Doctor of Philosophy, Miami University, 2024, Chemistry and Biochemistry

Pancreatic cancer remains a devastating disease with exceptionally poor prognoses due to inability to detect the disease when it is still surgically resectable and due to largely ineffective treatment modalities. Here, three engineered adenovirus vectors were developed to specifically target and suppress oncogenic activity of the HMGA1 architectural transcription factor that plays a crucial role in tumorigenesis, chemotherapy resistance and transformation of pancreatic cancer cells into pancreatic cancer stem cells. HMGA1 plays a similarly crucial role in many human cancers, including breast cancer. HMGA1 is an especially insidious oncoprotein because it is intrinsically disordered and therefore cannot be targeted by conventional small molecule drug therapy. Here, alternative adenoviral-mediated therapy approaches were designed and developed to suppress HMGA1 oncogenic activity in cancer cells and tested in MIA PaCa-2, PANC-1 and BxPC-3 human pancreatic cancer cell lines and in the ZR-75 breast cancer cell line. The engineered viruses characterized included: 1) a virus engineered to sequester overexpressed HMGA1 in cancer cells, 2) a virus engineered to express an artificial HMGA1 cis-antisense transcript and 3) a virus engineered to express an HMGA1-targeted shRNA transcript, the latter two viruses designed to suppress translation of HMGA1 mRNA into HMGA1 protein. Cancer cell characteristics after viral infection were evaluated using viability, toxicity, proliferation and wound healing assays and effects on HMGA1 mRNA transcript levels and HMGA1 protein levels were quantitatively evaluated. It was found that the virus designed to sequester HMGA1 proved more effective in suppressing HMGA1 oncogenic activity than the viruses designed to suppress HMGA1 translation.

Doctor of Philosophy, The Ohio State University, 2024, Microbiology

Respiratory pathogens entail many bacteria and viruses that cause upper or lower respiratory tract diseases. The severity and transmissibility of respiratory pathogens vary widely, with some agents able to cause large outbreaks of severe diseases and even pandemics. Two examples of such pathogens that have recently gained global attention are SARS-CoV2, a newly emerging zoonotic coronavirus that caused the COVID-19 pandemic, and Bordetella pertussis, the causative agent of whooping cough, recently designated as an emerging pathogen by the National Institute of Allergy and Infectious Diseases (NIAID). Vaccines have been one of the most important measures to counteract respiratory pathogens. Multiple elements are involved in vaccine design to maximize the benefits and minimize the potential side effects. That includes the use of adjuvants that induce a strong favorable immune response, selecting the right antigens that are likely to be protective and also selecting the route of immunization that would induce immune response at the mucosal entry sites. I addressed all this strategies in my thesis. The adjuvant Bordetella colonization factor A (BcfA) is an outer membrane protein derived from Bordetella bronchiseptica. Previously, in our lab, we combined BcfA and Alum in a subunit vaccine formula against B. pertussis, and the vaccine led to better protection and a TH1/TH17 polarized immune profile. However, the molecular mechanisms underlying this phenotype still need to be elucidated. In the first part, I investigated the mechanism of action of BcfA. Initial pattern recognition receptors (PRR) screening identified TLR2 and TLR4 as potential BcfA receptors. Ex vivo analysis using murine bone marrow-derived dendritic cells (BMDCs) further confirmed that finding as it showed the ability of BcfA to induce the expression of costimulatory molecules and the secretion of innate cytokines, it also indicated a greater dependence of B (open full item for complete abstract)

