MS, University of Cincinnati, 2021, Medicine: Molecular and Developmental Biology

Systemic Lupus Erythematosus (SLE) is a debilitating autoimmune disorder characterized by system-wide inflammation and rampant production of autoantibodies. The most prominent and highly replicated set of dysregulated genes in the immune cells of patients with SLE are the type I interferons (IFN-I) and IFN-responsive genes. Plasmacytoid dendritic cells (pDCs) are the chief producers of IFN-I, and their ability to modulate IFN-I expression is directly regulated by the transcription factor interferon regulatory factor 7 (IRF7). While IRF7 is an established SLE risk locus, the variants responsible for disease pathology remain unknown. We hypothesized that an amino-acid changing SLE risk variant in the inhibitory domain of IRF7 (rs1131665) alters expression of disease-relevant IFN-I in clinically relevant cells to increase SLE risk. The functional consequences of the SLE-associated variant were assessed in human cell lines and in genome-edited mice with an introduced SLE-risk variant at Irf7. Our data demonstrate greater than 2-fold genotype-dependence in IFN-stimulated response element-driven luciferase activity and inflammatory cytokine secretion detected in the supernatant after toll-like receptor-7 stimulation. Gene expression differences in cells with IRF7/Irf7 risk variants are consistent with those dysregulated in SLE patients. In the present study, we demonstrate the functional consequences of a missense mutation in a critical type I interferon regulator. Understanding these mechanisms will enhance development of more effective clinical practices for autoimmune patients expressing the risk variant for IRF7.

Committee: Leah Claire Kottyan Ph.D. (Committee Chair); Brian Gebelein Ph.D. (Committee Member); Emily Miraldi Ph.D. (Committee Member); Stephen Waggoner Ph.D. (Committee Member); Matthew Weirauch Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member) Subjects: Genetics

Doctor of Philosophy, Case Western Reserve University, 2021, Molecular Medicine

Systemic Lupus Erythematosus (SLE) is an autoimmune disease that can present with in a variety of ways, often with chronic inflammation, pain, and fatigue. Unfortunately, the extreme variety of symptoms with which SLE patients present has proven to be a barrier for the successful development of targeted treatments, with only a select few therapies progressing through clinical trials. Interferon α (IFNα) is one of the main cytokines elevated in a majority of SLE patients [1]. IFNα has been also shown to be important for disease development in animal models, however how IFNα is contributing to disease pathogenesis via the direct stimulation of specific immune cells such as B cells, T cells, and myeloid cells remains largely unknown. My research shows that IFNα stimulation of B cells leads to increased B cell activation, increased germinal center B cell survival, increased populations of antibody producing cells, and auto-antibodies. This research also shows that IFNα stimulation of T cells leads to a reduction in CD8+ FoxP3+ Tcregs. Additionally, IFNα stimulation of myeloid cells was found to lead to a decrease in marginal zone macrophages and myeloid-derived suppressor cells that normally provide tolerance. Finally, my research highlights that the action of IFNα is directly tied to the ability of apoptotic pathways to signal correctly. In summary, these findings provide critical insight into the progression of SLE, in addition to identifying new potential therapeutic targets.

Committee: Trine Jorgensen PhD (Advisor); William Baldwin MD PhD (Committee Chair); Ganes Sen PhD (Committee Member); Christine O'Connor PhD (Committee Member); Howard Smith MD (Committee Member) Subjects: Animal Diseases; Health Sciences; Immunology; Molecular Biology

MS, University of Cincinnati, 2020, Medicine: Molecular, Cellular and Biochemical Pharmacology

Systemic Lupus Erythematosus (SLE) is a complex, chronic autoimmune disease involving multiple organ systems, which can range in severity from mild to life threatening commensurate with organ system involvement. Research suggests that an increase in production of pro-inflammatory cytokines, particularly, Type I Interferon (IFN-I) is a key mediator in the pathogenesis of SLE. This process is critically regulated by Interferon Regulatory Factor 7 (IRF7), a transcription factor that finely regulates transcription of interferon stimulated genes, and therefore production of IFN-I. Faulty regulation of IFN-stimulated genes by IRF7 leads to aberrant production of IFN-I, and therefore plays an essential role in the pathogenesis of SLE. This thesis highlights the importance of rs1131665, a genetic variant at position 412 within the putative inhibitory domain of the IRF7 gene, changing a glutamine (412Q, risk) to an arginine (412R, protective). This variant is statistically associated with the risk of developing SLE. A Luciferase Reporter Assay confirms increased interferon stimulated response element (ISRE) reporter activity in the risk IRF7 relative to the protective IRF7 in vitro. Enzyme-linked immunosorbent assay (ELISA) assays show genotype dependent production of all IFN-a subtypes, with increased IFN-a production demonstrated by the risk variant at 3 and 24 hour post-stimulation with resiquimod. RNA-sequencing analysis shows increased IFN-a gene expression, and interferon-responsive gene expression in the IRF7 risk variant, confirming the role of the IRF7 (412Q) risk variant in the pathogenesis of SLE. Due to the lack of curative treatment for SLE patients, this thesis highlights the importance of IRF7, and serves as a plausible foundation for future targeted therapy against IRF7, and IFN-I.

