Doctor of Philosophy, The Ohio State University, 2009, Veterinary Biosciences

Porcine circovirus 2 (PCV2) and porcine torque teno virus (TTV) are single stranded DNA (ssDNA) viruses that have the ability to cause disease in conjunction with other pathogens or sources of immunomodulation and are emerging pathogens of swine. PCV2 is the necessary infectious cause for the production of post-weaning multisystemic wasting syndrome (PMWS), and the sudden global emergence of epidemic PMWS remains unexplained. Immunohistochemical and in vitro culture-based methodologies have suggested that macrophages are likely sites of viral replication; however the tissues and cells involved in PCV2 replication have not been fully delineated. Current immunohistochemical (IHC) methodologies identify PCV2 antigens but are not capable of differentiating replicating virus from non-replicating virion particles in vivo. In Chapter 1, a combination of IHC with commercial monoclonal antibodies specific for single stranded (ss) and double stranded (ds) DNA and PCV2 specific in situ hybridization (ISH) was used to show the specificity of the former for PCV2 DNA in tissue sections from PCV2-infected gnotobiotic pigs. Cold-ethanol-fixed tissue sections were superior to formalin-fixed tissues for detection of PCV2 DNAs, presumably due to the lack of protein cross-linking in the former. These data demonstrate that conventional IHC detects PCV2 DNA forms in mononuclear cells of experimentally infected, PCV2-positive gnotobiotic porcine tissue sections that are minimally compromised by either formalin fixation or the harsh conditions needed for ISH. In chapter 2, we investigated a PCV2 reference pathogenic isolate engineered to contain a unique genotype identified in archival porcine clinical samples collected 26 years prior to the first reports of epidemic PMWS. This three amino acid threonine-glycine-asparagine to alanine-threonine-alanine (Thr-Gly-Asn to Ala-Thr-Ala) sequence in the C-terminus region of the nucleocapsid protein distinguishes archival PCV2 from contemporary PC (open full item for complete abstract)

Committee: George Krakowka PhD DVM (Advisor); Charles Brooks PhD (Committee Member); John Ellis PhD DVM (Committee Member); Michael Lairmore PhD DVM (Committee Member); Michael Oglesbee PhD DVM (Committee Member) Subjects: Animal Diseases; Livestock; Microbiology; Molecular Biology; Pathology; Virology

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

The lens is the pivotal tissue of the eye allowing accommodation, the process by which the eye adjusts focal distance. Presbyopia and cataract, age-associated lens dysfunctions, prevent proper accommodative function and focusing of light as it passes through the lens. As the eye ages, lenses continue to grow in size and stiffen. As these properties change with age, lenticular dysfunctions arise. Presbyopia is incredibly common, impairing near vision in nearly all people by 40 to 50 years of age. A significant portion of the population lives with imperfect near vision due to presbyopia. The exact causes of presbyopia are yet to be explained, and additionally, there remain no effective therapies capable of restoring, or preventing the loss of, full accommodative function in aged lenses. It is now understood that both lens stiffening and lens geometric change due to continual lens growth, lead to presbyopia. Recent studies have demonstrated that mechanical force transduction, through the zonular fibers to the lens capsule, increases lens epithelial cell proliferation. These findings offer an avenue of study that could reveal mechanisms governing lens growth and guide future lens treatment options. Some clinical practitioners consider presbyopia and cataract to be entirely treated through the use of spectacles and implanted artificial intraocular lenses; however, no preventative therapy exists and no current treatment is capable of restoring accommodation. Advancement in the understanding of lens cell biology, mechanics, and the lens epithelial cell (LEC) mechanobiological response is necessary for the improvement of clinical treatment for age-associated lens dysfunction. This dissertation seeks to describe a new branch in the field of lens study with hopes of expanding fundamental knowledge focusing on LEC growth and mechanobiology. First, a novel method of simulating accommodation-like forces in a murine eye model is detailed. The methods described here demo (open full item for complete abstract)

Committee: Katelyn Swindle-Reilly (Committee Member); Cynthia Roberts (Committee Member); Heather Chandler (Committee Member); Matthew Reilly (Advisor) Subjects: Biology; Biomechanics; Biomedical Engineering; Biomedical Research; Ophthalmology; Optics

PhD, University of Cincinnati, 2023, Engineering and Applied Science: Mechanical Engineering