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2024, Biological Sciences

West Nile virus (WNV) is an arbo-Orthoflavivirus belonging to the Japanese Encephalitis Complex that was first discovered in a febrile woman of the Uganda district in Africa. Following its initial discovery WNV has been detected on every major continent, except Antarctica, officially being detected in the Western hemisphere in the fall of 1999 in the Queens District of New York City. Since entering the western hemisphere 25 years ago, WNV has been reported in over 300 different avian species. In addition to avian cases, WNV has been detected in humans within all 50 states totaling 58,981 cases (both neuroinvasive and non-neuroinvasive) and 2,776 deaths. An RT-PCR protocol was developed and optimized to reproducibly generate cDNA for sequence analysis. Forty cDNA sequences were assembled from RT-PCR products of WNV origin, and the corresponding amino acid sequences predicted. Sequence variations at the nucleotide level were evident as 173 polymorphic sites; when translated these polymorphisms resulted in 18 polymorphic amino acid residues relative to the original New York reference strain. There were 42 total (36 different sites) instances where nucleotides present were ambiguous, suggesting that some of the samples analyzed contained material from more than one virion. Predicted polypeptide sequences indicated high conservation of specific envelop protein regions. These conserved regions were concentrated in envelope domain II, a region recognized to be essential for productive infection of host cells. My results suggest the number of WNV genomes recovered for analysis varied relative to local weather conditions during the 2018 collection season. Together these observations support the contention that environmental conditions have the potential to select variant genotypes of WNV.

Doctor of Philosophy, The Ohio State University, 2024, Microbiology

Microbes are engines of ocean biogeochemical processes. Viruses influence and shape microbial communities via lysis, horizontal gene transfer, and metabolic reprogramming. Viral lysis facilitates the export of carbon from the surface into the deep ocean via aggregates of sinking particles. In fact, they outperform prokaryotes and eukaryotes as the strong predictor for carbon fluxes in the oligotrophic ocean. Viruses also impact the gene flow of their hosts, and the genes transferred from virus-host interactions can be fixed in viral genomes. Viruses are known to carry and express host-derived auxiliary metabolic genes (AMGs) that directly reprogram metabolisms within virus-infected cells, termed virocells. However, viral communities are poorly characterized in the oligotrophic ocean, and their AMG-driven metabolic reprogramming lacks systematic descriptions from the global oceans. The Sargasso Sea is highly stratified and nutrient-depleted each year in the summer months. This seasonal pattern makes the Sargasso Sea one of the ideal model ecosystems to study oligotrophic oceans. In the Sargasso Sea, abundance of viral-like particles has seasonal and depth-associated structuring patterns. Here, to better survey the Sargasso Sea viruses, we apply sequencing approaches to characterize viral communities via metagenomics and uncover their biogeographical and ecological structures locally and globally in the ocean. As described in Chapter 2, comparison with global viral metagenomics revealed that Sargasso Sea viruses were similar across warm oligotrophic oceanic regions but not represented globally. They form discrete populations in the viral and cellular fractions at the viral maximum (80m) and mesopelagic (200m) depths. Inclusion of long-read data captured 1,257 viral genomes in addition to the 1,044 viral genomes derived from short-read assemblies, resulting in the identification of ecologically important and microdiverse viral genomes. Having established lo (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Veterinary Preventive Medicine

Viral hepatitis is primarily caused by five unrelated hepatotropic viruses, hepatitis A, B, C, D, and E. While hepatitis A through C are commonly recognized as causing significant liver disease by general public due to successful public health campaigns, most are not aware of hepatitis E. Despite being unheard of in the public, hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide with an estimated 20 million cases annually. Hepatotropic viruses are notoriously tricky, utilizing differing mechanisms to avoid detection and elimination by the host organism. While hepatitis B and C infections often produce few symptoms in the host while becoming chronic and spreading silently to new hosts, HEV utilizes a different strategy to continue circulating in its hosts. HEV's long incubation period and ability to self-resolve in many infected individuals coupled with animal reservoirs that show little disease upon infection allow HEV to transmit to humans through the food chain. Endemic human strains have similar strategies, circulating at low levels within the populace waiting for conditions associated with socio economic turmoil when sanitary conditions decrease allowing for massive outbreaks through contaminated water. This virological game of hide and seek ensures the continued survivability and transmission of the pathogen. While most otherwise healthy individuals will be able to self-resolve HEV infections, people with underlying comorbidities, immunocompromised individuals, and pregnant women in their third trimester are at much greater risk of succumbing to hepatitis E. There is still much work to be done to unravel the nuances of HEV's deadly hide-and-seek game so that humans may rid themselves of this malady. Recently, the new emerging zoonotic rat HEV has been a hot topic in the field of HEV. The first rodent-associated hepevirus was discovered in 2010 from fecal and liver specimens of rats in Germany. Based on sequence divergence, this (open full item for complete abstract)

