Cell Biology & Molecular Genetics Theses and Dissertations
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Item THE ROLE OF GENOME ORGANIZATION AND FILAMENTOUS BACTERIOPHAGE ON GONOCOCCAL BIOLOGY AND PATHOGENICITY(2024) Kopew, Jessica; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (GC), represents a significant global health concern as it is the second most common bacterial STI and has a rising rate of antimicrobial resistance. The first study of this thesis aims to elucidate the causes and consequences of gonococcal genome reorganization. Here I found that when looking at many GC strains’ genomes, each GC strain had a unique genome organization including both inversions and translocation events. I also saw a consistent pattern of DNA high sequence similarity on both sides of the translocation or inversions, consistent with homologous recombination driven reorganization. PCR analysis of inversion events suggests that these large-scale reorganization events are both stable and rare. Growth curve analysis demonstrates a wide variability in growth rate between strains. Proteomic analysis suggests reorganization driven changes to replication termination location leads to upregulation of many kinds of proteins including energy metabolism and antimicrobial resistance associated genes. This study suggests that homologous recombination driven genome reorganization can have large impacts on gonococcal biology and pathogenicity. This study demonstrates the need for future gonococcal studies to use multiple GC strains from a diverse background to capture the wide variability in GC phenotypes. The second study of this thesis sought to uncover the role filamentous bacteriophage play in GC biology. I found that every GC strain currently in the NCBI database at the date of this study contains four filamentous bacteriophage gene regions in the GC genome. I found that FA1090Δfil (a GC strain lacking all four filamentous bacteriophage gene regions) grew poorly at 37⁰C both in broth and on agar, as compared to wild type FA1090. However, there was no difference when the strains were grown at 34⁰C or when grown without shaking, demonstrating the condition dependent nature of this growth advantage. FA1090Δfil formed larger bacterial aggregates than FA1090 WT. When these strains were analyzed for their ability to produce biofilms, no differences were seen in the overall biofilm’s biomass, yet the overall structure of the biofilms were different, with FA1090Δfil producing taller and rougher biofilms. Previous unpublished research in the Stein Lab demonstrates that filamentous phage derived proteins are capable of deteriorating the integrity of epithelial cell cultures and cervical tissue explants. The data from this chapter suggests that filamentous phage provide the gonococcus with a growth advantage, inhibit bacterial aggregation, alter the structure of the GC biofilm, and that phage proteins can lead to loss of the integrity of the epithelium. Taken en toto, these studies demonstrate that both alterations in bacterial genome organization and contributions from filamentous bacteriophage genomes can impact gonococcal biology and pathogenicity, which could be key to preventing and treating GC infections.Item INITIAL LOCAL CYTOKINE RESPONSES AGAINST NEISSERIA GONORRHOEAE INFECTIONS IN THE HUMAN CERVIX(2024) Dai, Yiwei; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) is a human-specific pathogen that causes gonorrhea, a common sexually transmitted infection. In women, GC initiate infection by colonizing the cervix. Although GC colonization can cause cervicitis, most female infections are asymptomatic. Asymptomatic colonization of the cervix increases the risk of transmission and progression to severe complications, including pelvic inflammatory disease and infertility. Despite its clinical significance, the mechanisms underlying GC asymptomatic colonization remain unclear. Using a human cervical tissue explant model, which can mimic GC infection in vivo, my Ph.D. research examined the early local cytokine responses to GC cervical colonization, a determining factor for asymptomatic and symptomatic clinical outcomes. Luminex and spatial transcriptomic analyses found that cervical tissue explants constitutively secrete and express a broad spectrum of cytokines, with particularly high levels of the IL-1 receptor antagonist IL-1RA, the anti-inflammatory cytokines IL-10, and the multi-functional cytokine IL-6. During the first 24-h inoculation, GC strain expressing an opacity-associated protein binding to the host receptor CEACAMs (MS11OpaCEA) increased the secretion and transcript levels of both pro-inflammatory, like IL-1α/β, and the anti-inflammatory cytokine IL-10, as well as multi-functional cytokines, like IL-6 and CFS3, but MS11 lacking Opa (MS11∆Opa) induced much less. Notable, the cervix secreted IL-1RA at 100-fold higher levels than IL-1α/β. Cervical secreting levels of soluble IL-6 receptors, required for activating IL-6 inflammatory functions, were 10,000-fold less than IL-6. These results support an anti-inflammatory-dominated cytokine environment of the human cervix, and GC further push it in the anti-inflammatory