Committee: Leah Claire Kottyan Ph.D. (Committee Chair); Terry Kirley Ph.D. (Committee Member); Jo El Schultz Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2016, College of Sciences and Health Professions

Pneumonia Virus of Mice (PVM) and Respiratory Syncytial Virus (RSV) are negative sense, single-stranded, enveloped RNA viruses from Pneumovirus genus, Paramyxoviridae family. RSV is the leading cause of respiratory diseases in infants. PVM causes similar respiratory illness in mice. PVM is used as an animal model to study RSV pathogenesis because of its similarity with RSV infection. Viral infection induces type I interferon (IFN) response as an antiviral strategy. PVM and RSV both have two non-structural (NS) proteins that are known to be IFN antagonists. While RSV can target different signaling components of IFN pathway, the mechanism of IFN suppression for PVM was unknown. We have identified that PVM can also target different signaling components of IFN pathway to circumvent the host immune system. Our observations showed that PVM NS proteins facilitate proteasome-mediated degradation of RIG-I, IRF3, STAT2 in IFN pathway by direct interactions with them. Production of several Interferon Stimulated Genes (ISGs) is the distal part of the IFN pathway. We have identified that NS proteins of PVM can also target a few of them such as TRAFD1, IFITM1, ISG20, and IDO for complete suppression of the host immune system. RSV NS proteins play a similar role to suppress IFN pathway by targeting TBK1, RIG-I, IRF3, IRF7, and STAT2. Our study has identified one ISG, OASL, that has antiviral properties against RSV and documented that to counteract this antiviral property of OASL, RSV NS proteins can degrade OASL in a proteasome-dependent way. These above observations help us to delineate the complete suppression mechanism for the whole Pneumovirus genus, both for PVM and RSV by providing the first experimental evidence of signaling components from the IFN pathway targeted by PVM to suppress the IFN response. PVM is a clinically relevant animal model that will help us to find new therapeutic strategies against Pneumovirus infection. RSV study with one of those important ISGs, OA (open full item for complete abstract)

Committee: Sailen Barik Ph.D. (Advisor); Barsanjit Mazumder Ph.D. (Committee Member); Roman V Kondratov Ph.D. (Committee Member); Cornelia Bergmann Ph.D. (Committee Member); Aaron F Severson Ph.D. (Committee Member); Aimin Zhou Ph.D. (Committee Member) Subjects: Biology; Molecular Biology; Virology

Doctor of Philosophy, Case Western Reserve University, 2010, Pharmacology

Bax is a pro-apoptotic protein that mediates intrinsic cell-death signaling. Using a yeast-based functional screening approach, interferon gamma receptor beta chain (IFNγR2) and the DNA repair protein Ku70 were identified as Bax suppressors. IFNγR2 is a component of the IFNγ receptor complex along with the IFNγR alpha chain (IFNγR1). Upon IFNγ binding, a conformational change in the receptor complex occurs that activates the Jak2/STAT1 signaling cascade. The Bax binding domain is located in the C-terminal region IFNγR2296-337. This portion does not contain the Jak2-binding domain; therefore, the antiapoptotic function of IFNγR2 is independent of JAK/STAT signaling. IFNγR2296-337 rescued human cells from apoptosis induced by Bax overexpression but not Bak. IFNγR2 inhibited apoptosis induced by the BH3-only protein Bim-EL, suggesting that IFNγR2 inhibits Bax activation induced by a BH3-only protein. Bax and IFNγR2 were co-immunoprecipitated from cell lysates prepared from HEK293 and DAMI cells. Addition of recombinant Bcl-2 protein to cell lysates significantly reduced the interaction of IFNγR2 and Bax, suggesting that Bcl-2 and IFNγR2 bind a similar domain of Bax. The C-terminal fragment (cytoplasmic domain) of IFNγR2 is expressed in human cancer cell lines of megakaryocytic cancer (DAMI), breast cancer (MDA-MD-468), and prostate cancer (PC3 cells), this fragment may confer cancer cells resistance to apoptotic stresses. From the Bax binding domain of Ku70 and IFNγR2 a series of cell penetrating cytoprotective penta-peptides were developed, these peptide are known as Bax Inhibiting Peptides (BIPs), e.g. VPMLK, VPTLK, and VPALR. Another cell penetrating peptide KLPVM that does not inhibit Bax-mediated apoptosis was also developed as a control. All the five amino acids cell permeable peptides (whether they inhibit Bax or not) were named: cell penetrating penta-peptides (CPP5s). The CPP5s VPTLK and KLVPM penetrated in mammalian cells in a dose dependent manner, and did n (open full item for complete abstract)