Master of Science, The Ohio State University, 2024, Immunology and Microbial Pathogenesis

Influenza B virus circulates seasonally in humans and primarily affects young children, the elderly, and the immunocompromised with 290,000-650,000 influenza B related deaths annually. The cellular protein interferon induced transmembrane protein 3 (IFITM3) is known to restrict several virus infections by altering endosomal membrane properties in a manner that prevents virus-to-cell membrane fusion reactions. IFITM3 is particularly beneficial in limiting the severity of influenza A virus infection in humans and mice, though whether it inhibits influenza B virus infections of cells or pathogenesis in vivo is not well studied. We thus sought to investigate the role of IFITM3 in influenza B virus infections in human cells and using the mouse model. From my work using influenza B virus in vitro with IFITM3 deficient and IFITM3 overexpressing cell models, and in vivo with wild type and IFITM3 knockout mice, we conclude that IFITM3 restricts influenza B virus infections of cells and pathogenesis in vivo.

Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Medicine

Understanding the viral infection and lifecycle of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) and Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is important in improving disease outcomes and reducing viral prevalence. In our SFTSV study, we discovered specific cytokine profiles associated with the severity of clinical symptoms. We used single cell RNA sequencing (scRNAseq) on patient blood samples to identify a unique expansion of the B cell population in SFTSV-induced fatal cases which indicated that plasma B cells are a primary reservoir of SFSTV replication. These findings present a potential method of reducing the severity of SFTSV infection, especially in aged patients who are more susceptible to adverse outcomes. In our KSHV study, we developed a novel oral 3D infection model and demonstrated that KSHV can only infect exposed basal epithelial cells in the oral epithelia. We used scRNAseq to show that keratinocyte differentiation and cell death pathways were affected by KSHV infection, suggesting that epithelial differentiation could contribute to KSHV reactivation through changes in epigenetic regulation. In addition, we found a unique population of infected cells with limited early lytic gene expression and a unique gene expression profile, which we termed latent-2 cells. These findings demonstrate that our in vitro 3D epithelial ALI culture model should be a valuable tool to further understand oral KSHV infection for the development of future anti-viral therapeutics to block KSHV transmission.

Master of Science, The Ohio State University, 2024, Animal Sciences

Bovine coronaviruses (BCoVs) are important members of the Betacoronavirus genus, Embecovirus subgenus in Orthocoronavirinae subfamily within the Coronaviridae family. They can cause enteric or/and respiratory diseases in cattle. However, the mechanisms for BCoV tissue tropism and pathogenesis are still unknown and could be due to interactions of viral, host, and environmental factors. There are limited studies to investigate whether co-infection with other bovine pathogens, environmental factors (such as season of the year) and host factors (such as beef vs dairy; age, such as pre- vs post-weaning; and the level of pre-existing BCoV antibodies) contribute to enteric or respiratory tropism. In our study, we isolated recent BCoV strains and compared them with historical strains to study the evolution of BCoVs. Bovine fecal samples were collected from dairy calves, dairy cows, and beef cattle in Georgia by Dr. Palomares Lab at the University of Georgia. Bovine fecal and nasal samples from asymptomatic veal calves from an Ohio farm were provided by Dr. Habing's Lab at The Ohio State University. We detected BCoVs from both nasal (n=10) and fecal samples (n=156) and isolated BCoV strains from enteric samples. The BCoV genomic sequences of five strains BC7, BC8, BC9, BC39, and BC47 were characterized, and they all belong to genogroup II by phylogenetic analyses. Sequence analyses were performed to compare the mutations between the historical and current viruses and between the viruses isolated from respiratory and enteric samples. We found that one pair of samples BC8 and BC18 from the same calf, but different collection sites (fecal and nasal sites), showed two amino acid differences in the spike (S) protein; We predicted the locations of these two amino acid residues in the 3D protein structures. Reverse genetics is a state-of-the-art technology to study viral factors, but until now no infectious clones have been generated for BCoVs, and this has hampered in-dept (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Medicine