direction. Using isogenic GC strains and inhibitors, the mechanism underlying GC cytokine induction and the impact of GC-induced cytokines on GC infection were examined. My research found that GC-induced inflammatory cytokine production involved NF-κB activation in both epithelial and subepithelial cells. GC-induced IL-10 production depended on the activation of CEACAM-downstream signaling molecule SHP1/2. Reductions in inflammatory cytokines, TNF-α and IL-1β, by an NF-κB inhibitor did not significantly affect GC colonization, epithelial cell-cell junctions, or epithelial shedding. In contrast, neutralizing IL-10 or blocking its receptor reduced GC colonization and increased ectocervical epithelial shedding and disassembly of epithelial cell-cell junctions. Thesis results suggest that IL-10 plays critical roles in strengthening the cervical epithelium and suppressing the epithelial cell-cell junction disrupting function of inflammatory cytokines, and that GC further elevate the local IL-10 level to prevent bacteria from shedding off with epithelial cells, enhancing colonization.Immunofluorescence and spatial transcriptomic approaches were utilized to identify the types of cervical cells contributing to the local cytokine response to GC infection. Cervical epithelial cells and macrophages are two of the major contributors. IL-1RA protein and mRNA were primarily detected at the ectocervical epithelium. IL-6 protein and mRNA were also detected in ectocervical epithelial cells. MS11OpaCEA colonization increased IL-1RA transcript levels, while MS11ΔOpa switched ectocervical epithelial cells from IL-1RA- to IL-8/IL-6-expressing. GC inoculation did not alter the transcriptomic program of CD68+ macrophages adjacent to the ectocervical epithelium, maintaining the tissue-repair signature. However, GC changed the transcriptomic profiles of macrophages at the explant tissue side, exposed to media and inoculated GC, leading to increased expression of either inflammatory M1- or anti-inflammatory M2 signature genes. These results suggest that the human cervix utilizes high levels of epithelial-secreted IL-RA, low levels of soluble IL-6 receptor release, and tissue-repairing macrophages at the subepithelium to control inflammation induced by colonizing GC when the epithelium prevents GC from entering the tissue. Overall, my research results suggest that GC exploit the local cytokine response of the human cervix, dominant by anti-inflammatory IL-1RA, IL-10, and IL-6, to facilitate colonization and desensitize immune detection, promoting asymptomatic colonization.Item Biomechanical regulation of T cells: The cytoskeleton at the nexus of force and function(2024) Pathni, Aashli; Upadhyaya, Arpita; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The adaptive immune response is a sophisticated and multi-pronged defense mechanism that provides specific and long-lasting protection against infections and cancer. Central to this response are T lymphocytes - immune cells that orchestrate the immune response and directly eliminate infected or malignant cells. T cell function is intricately linked to their cytoskeleton, a dynamic network of protein filaments, consisting of actin, microtubules, and intermediate filaments, which provides structure, facilitates movement, and regulates intracellular transport. While the biochemical aspects of T cell function have been well-studied, recent advances have highlighted how mechanical forces influence T cell behaviors such as activation, migration, and effector functions—all processes driven by dynamic cytoskeletal remodeling. However, the mechanisms by which cytoskeletal dynamics, forces and mechanical stimuli drive T cell function remain poorly understood. This dissertation investigates this interplay, focusing on cytotoxic T lymphocytes (CTLs), a subtype of T cells that directly kill infected or cancerous cells. To launch a killing response, naïve CD8+ T cells must be activated by antigen-presenting cells (APCs) in lymph nodes, following which they proliferate and differentiate into an effector CTL population. CTLs eliminate targets via a specialized interface called the immunological synapse (IS), where they release lytic granules containing cytotoxic molecules and exert cytoskeletal forces to induce target cell death. A key event in IS formation is polarization of the centrosome, or the microtubule-organizing center, facilitating directional release of lytic granules. We first examined how biochemical signals provided by APCs modulate the cellular cytoskeleton. APCs provide not only antigenic stimulation, but also co-stimulatory signals required for full activation. Inflammatory cytokines such as interleukin-12 (IL-12) act as a third signal, enhancing CTL proliferation and cytotoxicity. Our findings demonstrate that CTLs activated in the presence of IL-12 exhibit enhanced IS formation, altered actin dynamics and microtubule growth, and generate greater mechanical forces, thus highlighting how activation signals can shape T cell mechanics, dynamics and function. Next, we investigated how the mechanical properties of target cells influence CTL function. Employing a biomimetic hydrogel