. The scope of the amplifier model remains controversial in part because studies of MYC largely consist of cell population-based measurements obtained at fixed timepoints, which makes distinguishing direct from indirect consequences on gene expression difficult. A high-temporal, high-spatial precision viewpoint of how MYC acts in single living cells does not exist. To evaluate the competing hypotheses of MYC function, we developed a single-cell assay for precisely controlling MYC and interrogating the effects on transcription in living cells. We engineered `Pi-MYC,' an optogenetic variant of MYC that is biologically active, can be visualized under the microscope, and can be controlled with light. We combined Pi-MYC with single-molecule imaging methods to obtain the first real-time observations of how MYC affects RNA production and transcription factor mobility in single cells. We show that MYC increases the duration of active periods of genes population-wide, and globally affects the binding dynamics of core transcription factors involved in RNA Polymerase II transcription complex assembly and productive elongation. These findings provide living, single-cell evidence of MYC as a global amplifier of gene expression, and suggests the mechanism is by stabilizing the active period of a gene through interactions with core transcription machinery.Item ISOTOPIC LABELING STRATEGIES AND NMR METHODOLOGIES TO FACILITATE RNA STRUCTURAL AND DYNAMICS STUDIES: APPLICATIONS TO A LONG NON-CODING RNA FROM KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS(2020) Becette, Owen; Dayie, Kwaku T; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNAs are essential components of biological pathways that result in human disease making them attractive therapeutic targets. Currently, NMR spectroscopy is the only high-resolution technique capable of probing RNA interactions in solution. Although NMR spectroscopy is well-suited to characterize macromolecular interactions at atomic-level detail, the currently available isotopic labeling strategies and NMR methodologies are limited to relatively small RNAs (~ 30 nts, ~ 10 kDa). This size limitation is due to poor sensitivity and limited spectral resolution both of which worsen with increasing size. Here I present novel isotopic labeling schemes and NMR experiments to help expand the size limitations of NMR. These new technologies are then applied to characterize the structure and dynamics of a non-coding RNA from Kaposi’s sarcoma-associated herpesvirus (KSHV) that causes cancer in AIDS patients.Item THE DEVELOPMENT AND APPLICATION OF SITE-SELECTIVELY ISOTOPICALLY LABELED NUCLEOTIDES TO PROBLEMS IN NMR SPECTROSCOPY:STRUCTURAL INSIGHTS INTO THE EPSILON RNA AND TARGET COMPOUND INTERACTIONS(2017) Longhini, Andrew Paul; Dayie, Theodore K; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA plays a central role in a multitude of cellular processes. Understanding the complex interplay between its structure and function is a requisite for understanding these cellular roles mechanisms of action. Herein we describe technologies that we have developed to help better study RNA structure and function via NMR. Our development of site-selectively isotopically labeled pyrimidine and purine nucleotides has reduced spectral crowding, eliminated problems associated with scalar coupling, and led to novel assignment protocols. We have applied these labels to a 61-nucleotide viral RNA element, HBV-ε. This RNA is central Hepatitis B’s viral life cycle. Its NMR resonances have been assigned and initial structure calculations have begun to show how it interacts with compounds screened to bind to an internal six nucleotide bulge.Item Analysis of Genetic Regulatory Mechanisms that Control Ethanolamine Utilization in Enterococcus faecalis(2017) Gebbie, Margo Page; Winkler, Wade C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this project, we studied the genetic regulatory mechanisms that affect utilization of ethanolamine, an abundant compound in the gastrointestinal environment. In Enterococcus faecalis, the ethanolamine utilization (eut) gene cluster encodes for a two-component regulatory system (TCS), comprised of a histidine kinase, EutW, which autophosphorylates upon sensing EA, and a cognate response regulator, EutV, which dimerizes upon receiving the phosphoryl group from EutW and binds the nascent transcript to prevent premature transcription termination. This TCS is responsible for coupling sensing of ethanolamine to production of eut transcripts. However, clues from other organisms had previously suggested that adenosylcobalamin (AdoCbl) might also be an important genetic regulatory signal for the E. faecalis eut genes. Indeed, we discovered a novel trans-acting noncoding RNA (EutX) that contained an AdoCbl-responsive riboswitch. Our data demonstrated that the riboswitch promotes a shortened form of EutX when cellular AdoCbl levels are replete. In contrast, a longer form is synthesized when AdoCbl levels are depleted. We demonstrated that structural motifs contained in the longer form of EutX act to sequester the EutV protein, preventing it from promoting transcription elongation of eut transcripts. These unexpected data revealed an important new type of regulatory mechanism for riboswitch RNAs. In support of this overall genetic regulatory model, we recapitulated the full genetic circuitry in a heterologous host. Using this system, we employed extensive site-directed mutagenesis to examine the functional importance of highly conserved EutV residues. This led to the identification of a cluster of positively charged residues, which we speculated are important determinants for RNA-binding activity. Consistent with this hypothesis, mutations of these residues resulted in loss of RNA-binding activity. Furthermore, we also explored whether the eut gene cluster was affected by additional genetic regulatory mechanisms. From these efforts, we concluded that oxygen is not a genetic regulatory feature of eut genes, in contrast to previously published speculation. However, we did find that it is likely to be repressed under conditions of high glucose. Therefore, these aggregate studies revealed new mechanisms of post-initiation genetic regulation, and showed how E. faecalis specifically controls expression of ethanolamine catabolism genes.Item QUANTITATIVE ANALYSIS OF INTACT PROTEINS AND RNAS CARRIED BY IMMUNOSUPPRESSIVE EXOSOMES(2016) Geis