Committee: Shigemi Matsuyma PhD (Advisor); Anthony Berdis PhD (Committee Chair); Yu-Chung Yang PhD (Committee Member); George Stark PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology; Pharmacology

Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

Viral pandemics are caused by viruses spilling over from animal reservoirs and subsequently adapting to efficiently infect, replicate, and spread in human hosts. Given that coronavirus and avian influenza virus outbreaks have occurred in recent years in geographic regions in which human deficiencies in the Interferon-Induced Transmembrane Protein 3 (IFITM3) antiviral protein are common, we investigated whether IFITM3 may play a role in interspecies virus infection and adaptation. We found that both IFITM3-deficient mice and human cells could be infected with low doses of avian influenza viruses that failed to infect WT counterparts, identifying a new role for IFITM3 in controlling the minimum infectious viral dose threshold. Additionally, we used a panel of 11 diverse avian influenza viruses and 3 swine viruses, 2 of which are known to have transmitted to humans, to infect human cells with or without IFITM3. Each animal virus showed increased infection of human cells lacking IFITM3 as compared to controls, even upon interferon treatment. Remarkably, influenza viruses passaged through Ifitm3-/- mice exhibited enhanced host adaptation, a result that was distinct from passaging in mice deficient for interferon signaling, which caused virus attenuation. Passaging of a SARS-CoV-2 beta strain and an Omicron BA.4 strain through WT or Ifitm3-/- mice both resulted in elimination of tissue culture adaptations prevalent in the parent stocks of these viruses. Further, virus passaged 20 times through Ifitm3-/- animals gained >1-log replicative advantage and ability to induce weight loss in WT mice while WT-passaged virus did not significantly change in its ability to replicate or induce weight loss. Mouse adaptation of both influenza virus and SARS-CoV-2 was associated with discrete changes in the viral genomes resulting in amino acid substitutions, suggesting that enhanced virus replication in the absence of IFITM3 may facilitate adaptive mutations. Our data demonstra (open full item for complete abstract)

Committee: Jacob Yount PhD (Advisor); Shan-Lu Liu MD, PhD (Committee Member); Adriana Forero PhD (Committee Member); Andrew Bowman DVM, PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology; Immunology; Microbiology; Virology

Doctor of Philosophy, Case Western Reserve University, 2024, Epidemiology and Biostatistics

Tuberculosis (TB) continues to be a major public health problem globally. In 2022, TB was responsible for over 1.3 million deaths. One of the World Health Organization's (WHO) End TB Strategy goals is to reduce the incidence and mortality of TB by identifying those who are at the highest risk of developing TB and promptly treating them. Recently, the focus of research has turned to converters or individuals who have acquired Mtb within the past two years (acute Mtb infection). These converters have the highest risk for disease progression and being able to identify them before they develop TB and become contagious would be a crucial step in achieving the elimination of this disease. The only diagnostic tools available to identify converters, the tuberculin skin test (TST) and interferon gamma release assay (IGRA), have several limitations. The objective of this project is to identify factors associated with conversion of both the TST and/or IGRA, examine whether quantitative TST and/or IGRA measures further clarify differences, and to evaluate whether these factors are also associated with progression to incident TB. This project is one of the few in the world that collects both IGRA and TST on household contacts of TB cases, providing a unique opportunity to examine both diagnostic tests together. This question is critical as many TB research studies globally are transitioning to the exclusive use of IGRA. A clear definition of conversion would be necessary to obtain more meaningful results from research that targets those individuals at higher risk for progression to TB. It would also be an important step in improving the effectiveness of preventive therapy.

