Cell Biology & Molecular Genetics Theses and Dissertations
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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 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 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 CHARACTERIZATION OF THE SHEDDASE-MEDIATED RELEASE OF A DROSOPHILA MEMBRANE-ANCHORED FGF THROUGH CYTONEMES(2023) Li, Yujia; Roy, Sougata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)During tissue morphogenesis, cells must precisely coordinate their functions over distance by communicating with secreted paracrine signaling proteins, such as the fibroblast growth factors (FGFs). The spatiotemporal regulations of FGF signaling are critical for development and homeostasis in all organisms. However, the mechanisms that control the location, timing, and level of FGF secretion and ensure its target-specific delivery over distance are poorly understood. This thesis has addressed this fundamental question by using a Drosophila FGF Branchless (Bnl) during tracheal development. Bnl is produced in a small group of wing-disc cells. Bnl regulates the development of a disc-associated tracheal branch, the air-sac-primordium (ASP), a vertebrate lung bud analog. Although FGFs are secreted diffusible signals, previous work has shown that the inter-organ dispersion of Bnl from the source to the ASP is highly polarized, target-specific, and is mediated over distance via long polarized actin-based filopodia, named cytonemes. The contact-dependent cytoneme-mediated Bnl delivery is programmed by the glycosylphosphatidylinositol (GPI) anchoring of Bnl. A GPI anchor tethers Bnl to the outer leaflet of the source cell membrane and inhibits free/random Bnl secretion. Surprisingly, GPI anchoring also facilitates Bnl’s release, but only through the cytoneme contact sites, thereby activating MAPK signaling exclusively in those recipient cells that establish contact with its source. However, the molecular mechanisms that might spatiotemporally activate the release of the membrane-anchored Bnl at the cytoneme contact sites remained unknown. This work addresses this knowledge gap by using a combination of cell biological, biochemical, and genetic analyses. Visualization of the distribution of a fluorescently-tagged Bnl revealed that a proteolytic cleavage within the Bnl backbone is essential for its target-specific release. Phenotypic analyses of an uncleavable Bnl mutant generated by genome editing suggested that the proteolytic event that releases Bnl is essential for Bnl signaling and morphogenesis. An in vitro cell-culture-based screening for candidate sheddases identified five membrane-localized enzymes that specifically shed Bnl from the cell surface. These enzymes include two matrix metalloproteinases, MMP1 and MMP2, a pro-protein convertase, Furin1, and two ADAM family metalloproteases, Meltrin and Tace. An inherently disordered region within the Bnl backbone was identified as the substrate for the proteolytic cleavage. Serial mutagenesis in this region revealed four minimal zones required for enzymes to act on Bnl. Interestingly, one of the minimal regions at the juxta-membrane position harbors the Bnl cleavage site, and the other three zones apparently play regulatory roles. Further work on MMPs using mosaic gain-of-function analyses confirmed MMP-mediated Bnl shedding in vivo. However, MMPs are not expressed in the Bnl source. Consequently, Bnl is not freely released. In contrast, MMPs are expressed in the ASP and are localized on the recipient ASP cytonemes. in vitro cell culture experiments that reconstituted contact-dependent Btl-Bnl interactions between Bnl-source and recipient cells confirmed that MMPs are selectively recruited by the recipient cells to the signaling contact sites and activate Bnl shedding in trans. These results explained why and how Bnl is released only via the cytoneme contact sites. Notably, MMP1 and MMP2 are GPI-anchored proteins but are also catalytically active in secreted soluble forms. Therefore, how is Bnl prevented from non-specific shedding by the extracellular soluble MMPs? in vitro and in vivo experiments using GPI-modified and non-GPI-modified MMPs revealed that only the membrane-tethered MMPs that are retained on the cell surface could efficiently shed cell surface Bnl. These findings supported a working model suggesting that recipient ASP cells extend receptor-containing cytonemes and establish contact with the Bnl-source via the receptor-ligand interaction. Due to the GPI-anchoring, MMPs are presented on the surface of ASP cytonemes and are delivered via these cytonemes to their contact sites, leading to the contact-dependent target-specific Bnl trans-shedding. These results provide new insights into how contact-mediated signal dispersion via cytonemes can be modulated by cell surface sheddases. Additional work on Furin1 and ADAM proteases showed that Tace and Meltrin shed Bnl in a cell-non-autonomous manner, like MMPs. In contrast, Furin1 is required only in the source cells for Bnl shedding. How and under what contexts Furin1 and ADAM proteases are activated to shed Bnl needs to be explored in the future. Collectively, this work characterized a novel enzymatic Bnl shedding mechanism and provided insights into how Bnl sheddases might be controlled in space and time to ensure cytoneme-mediated Bnl exchange.Item Molecular and Biophysical Bases of Intracellular Electric Fields in Pollen Tubes(2022) Oliveira Nunes, Custódio; Feijό, José A.