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus causing hemorrhagic fever with mortality rate ranging up to 30%. However, licensed vaccine or targeted therapy against SFTSV is yet to be developed. In this study, we developed a protein subunit vaccine employing 24-mer self-assembling ferritin (FT) nanoparticles as a platform to present the head region of the SFTSV Gn (GnH). The purified GnH-FT nanoparticles preserved structural integrity and induced robust humoral and cellular immunity against SFTSV Gn. Furthermore, aged ferrets that received immunization with GnH-FT nanoparticles exhibited complete protection from lethal SFTSV challenge and symptoms of body weight loss, viremia, fever, thrombocytopenia, leukopenia, and mortality. We also developed mRNA vaccines encoding GnH and GnH-FT, which potently induce immunity against SFTSV and provides full protection against lethal SFTSV infection in mouse model. Furthermore, we repurposed the GnH-FT nanoparticle to develop therapeutic antibody against SFTSV. We identified five unique antibodies specifically binding SFTSV, including two candidates with sub-nanomolar KD. Our study suggests two vaccine candidates and potential therapeutic antibodies against SFTSV.

PhD, University of Cincinnati, 2023, Medicine: Molecular Genetics, Biochemistry, & Microbiology

The human immunodeficiency virus (HIV) is the viral pathogen underlying the ongoing AIDS pandemic. Current estimates place the number of infected individuals worldwide at nearly 40 million people, and the number of infected is still growing every year. Although modern antiretroviral therapy (ART) regimens are effective at halting the progression of disease, there is still no cure or vaccine available. The only viral antigen present on the surface of particles is the HIV Envelope glycoprotein (Env), and its expression on the surface is tightly regulated. Surprisingly, Env is endocytosed rapidly upon reaching the plasma membrane (PM), requiring recycling pathways in order to return to the PM site of particle assembly. Understanding how Env navigates the endosomal recycling compartment (ERC) could lead to development of novel therapeutics and improved vaccine design. The mechanisms underlying Env trafficking to the site of assembly following endocytosis are poorly defined. The cytoplasmic tail (CT) of Env contains structural determinants required for incorporation in physiologically relevant cells. The CT contains a long unstructured region followed by three amphipathic helices, and mediates interactions with cellular trafficking machinery including clathrin, clathrin adaptor protein, retromer, and Rab11 family interacting protein-1C (FIP1C). We created a series of truncation and point mutants in the CT in order to assess their ability to direct Env trafficking and particle incorporation. We initially utilized a truncated form of FIP1C that collapses the ERC as a marker of Env trafficking to this compartment. Trafficking to the ERC required specific tryptophan-based motifs in the LLP3 region of the CT. Disruption of these motifs eliminated ERC trafficking and rendered Env defective for incorporation into particles. This work was then extended to examine the trafficking of Env to the tubular recycling endosome (TRE). This work identified for the first time a prominent a (open full item for complete abstract)

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)

Doctor of Philosophy, The Ohio State University, 2023, Comparative Biomedical Sciences

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic deltaretrovirus estimated to infect 5-10 million individuals globally. Approximately 5-10% of infected people develop disease after a prolonged clinical latency period of up to several decades. Diseases caused by HTLV-1 include the highly aggressive CD4+ T-cell malignancy known as adult T-cell leukemia/lymphoma (ATL) and the debilitating neurodegenerative disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Unfortunately, limited therapeutic options are available, resulting in dismal prognoses for individuals with these diseases. Research efforts aimed at unraveling HTLV-1 pathobiology are crucial in identifying novel therapeutic targets and devising successful medical interventions. Chapter One reviews our current knowledge of HTLV-1 transmission, life cycle, and pathogenesis. Two viral genes, Tax and Hbz, are individually linked to oncogenic transformation and play an important role in HTLV-1 pathogenesis. Tax is essential for de novo infection and cellular immortalization while Hbz promotes proliferation and survival of infected cells in both its protein and mRNA forms. Hbz is uniquely encoded on the antisense strand of the proviral genome and is the only viral gene constitutively expressed in ATL patients. However, recent research suggests that sporadic and transient Tax expression is still required for survival of the HTLV-1 leukemic cells. Transcription of Tax and Hbz relies on promoter elements within the long terminal repeats (LTRs) at the 5′ and 3′ ends of the integrated genome. Consequently, regulation of HTLV-1 gene expression is a central feature in the viral life cycle and directly contributes to its pathogenic potential. In Chapter Two, we investigated interactions between viral and host proteins to enhance our understanding of HTLV-1 transcriptional regulation. Specifically, we identified a host transcription factor, Y-box binding protein-1 (YBX1), that (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2023, Molecular Genetics