Committee: Catherine Stein (Advisor); W. Henry Boom (Committee Member); Scott Williams (Committee Member); Daniel Tisch (Committee Chair) Subjects: Epidemiology

PhD, University of Cincinnati, 2024, Medicine: Immunology

Natural killer (NK) cells are a subset of innate lymphocytes characterized by their ability to eliminate infected or non-self cells. In addition, NK cells play a noncanonical role in regulation of adaptive immune responses. After certain viral infections and vaccinations, NK cells kill a subset of activated T cells in a perforin-dependent manner. The clearance of these T cells early in the immune response negatively affects downstream antiviral immunity. Ergo, NK cell suppression of adaptive immunity dictates the outcome of viral infections and efficacy of vaccinations. However, the mechanisms driving immunoregulatory activity of NK cells remain incompletely defined. The established requirement for perforin in NK cell immunoregulation suggests the necessity for cell-to-cell contact between NK cells and their targets. At homeostasis, NK cells and T cells localize in different tissue compartments. Chapter II explores re-localization of NK cells to T-cell rich sites after infection, a phenomenon shown to be dependent on the dose of virus given. This is shown to be associated with reduced induction of type I interferons during the lower doses of virus, which limits upregulation of the necessary chemokine ligands involved in NK cell enrichment within T cell rich regions of lymphoid tissues. Localization of NK cells and subsequent regulation of T cells is dictated by the pattern of type I interferon expression. However, pleiotropic effects of interferon on NK cells, T cells, and other immune cells precluded clear dissection of the precise dose-dependent governance of NK-cell immunoregulatory capacity. Cell transfer experiments complement current strategies to study NK cell immunoregulation but are predominantly used in anti-tumor models or in mice with incomplete immune systems. The conventional need for proliferative expansion of NK cells in a less competitive, immunodeficient environment when performing adoptive transfer studies precludes use of such models to e (open full item for complete abstract)

Committee: Stephen Waggoner Ph.D. (Committee Chair); David Hildeman Ph.D. (Committee Member); Grant Schulert M.D. Ph.D. (Committee Member); Leah Claire Kottyan Ph.D. (Committee Member); Koichi Araki D.V.M. Ph.D. (Committee Member) Subjects: Immunology

Master of Science (MS), Bowling Green State University, 2024, Biological Sciences

Tripartite motif (TRIM) proteins are a vast family of ubiquitin ligase proteins with a prominent role in innate immunity, with specific members possessing correlations to the production of Interferons (IFNs). The IFNs comprise a family of cellular signaling proteins involved in cellular defense against a viral infection. TRIM protein expression is correlated with IFN production and antiviral activity via IFN activated gene expression. In turn, IFN activation is associated with retroviral infection of the host due to sensing of viral nucleic acids or proteins. A notable example of retroviral induced antiviral responses comes from that of TRIM5α in the rhesus macaque. In the macaque, TRIM5α is known to interfere with the retroviral replication cycle of HIV-1 by inducing the premature dismantling of the viral capsid, thus blocking the virus from integrating into the genome of the host. Due to this capability, TRIM5α has been heavily studied with the hopes of applications towards combating retroviral infections in humans. Surprisingly, the canine lacks a functional TRIM5α due to disruption of the gene via a pseudogene (PNRC1) insertion. Due to the loss of TRIM5α, the canine model has been neglected regarding TRIM protein research. The disruption of TRIM5 in the canine model suggests that the function of antiretroviral activity is compensated by processes encoded by other TRIM proteins, such as the TRIM5 orthologues TRIM21 and TRIM68. For example, evidence of episodic selective pressures in primates depicts the relationship of TRIM5α and another ortholog TRIM22 for a positive selection of TRIM5α specifically over TRIM22. While others in the Halo lab have begun to explore TRIMs, TRIM21 and TRIM68 have not yet been explored. In humans and primates, TRIM21 is known to elicit immune stimulation properties and antiviral functions, while also possessing structural similarities to TRIM5α. This prompts the investigation of TRIM21 and TRI (open full item for complete abstract)

Committee: Julia Halo Ph.D. (Committee Chair); Raymond Larsen Ph.D. (Committee Member); Daniel Pavuk Ph.D. (Committee Member) Subjects: Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2023, Comparative and Veterinary Medicine

5' methylcytosine (m5C) refers to the addition of a methyl group onto the fifth carbon of the cytosine ring. Among more than 180 types of RNA modifications, m5C is one of the most prevalent modifications and is widely distributed in many RNA species. It plays important roles in RNA metabolism, nuclear export, and translation. The m5C methylation is catalyzed by m5C methyltransferase (MTases) including seven members of the NOL1/NOP2/SUN domain (NSUN) family (NSUN1-7) and DNA methyltransferase-like 2 (DNMT2). Currently, the potential role of these m5C MTases and RNA m5C methylation in innate immunity and virus infection remains poorly understood. To begin to understand the role of m5C MTase in virus infection, we depleted the NSUN2 from A549 cells and examined its impact on virus replication. We found that depletion of NSUN2 significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses including human respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV), human metapneumovirus (hMPV), and Sendai virus (SeV), and herpes simplex virus (HSV). Importantly, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN-I) response upon NSUN2 depletion. To understand the mechanism by which NSUN2 modulates IFN-I response, we recapitulated the IFN-I signaling pathway and found that cytosolic RNA sensor RIG-I but not MDA5 is involved in IFN-I activation. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant non-coding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially upregulated, leading to an increased level in unshielded 7SL RNA in cytoplasm, which served as direct ligands for the RIG-I mediated IFN response. In NSUN2 de (open full item for complete abstract)