system that mimics the stiffness of target cells, we demonstrate that substrate stiffness modulates multiple aspects of CTL responses. CTLs interacting with stiffer substrates exhibit enhanced spreading, accelerated actin ring formation, increased contractile forces, and more efficient centrosome polarization. Mechanical cues also influence lytic granule release and the nuclear translocation of mechanosensitive transcription factors. This work underscores the importance of mechanical cues in regulating immune responses. Given that coordinated cytoskeletal interactions are crucial for T cells to effectively respond to environmental cues, we further examined this crosstalk with a focus on intermediate filaments, the third, often understudied component of the cytoskeleton. Our characterization of the vimentin intermediate filament network reveals an expansive structure complementary to and dependent on other cytoskeletal components. We study the dynamics and organization of the vimentin network and find a close association of this network with the centrosome. Our results suggest a structural role for vimentin in supporting IS formation. Throughout this work, we use advanced imaging techniques and analysis approaches to probe various facets of T cell function. By bridging immunology, cell biology, and biophysics, this research contributes to our understanding of how physical forces shape immune responses.Item Roles of Female Sex Hormones in Regulating Neisseria gonorrhoeae Colonization of the Human Cervix(2024) Di Benigno, Sofia; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) is a human-exclusive pathogen that infects the genital tract. Gonococcal infection may present with or without symptoms and can lead to a variety of serious sequelae if left untreated, especially in female patients. Despite this, there are few models that can effectively mimic GC infection in the female reproductive tract (FRT); of these, even fewer consider the impact of the menstrual cycle, an important feature of the FRT, on GC infection. I used the human cervical tissue explant model previously developed in our lab, which can recapitulate GC infection in vivo. Tissue explants were treated with the sex hormones estradiol and progesterone to mimic various stages of the menstrual cycle and examine its impact on GC infectivity. Estradiol was used to mimic the late proliferative phase, and a combination of estradiol and progesterone was used to mimic the middle of the secretory phase. The effects of hormones on GC infectivity were examined after 72 total hours of hormone treatment and 24 hours of inoculation with GC of strain MS11. My results show that treatment with estradiol and with a combination of estradiol and progesterone both increase the level of GC colonization on the endocervix, but not on the ectocervix, compared to controls that were not treated with hormones. However, the hormone treatment did not affect GC penetration of the cervical epithelium. Both hormone treatments increased the number of GC colonies on the endocervical epithelium, and a combination of estradiol and progesterone produced an additional population of large GC colonies, leading to an increase in the average colony size. These increases in colony number and size were not associated with an increase in the expression of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which are the host receptors for GC Opa proteins. In contrast, treatment with estradiol induced a redistribution of CEACAMs from the luminal surface to the inside of epithelial cells. Additionally, estradiol altered the morphology of endocervical epithelial cells from columnar to cuboidal, but the integrity of cell-cell junctions was unchanged. The increase in colonization under high estradiol conditions was correlated with a decrease in levels of certain pro-inflammatory cytokines and chemokines, but this decrease was not sufficient to fully explain the increase in colonization. Next, I investigated the impact of cervical mucus on GC infectivity and interactions, as gel-forming mucin MUC5B but not MUC5AC increases with estradiol at the proliferation phase. Under both hormone treatment conditions, GC were able to establish close interaction with the luminal surface of the endocervical epithelial cells, displacing membrane-spanning mucin MUC1 in the membrane. Furthermore, GC were able to diffuse through an artificial mucin hydrogel and diffused more efficiently through a MUC5AC-dominant than a MUC5B-dominant hydrogel. Gel-forming mucins collected from cervical tissue explants enhanced GC aggregation in vitro, even at very low concentrations. However, mucins collected from estradiol-treated tissues showed less impact on GC aggregation than those collected from untreated tissues or tissues treated with both estradiol and progesterone. MUC5B and MUC5AC purified from cows and pigs also increase GC aggregation in vitro with GC aggregating more in a MUC5AC- than a MUC5B-dominant mucin mixture. Taken together, my research reveals for the first time that female sex hormones regulate GC colonization at the human cervix by changing the composition of the cervical mucus, providing a mechanism