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Pollen tubes are the male gametophyte of flowering plants. They are arguably one of the fastest-growing cells in nature and inherently an excellent model for studying cellular processes like apical growth, polarity, and chemotropism. Pollen tube development is associated with a unique choreography of ion fluxes and cytosolic ion gradients of Cl-, Ca2+, H+, and K+, creating a unique electrochemical environment, where alternating depolarizing ionic currents at their growing apex are spatially separated from hyperpolarizing currents in their shank. We hypothesize that these electrical differences generated by the opposite ionic patterns could sustain a standing membrane potential gradient at the growing apex. In agreement with evidence from other cellular electrotaxis phenomena, we further hypothesize that a standing electric field gradient could be mechanistic in terms of cell polarity and chemotropism of pollen tubes.Here we show, for the first time, the existence of a standing membrane potential gradient in pollen tubes, confirmed in three different species, thus suggesting a conserved role in apical growth. This conclusion was achieved using three complementary methods, two membrane potential dyes with opposite fluorescence kinetics, and a genetic probe for cytosolic potassium (K+). The K+ gradient is focused at the pollen tube tip, and is compatible with previous information on the individual ion features. Of relevance, K+ shows a negative gradient from the tip, the first ever described in a living cell, suggestive of K+ apical efflux that contributes to the depolarized state. Quantifications of the fluorescent dyes estimate an apical depolarization of approximately 30mV compared to the shank. Screening of ion-channel mutants inducing male-fertility phenotypes supports the hypothesis that this bioelectric oddity is mechanistic for pollen tube’s critical functions, fast invasive growth and chemotropism. Furthermore, we determined that anionic lipids determine the emergence of the pollen tube and correlate with the apical depolarization area, suggesting that they may act as physical determinants of the growing apex. These results open important questions in our understanding of the bioelectrical processes determining cell growth, polarity, morphogenesis, and chemotropic reactions.Item DEVELOPMENT OF AN ACCELERATED ALZHEIMER’S DISEASE IN VITRO MODEL WITH THE ADDITION OF PROGERIN(2023) Xue, Huijing; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Alzheimer’s Disease (AD) is one of the most common causes of dementia. Twopathological features of AD include amyloid plaques and neurofibrillary tangles. The mechanism underlying the disease's onset and progression remains unclear. Lamin A is an essential component of the nuclear lamina, and nuclear lamina plays a vital role in essential cell functions. Specific mutations in lamin A yield a truncated protein called progerin that causes Hutchinson-Gilford Progeria Syndrome (HGPS), a premature aging disease. Despite the low expression of lamin A in the brain, several studies reported abnormal lamin A accumulation in patients' hippocampus through the different stages of AD. Besides, there are a lot of common phenotypes between AD and HGPS. Meanwhile, one of the challenges of studying AD is the model issue. It is difficult to recapitulate all AD pathology in a single model, and most models are time-consuming. This dissertation focuses on goals: (1) exploring the potential role of lamin A in AD and (2) facilitating the AD model development. To investigate the potential role of lamin A in AD, I overexpressed either lamin A or progerin in neural cells and checked the phenotypes in Chapter II. Early cell death is closely associated with neuronal loss in AD. After ectopically expressing lamin A in neural cells, early cell death was slightly increased. Progerin could worsen these phenotypes. Oxidative stress and cell cycle re-entry are early events in neurodegeneration and are associated with increased cell death. With the ectopic expression of lamin