Committee: Jianrong Li Dr. (Advisor) Subjects: Immunology; Virology

Doctor of Philosophy, University of Toledo, 2023, Biology (Cell-Molecular Biology)

Degrading cellular and viral RNAs required for viral replication is an evolutionarily conserved antiviral mechanism. Host response to a viral infection includes the production of type I interferon (IFN) and the induction of interferon-stimulated genes that have broad antiviral effects. One of the key antiviral effectors is the IFN-inducible oligoadenylate synthetase/ribonuclease L (OAS/RNase L) pathway, which is activated by double-stranded RNA to synthesize unique oligoadenylates, 2-5A, to activate RNase L. RNase L exerts an antiviral effect by cleaving diverse RNA substrates, limiting viral replication; many viruses have evolved mechanisms to counteract the OAS/RNase L pathway. RNase L activates different signaling pathways including: IFN production, inflammasome activation, inhibition of protein synthesis, autophagy, and apoptosis, to mount an antiviral response. My work has focused on the signaling role of RNase L-cleaved small RNAs and the impact of regulating RNase L activity on autophagy and apoptosis during viral infections. Host responses to viral infections, involve overlapping signaling pathways that regulate autophagy and apoptosis. How the crosstalk between these two evolutionarily conserved processes is integrated to determine the fate of a cell remains unclear and represents a critical gap in our understanding of host response to viral infections and its iv resolution. Ribonuclease L (RNase L) is a regulated endoribonuclease that is activated during viral infections and cleaves cellular including rRNAs in ribosomes and viral single-stranded RNAs, to generate small double-stranded RNAs. Double-stranded RNAs serve as pathogen associated molecular patterns (PAMPs) to stimulate interferon (IFN) production. The cytosolic Rig-like helicases (RLHs) and endosomal Toll- like receptors (TLRs), along with dsRNA-dependent protein kinase (PKR) and 2'-5'oligoadenylate synthetase (OAS) serve as dsRNA sensors in cells. Activation of RNase L induces autophagy via s (open full item for complete abstract)

Committee: Malathi Krishnamurthy (Committee Chair) Subjects: Biology; Immunology

Doctor of Philosophy, Miami University, 2022, Microbiology

The signaling lymphocyte activation molecule family (SLAMF) of receptors is crucial to the proper development and effector function of both lymphoid and myeloid cells. In this work, SLAMF9 expression across various lymphoid and non-lymphoid tissues was assessed to identify SLAMF9+ immune cell subsets, revealing a common CD11b+ CD11c+ MHC-IIhi phenotype in hepatic, renal, and cardiac mononuclear phagocytic cell (MPC) populations. In addition, it was determined that SLAMF9 expression is inducible in CD11b+ CD11c+ Ly6c+ bone marrow cells and CD11bhi CD11cint Ly6c- splenic cells specifically following Toll-like receptor (TLR) 9 signaling in vitro. Experiments were also carried out to determine the effect of SLAMF9 on the regulation of pro-inflammatory and type-I interferon (IFN)-mediated immune responses. Murine SLAMF9 appears to promote the production of pro-inflammatory cytokine tumor necrosis factor α (TNFα) in response to in vitro stimulation of TLR4 with lipopolysaccharide (LPS), mirroring the function of human SLAMF9 in immortal monocytoid THP-1 cells. However, no effect of SLAMF9 on the production of type-I IFNs or signaling through the type-I IFN receptor (IFNAR) in response to TLR9 signaling, or the type-I IFN-mediated clearance of murine cytomegalovirus (MCMV) infection, was detected. The possibility that SLAMF9 has a conserved function in the promotion of pro-inflammatory effector protein production across humans and mice necessitated inspection of the changes which have taken place across evolutionary time at the SLAMF9-encoding genes of mammalian species. Surprisingly, SLAMF9 in humans, Neanderthals, and Denisovans encodes unique N-terminal mutations not observed in the SLAMF9-encoding genes of chimpanzees, mice, or any other mammal examined. These N-terminal mutations lead to poor expression of human SLAMF9 in transfected immortal human cells and may contribute to instability of the protein, leading to questions about whether SLAMF9 is normally expressed i (open full item for complete abstract)

Committee: Dr. Timothy Wilson (Advisor); Dr. Luis Actis (Committee Member); Dr. Susan Hoffman (Committee Member); Dr. Joseph Carlin (Committee Member); Dr. Eileen Bridge (Committee Member) Subjects: Immunology; Microbiology; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2021, Systems Biology and Bioinformatics