Geminiviruses infect a wide variety of plants and encode very few viral genes. As such, they rely heavily upon host machinery to drive their replication and hijack host pathways to ensure a productive infection. In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but instead is directed to responsive promoters through interactions with host factors, most likely transcriptional activators or repressors. In the main chapter of this dissertation, we describe a plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb repressive complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, CLE mutation in Tomato golden mosaic virus (TGMV) and Cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest a model where TCP24 binds the CLE early in infection and directly or indirectly recruits PRC2, resulting in CP promoter repressio (open full item for complete abstract)

Doctor of Philosophy (Ph.D.), University of Dayton, 2023, Biology

Antibiotic resistance has been declared a global concern by the World Health Organization and is increasing the rate of mortality of once-treatable, common infections. Antibiotic resistance is conferred by multiple mechanisms both intrinsic (horizontal gene transfer) and extrinsic (production of biofilms). The eradication of biofilms produced by bacterial colonization remains a serious threat to human infections. Bacterial biofilms produce an extracellular matrix composed of proteins, polysaccharides, and extracellular DNA (eDNA). This matrix acts as a scaffold for growth and imparts a form of protection against predators, harsh conditions, and chemicals (e.g., bacteriophage, pH, and antibiotics). The biofilm-associated cells of Pseudomonas aeruginosa (PsA) are up to 1000-fold more resistant to antibiotics than planktonic cells. Additionally, PsA has been linked to many infections that can be mortally dangerous for individuals with compromised immune systems such as Cystic Fibrosis (CF). PsA colonization in individuals with CF causes a decreased quality of life. Thus, there is a search for alternative strategies for antimicrobial management. Our lab has produced a patented zinc-containing porphyrin, Zn(II)meso-5,10,15-triyl-tris(1-methylpyridin-1-ium)-20-(pentafluorophenyl) porphine tritosylate (ZnPor), which exhibits broad antibacterial activity against planktonic and biofilm-associated cells of PsA. ZnPor presents itself as a unique possible surrogate for traditional antibiotics by its interaction with eDNA of biofilms. ZnPor intercalates between base pairs and binds to the outside of the helix, resulting in a more porous biofilm that dissembles and detaches from substrata. Furthermore, ZnPor has potent photoactivity that increases both its bactericidal and viricidal properties when exposed to light. The ability to disrupt the inherent matrix structure makes biofilm-associated cells more accessible to other treatments such as antibiotics and bacteriophage (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2023, Molecular Medicine

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus residing latently in a majority of the population. While generally asymptomatic in healthy individuals, primary infection or reactivation of latent infection in the immunocompromised, immunosuppressed, and immunonaive can lead to end organ failure if left untreated. HCMV encodes four G protein-coupled receptors (GPCRs), which play roles in both lytic and latent infection by manipulating the host cell environment, in some cases through altering cell signaling. The objective of these studies was to gain further insight into the role the HCMV-encoded GPCRs, US28 and UL33, play in the molecular mechanisms of lytic and latent infection, and in turn viral dissemination and transmission. In the first study, we demonstrate a new mechanism by which US28 controls HCMV latency. Our lab previously showed US28 is necessary for the establishment and maintenance of HCMV latency. To gain a more complete understanding of the cellular factors US28 manipulates during latency, we evaluated cellular transcriptomic changes in response to US28 expression and found this viral GPCR upregulates the cellular gene, myeloid nuclear differentiation antigen (MNDA). Previous findings revealed MNDA increases DNA-binding of the cellular transcription factor, Yin Yang 1 (YY1), which is a repressor of the major immediate early (MIE) enhancer/promoter, a region whose regulation of immediate early gene expression, in part, controls the balance between HCMV latency and reactivation. We show US28 upregulates MNDA, leading to a US28-dependent interaction between MNDA and YY1. This complex binds the MIE enhancer/promoter, resulting in transcriptional silencing of this locus, thereby aiding in the maintenance of latent infection. These studies reveal a new mechanism by which US28 regulates the MIE enhancer, through the control of cellular proteins MNDA and YY1. Secondly, we evaluated the HCMV-encoded GPCR UL33 and its role in viral replication. P (open full item for complete abstract)