A, neural cells exhibited slightly elevated oxidative stress and significantly increased cell cycle reactivation. Both two events were significantly increased with exogenous progerin. These results provide insights into how lamin A is involved in neurodegeneration. Besides, progerin addition could further disrupt cellular homeostasis and therefore provide a potential environment for modeling late-onset disease. Most of the current cellular models for AD require several months to display AD phenotypic features, mainly because of the lack of an aging environment in the in vitro cell culture, which is an essential player in age-related neurodegeneration. To provide the aging environment for modeling AD, I examined the impacts of exogenous progerin expression on the neural progenitor cells carrying familial AD mutations (FAD) in Chapter III. Exogenous progerin could accelerate hallmark AD phenotype exhibition from 8-16 weeks to 3-4 weeks, including increased tau phosphorylation and Aβ42/Aβ40 ratio in 2D cell culture, and accumulation of amyloid plaques in 3D cell culture. Additional AD cellular phenotypes, including elevated cell death and cell cycle re-entry, were significantly increased after progerin intervention as well. Together, these results indicated that the approach with progerin expression could create an accelerated model for modeling AD development and future drug screening.Item Mechanisms by which the actin cytoskeleton switches B cell receptor signaling from the activation to the attenuation mode(2022) Bhanja, Anshuman; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The B cell-mediated humoral immune response is critical in fighting off invading pathogens and potentially harmful foreign substances. B cells detect antigens through the B cell receptor (BCR). The binding of cognate antigen to the BCR induces a signaling response, a critical initiation and regulatory step for B cell activation and differentiation. The actin cytoskeleton has been shown to play essential roles in BCR signaling. When encountering membrane-associated antigens, actin amplifies signaling by driving B cell spreading and BCR clustering, while promoting signal attenuation by causing B cell contraction. This signal attenuation is essential for curtailing the activation of autoreactive B cells. However, the mechanism by which the actin cytoskeleton switches BCR signaling from amplification to attenuation was unknown. My thesis research examined the mechanisms by which actin reorganization transitions B cells from spreading to contracting and B cell contraction switches BCR signaling from amplification to attenuation, using mouse splenic B cells, a functionalized planar lipid bilayer system, and total internal reflection fluorescence microscopy. Our results show that branched actin polymerized by Arp2/3 is required for B cell transition from spreading to contraction after driving B cell spreading. Ubiquitously expressed Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP), but not the haematopoietically specific WASP, activates the branched actin polymerization and generates inner actin foci from lamellipodial actin networks, by sustaining their lifetime and centripetal movement. N-WASP-dependent inner actin foci are necessary for recruiting non-muscle myosin II, creating an actomyosin ring-like structure at the periphery of the membrane contact region to drive B cell contraction. B cell contraction primarily increases the BCR molecular density in individual BCR-antigen clusters, measured by the peak fluorescence intensity. Inhibition of B cell contraction by Arp2/3 inhibitor and B cell-specific N-WASP knockout (cNKO) reduced the increasing rates of BCR molecular density. Increased molecular density caused by B cell contraction leads to decreases in the levels of phosphorylated BCR, the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. However, the levels of total Syk and SHIP-1 have a different relationship with BCR density in individual clusters: Syk does not decrease until a high threshold of BCR density, which can be achieved only by contracting B cells, but SHIP-1 consistently reduces with the increase in BCR molecular density. Inhibiting B cell contraction by cNKO reduces the molecular density of BCR clusters but does not affect the relationship of the Syk and SHIP-1 levels with BCR molecular density in clusters. Taken together, our results suggest that the actin cytoskeleton reorganizes from the lamellipodial branched actin networks to centripetally moving actin foci, enabling actomyosin ring-like structure formation, through N-WASP-activated Arp2/3. Actomyosin-mediated B cell contraction attenuates BCR signaling by increasing receptor molecular density in individual BCR clusters, which causes the dissociation of both stimulatory and inhibitory