Strategies to find an effective TB vaccine have relied on enhancing the natural immunity that occurs in a subset of the population in response to primary exposure or reinfection with Mycobacterium tuberculosis (Mtb). Although still controversial, there is good evidence that some people exposed to Mtb show early clearance of the infection and do not exhibit a specific T-cell response that is the hallmark of Mtb infection. At the same time, the majority of people who do become infected, never progress to an active TB state and remain healthy without symptoms for many years. New vaccine candidates will require better targeting of these natural mechanisms through increased knowledge of immune functions. The biological processes contributing to early clearance of infection can be investigated by comparing individuals who resist infection to those with stable latent infection. We compared lung and blood cell subsets in a Ugandan cohort and found subtle differences that may contribute to resistance. In addition, we were able to quantify lymphocyte memory cell populations in the lung and blood and found a greater percentage of effector memory cells in the lung. Recent vaccine candidates have induced a robust T-cell response to Mtb antigens as measured in the blood, and yet provide no significant protection against progression from infection to disease. One hypothesis is that measurement of vaccine biomarkers in the blood do not correlate with markers in the lung where infection occurs. In this study, proteomic profiles in alveolar macrophages and blood monocytes were measured to answer the question of whether there is a differential IFN-γ cytokine response in these compartments. We found greater overall protein expression in monocytes that was not explained by differences in proximal signal transduction. In order to further interrogate IFN-γ transcriptional processes, we performed a meta-analysis with MDM transcriptomic datasets and combined the results with STAT1 tra (open full item for complete abstract)

Committee: William Henry Boom MD (Committee Member); Catherine Stein PhD (Advisor); Gurken Bebek PhD (Committee Member); Richard Silver MD (Committee Member); Mark Cameron PhD (Committee Chair) Subjects: Bioinformatics; Biology; Biomedical Research; Biostatistics; Epidemiology; Immunology

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

Influenza virus is a leading cause of human mortality globally and presents a persistent pandemic threat. In addition to lung pathology, severe infections may be accompanied by extrapulmonary complications, including cardiac pathology. Influenza virus is recognized as a cardiotropic virus and an etiologic agent of viral myocarditis; thus, the virus can cause cardiac pathology in the absence of pre-existing cardiovascular disease. However, the underlying mechanisms of influenza-associated cardiac pathology are poorly understood. Influenza virus infections of extrapulmonary tissues have been reported for various mouse models of infection, but relevant models in which significant cardiac pathogenesis occurs and the animal's susceptibility is relevant to human infections are lacking. Single nucleotide polymorphisms in the IFITM3 gene are among the only genetic defects that have been reproducibly associated with severe influenza in humans. Given that numerous studies have linked IFITM3 defects to severe influenza virus infections, and that severe influenza virus infections may include cardiac complications, we sought to investigate whether IFITM3 plays a role in protecting the heart during infection. We showed that IFITM3 deficiency in a knockout mouse model exacerbated weight loss and mortality following influenza virus infections, and observed increased replication of virus in the lungs and hearts of IFITM3 KO mice. Infected IFITM3 KO mice developed aberrant cardiac electrical activity, which was accompanied by fibrosis of heart tissue, prior to succumbing to infection. These findings revealed an essential role for IFITM3 in limiting influenza virus replication and pathogenesis in heart tissue and established IFITM3 KO mice as a powerful model for studying influenza virus-induced cardiac dysfunction. We next sought to explore whether direct infection of heart tissue by influenza virus contributed to the observed cardiac pathologies following infection. We genera (open full item for complete abstract)

Committee: Jacob Yount PhD (Advisor); Emily Hemann PhD (Committee Member); Peter Mohler PhD (Committee Member); Brandon Biesiadecki PhD (Committee Member); Shan-Lu Liu MD, PhD (Committee Member) Subjects: Biomedical Research; Immunology; Virology