Master of Science (MS), Wright State University, 2023, Anatomy

This thesis tests the hypothesis that the spike protein of SARV-CoV-2 is under positive evolutionary selection. A consensus nucleotide sequence was generated for the most current SARS-CoV-2 variant, XBB1.5, by performing sequence alignments of recent isolates. The consensus nucleotide sequence was translated to obtain the amino acid sequence. The amino acid and nucleotide sequences were compared to the original Wuhan-hu-1 strain to identify mutations. Forty-three mutations were identified in the spike protein, and all but two changed the amino acid. The majority of mutations were found in the receptor binding domain of the spike protein, which is the region of the protein that interacts with the ACE-2 receptor on cells to allow viral entry. A novel mathematical formula was created to calculate random genetic drift. Using this formula, the percentage of mutations that changed the amino acid sequence due to random genetic drift was calculated for the Wuhan-hu-1 reference sequence and compared with the observed percentage for the Omicron XBB1.5 variant. There was a statistically significant difference between the observed number of mutations that changed the amino acid and the number expected from random genetic drift for the receptor binding domain and spike protein as a whole. It is concluded that the spike protein of SARS-CoV-2 is under positive evolutionary selection. This mathematical formula can be used to track future variants of SARS-CoV-2 and to analyze other emerging viruses and ongoing pandemic threats, such as highly pathogenic avian influenza A virus strains.

Doctor of Philosophy, The Ohio State University, 2023, Comparative Biomedical Sciences

Swine are a critical host species for influenza A virus (IAV) due to the public health risk posed by zoonotic transmission, which can result in pandemic IAV generation. Exhibition swine, raised by youth to be shown at agricultural fairs and livestock exhibitions, have been the primary source of zoonotic IAV infections in people in the USA. The structure of “jackpot” shows is distinct from county fairs, and they allow contact and comingling of numerous pigs from separate and unique locales year-round. To evaluate the role of this show network in IAV dissemination, we collected samples from 17,009 pigs attending 350 national, state, and local swine during 2016–2018. Jackpot shows had 4.3-fold higher odds of detecting at least one influenza-positive pig compared to county fairs. However, when influenza was detected at a county fair, almost half of pigs tested positive, clarifying why zoonotic infections occur primarily at county fairs. The IAV genotypes detected within the jackpot show network within a given year are the same genotypes detected at county fairs during those years. Because these jackpot shows occur in the early spring and summer, before peak county fair season in the Midwest, there is a clear pathway for IAV at these shows to seed the IAVs in county fairs that transmit from pigs to people. Next, we investigated how once IAV is introduced, the duration of these county fairs influences the high IAV prevalence in swine, which drives zoonotic transmission. We longitudinally sampled every pig daily for the full duration of 16 county fairs during 2014–2015 (39,768 nasal wipes from 6,768 pigs). We found that shortening duration drastically reduces IAV prevalence in exhibition swine at county fairs. Additionally, we found further evidence that altering the arrival procedures can substantially slow the potential IAV outbreak growth at these fairs further reducing zoonotic risk. Finally, we investigated the exposure and neutralizing protection of the human exhibit (open full item for complete abstract)

Master of Science (MS), Wright State University, 2023, Microbiology and Immunology