signaling molecules. My thesis research results reveal a novel negative regulatory mechanism for BCR signaling, an essential checkpoint for generating pathogen-specific and suppressing self-reactive antibody responses.Item MUCIN-MEDIATED AND INTERFERON-DRIVEN DEFENSE MECHANISMS AGAINST INFLUENZA VIRUS INFECTION IN HUMAN AIRWAY EPITHELIUM(2022) Iverson, Ethan; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The human airway epithelium represents the primary site of infection for many respiratory viruses, including influenza A virus (IAV). To safeguard this tissue and maintain the functionality of the lung, humans possess a two-layer, extracellular, mucus barrier composed predominantly of individual proteins termed mucins. Additionally, underlying epithelial cells produce interferons upon virus detection that promote the establishment of a local antiviral state through autocrine and paracrine signaling. However, despite these protective measures, IAV continues to cause significant annual morbidity and mortality across the globe. Therefore, we sought to further investigate how specific mucin molecules interact with IAV, and how interferon drives intrinsic antiviral defense in the context of a human airway epithelial (HAE) culture system. By utilizing fluorescently-labeled influenza virus particles we further elucidate the adhesive interactions between mucus and influenza virus while also detailing, for the first time, real-time IAV diffusivity within patient-derived mucus samples. These results reveal that the polymeric structure of mucus greatly influences the mobility of IAV within human secreted mucus. Additionally, we investigate the interaction between influenza virus and tethered mucin 1 (MUC1), finding that MUC1 expression is enhanced by virus-driven inflammation and interferon signaling. Moreover, by establishing a genetically-tractable airway epithelial model, we detail the protective role MUC1 plays in preventing the initial establishment and spread of influenza virus in HAE. Specifically, we find that the loss of MUC1 significantly enhances IAV uptake and spread. Finally, we observe that the directionality of IFN exposure at airway epithelial surfaces impacts the magnitude of protection against IAV and SARS-CoV-2. We then detail the cellular composition of our HAE culture system and define a shared IFN response profile across all HAE component cell types as well as cell type-specific interferon stimulated genes. Together our work provides novel insight into the innate and intrinsic anti-viral properties of the human airway epithelium.Item KEEPING IT IN FRAME: MONITORING RIBOSOMAL FRAMESHIFTING DURING TRANSLATION(2022) Kelly, Jamie; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Programmed -1 ribosomal frameshifting (-1 PRF) is a molecular mechanism that redirects translating ribosomes into a new reading frame. It is widely used by RNA viruses to conserve genome space while expanding the viral proteome and it can help regulate gene expression in eukaryotic cells. Strict regulation of both programmed and non-programmed frameshift events are essential to translational fidelity. This dissertation explores the -1 PRF element of SARS coronavirus 2 (SARS-CoV-2) and the -1 PRF inhibitor, Shiftless. We comparatively analyzed the structural and functional conservation of -1 PRF elements in SARS-CoV and SARS-CoV-2. Both -1 PRF structure and frameshift efficiency were highly conserved between the two viruses and a small molecule effective against SARS-CoV -1 PRF significantly decreased frameshift efficiency in SARS-CoV-2. This suggests -1 PRF is an attractive antiviral target and could be a useful tool to combat the SARS-CoV-2 pandemic or future outbreaks of similar coronaviruses. The innate immune system targets viral frameshifting using an interferon-stimulated -1 PRF inhibitor called Shiftless (SFL) that binds, arrests, and terminates translation of -1 frameshifted ribosomes. We found that SFL is not only expressed in response to interferon but that it may have a role in general translational fidelity. SFL is constitutively expressed at low levels in human-derived cell lines and its effects are not limited to -1 PRF signals. Disruption of SFL homeostasis results in reciprocal 2-fold changes to recoding efficiencies in a panel of human and viral-derived translational recoding signals, decreases reporter gene expression, and decreases mRNA steady state abundances. Additionally, SFL over or under expression combined with knockdown of prominent ribosome-associated protein quality control (RQC) proteins reveals that SFL is epistatic to RQC. These results suggest that SFL has a role in general translational fidelity monitoring for spontaneously frameshifted ribosomes in addition to its role as a member of the innate immune response.