Doctor of Philosophy, Case Western Reserve University, 2021, Pathology

Multifactorial diseases such as diabetes, allergy and asthma, inflammatory bowel disease and neurodegenerative disorders have increased in recent years. Genetic predisposition is unable to fully account for this sudden increase, yet our environment is a factor that has seem similar dramatic changes even in the last 100 years. It has since become apparent that the gene pool of endogenous microbes that populate our bodies might be more susceptible to these changes due to their shorter life span and generation turnover. Thus, examination of the microbiome has become a major focus in the hopes of identifying underlying changes in the microbiome which may give rise to or be compounded due to disease. Study of these interactions have revealed the involvement of the microbiome in many functions ranging from processing of nutrients, neuronal and immune system development. The host immune interactions with gut commensal microbes have demonstrated their importance in establishing tolerance and maintenance of homeostasis. Here, we utilized the capsular polysaccharide PSA produced by the gut commensal Bacteroides fragilis to better understand the impact on the host immune system locally and systemically. Prior work established that PSA is endocytosed, processed and presented by APCs to CD4+ T cells via MHCII molecules. This exposure lead to the expansion of a population of C45Rblow effector memory (Tem) cells capable of protecting from induction of inflammation. This protection was shown to be IL-10 dependent, the source of which was endogenous Tregs and not the PSA expanded cells. Through in vitro co-culture experiments with regulatory T cells, we demonstrated a novel T cell communication axis by which Tem cells secrete IL-2 and IL-4 to synergistically stimulate IL-10 production by Tregs. Furthermore, we demonstrated that PSA exposure results in potent interferon response, which results in the upregulation of immune-regulatory markers. These markers were primarily found (open full item for complete abstract)

Committee: Brian Cobb PhD (Advisor); Booki Min DVM, PhD (Committee Chair); Kristie Ross MD (Committee Member); Pushpa Pandiyan PhD (Committee Member); Clive Hamlin PhD (Committee Member) Subjects: Immunology

Doctor of Philosophy, University of Toledo, 2020, Biology (Cell-Molecular Biology)

Eukaryotes have evolved intricate responses to combat environmental stresses, particularly viral infections, including the innate immune response and the integrated stress response (ISR). The innate immune response is activated in response to viral infection through recognition of pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). The activation of the innate immune response, specifically the type I interferon (IFN) response, results in the synthesis and secretion of type I IFN protein which acts in a paracrine and autocrine fashion to induce the upregulation of interferon stimulated genes (ISGs). These ISGs impact a variety of cellular functions to put the cell into an antiviral state and protect from future infection. The integrated stress response aims to restore cell homeostasis through phosphorylation of the eukaryotic initiation factor 2α (eIF2α), which leads to a decrease in global protein synthesis. Activation of the ISR and the subsequent phosphorylation of eIF2α can result in the formation of cytoplasmic foci of translationally-stalled mRNAs and proteins called stress granules. Viral hemorrhagic septicemia virus (VHSV) is one of the deadliest infectious fish pathogens posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. VHSV isolates are classified into individual genotypes (I-IV) based on the geographic location and genomic similarities between the glycoprotein and nucleoprotein genes. Two important strains include VHSV Ia, which is endemic to Europe and is responsible for outbreaks in freshwater Rainbow trout farms, and VHSV IVb, which has been the cause of massive fish die-offs in the Great Lakes. Previous work showed that VHSV IVb suppresses host innate immune responses, but the exact mechanism by which this inhibition occurs remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is im (open full item for complete abstract)

Committee: Malathi Krishnamurthy (Committee Chair); Douglas Leaman (Committee Member); Travis Taylor (Committee Member); Scott Leisner (Committee Member); Saurabh Chattopadhyay (Committee Member) Subjects: Biology

Doctor of Philosophy, University of Toledo, 2020, Biology (Cell-Molecular Biology)

Virus infection leads to activation of the interferon (IFN)-induced endoribonuclease RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRRs), like retinoic acid-inducible I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), to further amplify IFN production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how cells coordinate RNA sensing with signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce the formation of antiviral stress granules (avSGs) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) and recruit the antiviral proteins RIG-I, PKR, OAS, and RNase L to avSGs. Biochemical analysis of purified avSGs showed interaction of a key stress granule protein, G3BP1, with only PKR and RIG-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production, but not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection, demonstrating the importance of avSGs in RNase L-mediated host defense. We propose a role during viral infection for RNase L-cleaved RNAs in inducing avSGs containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to mount an effective antiviral response.

Committee: Malathi Krishnamurthy (Committee Chair); Travis Taylor (Committee Member); Saurabh Chattopadhyay (Committee Member); Scott Leisner (Committee Member); Deborah Chadee (Committee Member) Subjects: Biology; Virology