of hormonal regulation underlying the varying susceptibility of female patients to mucosal GC.Item INVESTIGATING MECHANISMS UNDERLYING MLO’S ROLE AS A HOST FACTOR ESSENTIAL FOR PATHOGENESIS OF POWDERY MILDEW FUNGI(2024) Bloodgood, David; Xiao, Shunyuan; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Loss-of-function mutations in Mildew Locus O (MLO) family genes confer broad-spectrum resistance to powdery mildew (PM) fungi in various plant species. mlo-mediated resistance is invariably coupled with increased defense responses and early leaf senescence; hence the molecular basis of mlo-mediated resistance remains unresolved. A saturated genetic screen in the background of an Arabidopsis triple mutant where three essential immune components, EDS1, PAD4 and SID2 are mutated, led to the identification of five allelic mutations in MLO2, each of which results in compromised immunity yet poor infection (cipi) to PM. Further CRISPR-targeted mutagenesis of two functional homologs, MLO6 and MLO12 in a cipi mutant background result in complete lack of infection from PM fungi. The sextuple mutant, eds1pad4sid2mlo2mlo6mlo12 (epsm3) showed no early leaf senescence, ROS accumulation or expression of defense genes, indicating that MLO2, MLO6 and MLO12 are bona fide host susceptibility factors for PM. Expression of MLO2-GFP as a transgene in epsm3 restores susceptibility to PM and MLO2-GFP focally accumulates at the fungal penetration site. Thus, restoration of susceptibility to PM in the epsm3 background can be used as a sensitive reporter to assess whether other MLO family members share a conserved molecular function when expressed in leaf epidermal cells. The Barley MLO and Arabidopsis MLO7 enabled PM pathogenesis whereas MLO1, MLO3 and MLO4 could not, suggesting the existence of two distinct classes of MLO family members. Sequence alignment identified three conserved amino acid residues in the C terminal calmodulin-binding domain of MLO2, and MLO7, which are absent in MLO1, MLO3 and MLO4. This observation suggests that the C-terminal domain of MLO proteins could contribute to their functional divergence. Creation and functional assays of chimeric MLO2/MLO1 proteins by swapping their C terminal domains revealed that the C terminus determines the localization pattern of MLO proteins. The Feronia (FER) receptor-like kinase is required for localization of MLO7 in synergid cells; however, CRISPR-targeted mutagenesis of FER did not disrupt the localization of MLO2 to the fungal penetration site. Based on the results described above, it can be inferred that MLO2 localization to and possible stabilization of the plasma membrane at the fungal penetration site is essential for allowing PM fungi to penetrate the host cell and subsequently differentiate the haustorium. Further multiplexed CRISPR mutagenesis of other gene families suggests that SYP121 and SYP122, two closely related SNARE genes play essential roles in focal accumulation of MLO2 at the fungal penetration site.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 THE STANDALONE REGULATOR ROFA OF STREPTOCOCCUS PYOGENES EXHIBITS CHARACTERISTICS OF A PRD-CONTAINING VIRULENCE REGULATOR(2024) Hart, Meaghan Taylor; McIver, Kevin S; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Streptococcus pyogenes (Group A Streptococcus; GAS) is a human pathogen estimated to cause nearly 790 million cases of disease annually at diverse tissue sites. To successfully infect these sites, GAS must detect nutrient availability and adapt accordingly. One mechanism employed to detect and import carbohydrates is the phosphoenolpyruvate transferase system (PTS), which mediates both carbohydrate uptake and metabolic gene regulation. Gene regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several stand-alone regulators that contain PRDs, with corresponding regulons encoding both metabolic genes and important virulence factors. These regulators form a family called PRD-Containing Virulence Regulators (PCVRs). RofA is a putative member of this family and is known to regulate the expression of genes important for virulence. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate appropriate virulence gene expression. In this dissertation, the RofA regulon was determined in strain 5448, a representative strain of the globally disseminated M1T1 serotype. The pilus and capsule operons were consistently dysregulated across growth in the absence of RofA. This correlated with increased capsule production and decreased adherence to primary keratinocytes. Purified RofA-His was phosphorylated in vitro by the general PTS components EI and HPr, and phosphorylated species of RofA-FLAG were detected in vivo late in stationary phase in a glucose-dependent manner. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar detection and utilization with GAS virulence gene regulation. Additionally, a bioluminescent construct was generated for allelic exchange into any S. pyogenes strain. Allelic exchange of this construct into WT 5448 yielded strains that were highly bioluminescent, grew to a similar density as WT, and survived as well as WT when challenged with human neutrophils. This tool could be used to study the contribution of specific proteins on in vivo virulence in a non-invasive manner, including RofA and RofA phosphorylation.Item INHIBITION OF TYPE ONE INTERFERON SIGNALING THROUGH CROSSTALK WITH TOLL-LIKE RECEPTOR SIGNALING(2024) Shuster, Michael; Briken, Volker V; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Interferons (IFNs) are a class of cytokines that play a prominent role in host immunity. Type I IFN is broadly associated with antiviral immunity and susceptibility to bacterial pathogens, but others have shown that type I IFN can be beneficial in some bacterial infections. Additionally, some bacterial infections such as Mycobacterium tuberculosis and Legionella pneumophila can inhibit type I IFN signaling. Questions remain such as how these bacteria inhibit type I IFN signaling as well as if other bacterial pathogens, such as Salmonella enterica, can also inhibit type I IFN signaling. Additionally, type III IFN is a relatively new class of IFN, providing antiviral protection similar to and at times redundant to type I IFN. There are some important non-redundant differences from type I IFN though, such as type III IFN’s broader activity at epithelial surfaces (like those in the lungs) and its reduced proinflammatory effects. The role of type III IFN in bacterial infections as well if bacteria can inhibit this signaling pathway remains poorly understood.Here, we examined if Salmonella enterica can inhibit type I IFN signaling, the specificities of the previously observed inhibition with Mtb infection, and how these bacterial infections are inhibiting this signaling. We demonstrate that Salmonella Typhimurium infection inhibits type I IFN signaling through crosstalk with TLR4 signaling. We establish that TLR4 signaling results in reduced surface level type I IFN receptor, which dampens cellular responsiveness to type I IFN. We show that Mtb does not inhibit type III IFN signaling and that it inhibits type I IFN signaling independently of virulence, specifically EsxA and ESX-5. Additionally, this inhibition of type I IFN signaling seems specific to mouse cells as Mtb-infected human macrophages and dendritic cells did not have inhibited type I IFN signaling. We observed that other TLR signaling pathways result in specifically inhibited type I IFN signaling. Synthesizing a model from our results, there appears to be a mouse-specific crosstalk pathway between TLR signaling and type I IFN signaling, resulting in dampened responsiveness to type I IFN through downregulation of cell surface type I IFN receptor.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 Characterization of chromatin assembly dynamics mediated by the histone H3.3 chaperone HIRA and implications of innate immunity during Human Papillomavirus infection(2024) Della Fera, Ashley Nichole; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The circular double-stranded DNA genome of Human papillomavirus (HPV) is chromatinized throughout its viral lifecycle and relies on numerous host chromatin assembly processes, epigenetic modifications, and immune evasion to ensure genomic stability and productive infection. Despite its chromatinization, the HPV genome remains susceptible to innate immune pathways that sense and respond to foreign DNA. In this work next generation sequencing (RNAseq) was utilized to profile changes in the host transcriptome following cellular differentiation and HPV infection in keratinocyte cell lines. Global alterations in keratinocyte differentiation were observed upon HPV infection, and unexpectedly, upregulation of innate immune signaling upon differentiation. Recent findings indicate that packaged HPV genomes are enriched in histone H3.3. Notably, the replication-independent histone H3.3 chaperone HIRA has been implicated in several pro- and anti-viral responses, but its function during HPV infection has yet to be elucidated. Using in-situ approaches, the role of HIRA during the late phase of the HPV lifecycle was evaluated, which showed that HIRA and other chromatin assembly factors localize to sites of HPV replication. Here the requirements for this localization were further characterized, and the impacts of HIRA on HPV genome amplification and viral transcription during the late stage of the HPV life cycle were assessed. Moreover, histone H3.3 phosphorylated at serine 31 was shown to be highly associated with HPV replication factories. HIRA, in part through association with the PML nuclear body associated protein Sp100, has also been reported to promote innate immune responses following infection with other DNA viruses. Here, HIRA localization to PML-NBs was identified to increase following stimulation with IFN in an Sp100-dependent manner. However, while Sp100 is required for localization of HIRA at PML-NBs, it was not required for HIRA localization at sites of HPV replication. In summary, this work highlights the broad changes in the host transcriptome following cellular differentiation and HPV infection, elucidates a previously undescribed role for histone H3.3 chaperone HIRA during the late phase of the HPV life cycle, and further characterizes the relationship between HIRA and Sp100 at PML-NBs.