Asteggiante, Lucia Giorgina; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment of most cancer patients. They are a major obstacle to immunotherapy because they suppress both adaptive and innate immune responses. MDSCs collected from tumor-bearing mice release nano-sized vesicles, called exosomes, which carry biologically active molecules and participate in intercellular communication. Exosomes released by MDSC stimulate migration of other MDSC towards the tumor microenvironment and convert macrophages to a tumor-promoting phenotype. Among the proteins identified in MDSC-released exosomes, S100A8 and S100A9 are low-mass, highly abundant, pro-inflammatory mediators already known to contribute directly to the immune suppressive functions of MDSC. The aim of this work was to successfully interrogate the exosomal intact protein cargo using top-down proteomics, a strategy for protein analysis that has not previously been applied to exosomes of any kind. Several protein forms (proteoforms) were fully characterized, which is critical as post-translational modifications regulate protein functions, cellular location and protein interactions. Additionally, since the tumor promoting activity of MDSC is enhanced by inflammation, we focused on evaluating the effect of increased inflammation on the proteoforms relative abundance using current top-down label-free quantitation techniques (peak intensities and peak areas), and comparing them to our recently validated spectral counting approach. Using spectral counting we were able to estimate differences in abundances of both S100A8 and S100A9 proteoforms. Furthermore, it has been previously reported that exosomes can carry micro RNAs and messenger RNAs. In order to investigate if MDSC-derived exosomes also contain RNAs, a collaborative study was carried out entailing the qualitative and quantitative analysis of miRNAs, mRNA and proteins present in MDSC and their exosomes, and evaluate their changes due to heightened inflammation. The MDSC and exosome protein cargo was analysed by bottom-up proteomics in this case, and the RNA cargo by next generation sequencing. A large number of mRNA and miRNA species were found to be carried by MDSC-derived exosomes and, strikingly, their putative functions were associated to MDSC expansion and suppressive function, and cancer development.Item The conformational landscape of RNA translational regulators and their potential as drug discovery targets(2016) LeBlanc, Regan Michael; Dayie, Theodore K; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA is an underutilized target for drug discovery. Once thought to be a passive carrier of genetic information, RNA is now known to play a critical role in essentially all aspects of biology including signaling, gene regulation, catalysis, and retroviral infection. It is now well-established that RNA does not exist as a single static structure, but instead populates an ensemble of energetic minima along a free-energy landscape. Knowledge of this structural landscape has become an important goal for understanding its diverse biological functions. In this case, NMR spectroscopy has emerged as an important player in the characterization of RNA structural ensembles, with solution-state techniques accounting for almost half of deposited RNA structures in the PDB, yet the rate of RNA structure publication has been stagnant over the past decade. Several bottlenecks limit the pace of RNA structure determination by NMR: the high cost of isotopic labeling, tedious and ambiguous resonance assignment methods, and a limited database of RNA optimized pulse programs. We have addressed some of these challenges to NMR characterization of RNA structure with applications to various RNA-drug targets. These approaches will increasingly become integral to designing new therapeutics targeting RNA.Item NEW CHEMICAL TOOLS TO INVESTIGATE RNA FUNCTIONS(2013) Luo, Yiling; Dayie, Theodore K; Sintim, Herman O; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ribonucleic acid (RNA), as one of the essential macromolecules of life, plays an active role in gene regulation, catalysis, and signaling because of its ability to adopt complex 3D structures that can exist in multiple conformations. Until now, RNA preparation methods devised by most investigators utilized partially denatured the RNA. The mis-folding caused by denaturing - renaturing can seriously affect RNA structure and functional activity. To test this hypothesis, in PART I of this dissertation, we presented a simple strategy using `click' chemistry to couple biotin to a `caged' photocleavable guanosine monophosphate to synthesize native RNAs that are properly folded. We demonstrated that RNA ribozymes, ranging in size from 27 to 527 nt, prepared by our non-denaturing method form a homogenous population with superior catalytic activity than those prepared by traditional refolding methods. Having developed a method for in vitro RNA synthesis, we studied the riboswitch family of RNAs that require remolding their structure for function in PART II. C-di-GMP riboswitch, as the only currently known secondary messenger riboswitch, senses c-di-GMP using its aptamer domain to modulate the expression of genes which affects biofilm formation and virulence factors production in bacteria. To specifically target pathogenic bacteria in polymicrobial systems that control the RNA-mediated c-di-GMP signaling pathway through riboswitch regulation, different c-di-GMP analogs have been used as chemical tools to investigate the structure-activity relationship (SAR) of c-di-GMP binding to different c-di-GMP riboswitches. We demonstrated that different 2'-position modified c-di-GMP analogs could differentiate between two different classes of c-di-GMP riboswitches and even bind to one particular riboswitch in class I with different affinities. Specifically, Clostridium tetani (Ct-E88) RNA in class I c-di-GMP riboswitch was used for (structural dynamic) study to understand how ligand binding drives the conformational change to regulate the downstream genes within the expression platform. Our preliminary data obtained via different biochemical and biophysical tools - such as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy - demonstrated that Mg2+ ions accelerate ligand recognition by pre-organizing the RNA, and then rapid ligand binding folds the RNA into a compact structure for likely downstream gene regulation.