Doctor of Philosophy, University of Akron, 2020, Chemistry

The receptors embedded in cell membranes are essential for signaling in response to various stimuli or communicating with neighboring cells. In order to target these membrane proteins for drug discovery, it is essential to understand their interactions with each other in the live cell. Techniques such as structural studies, interactions in solution, and live cell fluorescence have all aided in determining the details of protein-protein complex formation. Here fluorescence cross-correlation spectroscopy (FCCS) is used to elucidate these events in live cells. This method allows analysis of fluorescent protein intensity fluctuations to gain insight on protein diffusion and oligomerization in the 2D cell membrane. The research presented here aimed to determine homotypic and heterotypic interactions for the plexin/neuropilin/semaphorin family of proteins. These membrane proteins are involved with tissue patterning in the developing embryo and play various roles in disease. While truncated, soluble domains have been studied in some capacity, the full-length receptor complexes have not been completely explored and FCCS allows insight to their network of interactions. Chapter I and II give an introduction to these receptors and an overview of the FCCS methods used. Chapters III and IV focus on the degree of interaction between Neuropilin-1 (Nrp1), Plexin A2, Plexin A4, and Plexin D1 when stimulated by Semaphorin 3A or Semaphorin 3C. These combinations have been implicated in various cancers and this work resolves interactions which may aid in the design of new therapeutic strategies. Chapter V looks at the oligomerization of the transmembrane domains of Nrp1, Plexin B1, Plexin B2, and Plexin D1 using FCCS and computational predictions. Most experiments agreed with the simulations, showing individual motifs which enhanced or disrupted helix interactions, while also revealing that competition in the live cell may prevent some of the interactions predicted in isolation. Chapte (open full item for complete abstract)

Committee: Adam Smith (Advisor); Leah Shriver (Committee Member); Michael Konopka (Committee Member); Sailaja Paruchuri (Committee Member); Nic Leipzig (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry

Master of Science, Miami University, 2020, Microbiology

SLAMF9 is a cell surface protein expressed on the surface of a wide variety of cells. It is a peculiar but compelling molecule due to its lack of a known receptor, ligand, or signaling adapter. While there is little known about this protein, there is evidence suggesting SLAMF9 enhances clearance of bacterial pathogens but makes the host more susceptible to viral pathogens. This dual phenotype is thought to be the result of inflammatory cytokine crosstalk. Stimulation of SLAMF9 Knockdown and Control THP-1 cells with lipopolysaccharide (LPS) mimicking an early response to infection resulted in higher proinflammatory cytokine production in cells with normal SLAMF9 expression. A proteomic analysis was performed to determine which proteins, other than cytokines, were differentially expressed between the SLAMF9 Knockdown and Control THP-1 cells. It was determined that the expression of SLAMF9 influences several components of Ras signaling, which is a complex network regulating proliferation, survival signals, and cytokine transcription. A combination of differential proteomics and a phosphosite analysis indicated other proteins of interest to pursue in future research. Determining new regulatory mechanisms may give rise to new potential drug targets to resolve inflammatory disease.

Committee: Timothy Wilson PhD (Advisor); Eileen Bridge PhD (Committee Chair); Xin Wang PhD (Committee Member) Subjects: Immunology; Microbiology

Doctor of Philosophy, The Ohio State University, 2020, Comparative and Veterinary Medicine

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is the first demonstrated human deoxynucleoside triphosphohydrolase (dNTPase) that reduces intracellular deoxynucleoside triphosphate (dNTP) levels. Homozygous mutations of SAMHD1 can cause autoimmune diseases, such as Aicardi-Goutieres syndrome (AGS). These patients have increased levels of type I interferon (IFN-I) or interferon-induced gene expression, suggesting that SAMHD1 is a negative regulator of innate immunity. IFN-I induction is also reported in SAMHD1 knockout (KO) mice, which further indicates the negatively regulatory function of SAMHD1 in interferon responses. SAMHD1 is also demonstrated to act at the replication fork to restart DNA replication, resulting in the prevention of IFN-I induction. However, the exact function and role of SAMHD1 in regulating the innate immune responses to viral infections and inflammatory stimuli remain unknown. Here, we report that SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by interacting with key proteins in both NF-kB and IFN-I pathways such as interferon regulatory factor 7 (IRF7); we also identify that the HD domain of SAMHD1 is required for its suppression of IFN-I induction, and that the dNTPase activity but not nuclear localization of SAMHD1 is important for its inhibition of innate immunity in differentiated monocytes. To investigate the role of SAMHD1 in regulating innate immune responses, we conducted siRNA-mediated SAMHD1 knockdown (KD) in human primary monocyte-derived macrophages (MDM), and infected the cells with Sendai virus (SeV). TNF-a, IL-6, IFN-a and IFN-b mRNA levels were quantified, and we found that SAMHD1 KD increased TNF-a, IL-6, IFN-a and IFN-b mRNA levels after SeV infection in MDMs, suggesting that SAMHD1 suppresses NF-kB activation and IFN-I induction to viral infection. Moreover, we demonstrated that in HEK293T cells the suppression of IFN-I induction by SAMHD1 is specific to IRF7 but (open full item for complete abstract)

Committee: Li Wu (Advisor); Patrick Green (Advisor) Subjects: Immunology; Molecular Biology; Virology