Adenovirus is a human pathogen that causes cold like symptoms in healthy individuals, which could be lethal in pediatrics and the immunocompromised. In polarized epithelia, adenovirus uses the eight-exon isoform of the Coxsackievirus and Adenovirus Receptor (CAREx8) on the apical surface, to facilitate infection. CAREx8 protein expression level is regulated by the scaffolding protein MAGI-1, using two of its PSD95/Dlg-1/ZO-1 (PDZ) domains: PDZ2 and PDZ4. We previously showed that CAREx8 is degraded after interaction with PDZ4 and rescued following interaction with PDZ2. We then developed a PDZ2 binding decoy molecule (E6) that blocks the interaction between CAREx8 and PDZ2, which increases degradation of CAREx8 and, consequently, decreases adenovirus infection. We then synthesized TAT, a cell penetrating peptide (CPP) to the N-terminus of E6 for easy delivery into cells. In comparison to a scrambled control peptide, TAT_E6 significantly reduced CAREx8 expression and adenovirus-5 transduction in MDCK cells that stably expressed human CAREx8. However, recent studies suggest that there may be other CPPs that when conjugated to E6, will decrease CAREx8 protein expression and subsequently AdV transduction with higher efficacy than TAT_E6. As such, I selected five new CPPs, conjugated them to E6, then tested their CAREx8 protein expression reducing capabilities via Western blot analysis. These results demonstrated that all five of the selected E6-conjugated CPPs reduced CAREx8 protein expression more significantly than TAT_E6.

PhD, University of Cincinnati, 2023, Medicine: Immunology

The global HIV/AIDS pandemic remains a serious disease with millions of individuals infected and no known cure. All current efforts to prevent the spread of the virus with vaccine protection have remained unsuccessful. The use of antiretroviral therapy (ART) continues to provide relief for those living with HIV by preventing high viral load and vastly improving patient prognosis. However, the ability of HIV to rapidly mutate and become resistant to antiretroviral drugs, as well as complications of lifelong use of ART, continues to support the discovery of new knowledge about the virus and thus the development of new classes of drugs to antagonize it. Research into the basic biology of Env and its interactions with host pathways is likely to illuminate new findings of importance to cell biology and host-pathogen interactions, and potentially could provide new targets for antiretroviral therapy. It is therefore important to understand how Env becomes incorporated into virions, what host cell factors are involved in the assembly process, and how Env is trafficked inside the cell to the site of particle assembly at the plasma membrane (PM). Previously, we identified the trafficking factor Rab14 as important for Env incorporation. This led us to hypothesize that a host molecular motor protein that traffics Rab14 dependent cargo, kinesin 16B (KIF16B), plays a role in Env trafficking and incorporation. In this study, we determined that Env is a cargo of KIF16B, and showed that KIF16B dramatically influences Env subcellular localization. Lack of KIF16B resulted in accelerated lysosomal degradation of Env, and shifted Env away from the plasma membrane site of particle assembly. Finally, KIF16B was shown to be important for Env incorporation, viral infectivity, and viral replication.

Master of Science (M.S.), University of Dayton, 2023, Interdisciplinary Studies

The SARS-CoV2 virus was responsible for the COVID-19 Pandemic, one of the most fatal international public health emergencies experienced in the past century. SARS-CoV2 induces symptoms like increased inflammatory response, severe acute respiratory syndrome (SARS), cognitive dysfunction like brain fog, and cardiovascular defects. Prolonged or long-term infection led to the emergence of Post-COVID-19 Syndrome, or PCS. PCS is characterized by chronic cardiovascular, autoimmune, and neurological manifestations and remains understudied. Individuals with pre-existing neurological insult like those with neuroinflammatory or neurodegenerative diseases are likely more vulnerable to such PCS effects. Furthermore, individuals with pre-existing neurological conditions often have comorbidities like obesity, hypertension, hyperlipidemia, and low activity levels. However, little is understood about the molecular effects of SARS-CoV2 on neuron in both healthy and neuro-compromised individuals. Currently, many individuals experiencing PCS-related neurological symptoms require management of their symptoms even though our knowledge in this area is still limited. Therefore, this study utilized an interdisciplinary approach to better understand how SARS-CoV2 impacts both neurons at a cellular level and clinically in neurologically compromised populations such as Multiple Sclerosis (MS). This interdisciplinary approach sheds light on how translational work is being done where basic science efforts complement efforts made clinically to make connections and identify relationships between observed effects and known science. To do so, SARS-CoV2 proteins were misexpressed in the Drosophila eye and through a forward genetic screen evaluated for changes to cellular structure or function. To corroborate these findings, SARS-CoV2 proteins were also transfected into Neuro-2a cells to assess how these proteins affected cellular functioning. Furthermore, SARS-CoV2 protein structure-function analys (open full item for complete abstract)