The knee meniscus is an integral part of the human knee. It performs the function of joint shock absorption and stabilization. It is physiologically complex and designed in a manner to be able to perform such important functions. However, the meniscus tear is a common injury observed in elderly people and people who perform intense physical activity. The meniscus has limited self-repair capabilities. Thus surgery is an eventual option for serious conditions. These surgical procedures may lead to complications and risks such as infection, nerve damage, persistent pain, etc. Given the complex function performed by the meniscus, it shows a complex material behavior at low strain rates. It exhibits basically hyperelastic material behavior of low strain rates and visohyperelasticity and damage of high strain rates. Thus, understanding the viscohyperelastic material properties of the meniscus is a basic step toward preventing meniscus damage. There is a need for models that can not only accurately capture the mechanical behavior of the meniscus but also be able to predict the damage and fracture behavior. In this study, a combination of experimental and computational methods are applied to characterize the mechanical behavior of knee meniscus and to establish mechanical damage criteria. To this end, the following are the specific aims of this study: -Specific aim 1: Design experiments and develop a finite element model to quantify the variation in mechanical properties of the meniscus under quasi-static deformation. -Specific aim 2: Develop novel constitutive models for high-strain rate behavior of the meniscus. These models describe the mechanical response under intensive sports and unexpected accidents. -Specific aim 3: Conduct experiments to model damage and fracture behavior of the meniscus. This would help in establishing damage criteria for the meniscus and help prevent potential meniscus injuries. Results from specific aim 1 d (open full item for complete abstract)

Committee: Chia-Ying Lin Ph.D. (Committee Chair); Yongfeng Xu Ph.D. (Committee Member); Woo Kyun Kim Ph.D. (Committee Member); Kumar Vemaganti Ph.D. (Committee Chair) Subjects: Mechanical Engineering

Master of Science (MS), Wright State University, 2023, Pharmacology and Toxicology

The skin is exposed to a variety of external stressors, from irritants to toxic agents to ultraviolet radiation (UVR). While mice and human volunteers can be used for research studies, there are limitations in the range of environmental stressors that can be studied using these models. Therefore, our research team has been investigating the use of human skin explants as a model to study the immediate effects of environmental stressors ex vivo. Although this method has been successful, obtaining fresh human skin for these studies is difficult due to limited availability. To overcome this issue, we are evaluating the possibility of using porcine skin explants in our model systems. The primary advantage of using porcine skin is that it is readily available in large quantities, and its structure bears many similarities to that of human skin. In our research, we have found that various stressors, including UVB radiation, can trigger the production of Platelet-Activating Factor (PAF) in the skin. This, in turn, activates the enzyme acid sphingomyelinase (aSMase), leading to the shedding of subcellular microvesicle particles (MVP). In our research studies, we have used various models, such as cell lines, mice, human skin explants, and limited studies in human volunteers. Currently, we are examining the possibility of using porcine skin explants as a model to study MVP release in response to different types of stressors that are relevant to the skin. The application of the carbamyl PAF agonist PAF (CPAF) or a phorbol ester topically to the skin leads to an increase in MVP release. Similarly, exposing the skin to different UVB fluences also results in a higher release of MVP. Our experiments on porcine skin explants show similar results to human skin explants in terms of MVP release, although porcine skin appears to be less reactive to these agents. Although porcine skin shares many structural similarities with human skin, unlike human skin and cell lines, the combined impac (open full item for complete abstract)

Committee: Jeffrey B. Travers M.D., Ph.D. (Advisor); David Cool Ph.D. (Committee Member); Yangfang Chen M.D., Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science, The Ohio State University, 2022, Veterinary Preventive Medicine

Rotavirus A (RVA) is the primary cause of acute viral gastroenteritis in children and young animals globally; however, its replication and pathogenesis remain poorly understood. We have previously demonstrated contrasting modes of interactions with the host cell glycans for two prevalent porcine RVA strains: OSU G5P[7] (historically associated with severe disease in piglets) and G9P[13] (globally emerging variant in humans and swine). Specifically, OSU G5P[7] and G9P[13] strain replication was significantly decreased and significantly increased, respectively, following removal of terminal sialic acids (SA) by neuraminidase (NA) treatment. The latter contrasting effects coincided with the presence of distinct mutations found in the VP4 fusion region of these strains. In our first study, to clarify cellular mechanisms associated with these differential mechanisms of cell attachment/entry we conducted transcriptome analysis of porcine small intestinal enteroids (PIEs) infected with the two RVA strains with and without NA treatment. NA treatment of porcine intestinal enteroids alone, before individual RVA G9P[13]/OSU G5P[7] infection resulted in altered expression of genes associated with biological regulation, transporter activity, protein binding, and multicellular organismal processes. This was shown with significant contradicting impacts, with G9P[13] being significantly enhanced, and OSU G5P[7] replication being significantly inhibited. Cholesterol (a key component of the host plasma membranes) has been shown to play a critical role in RVA replication. To further improve our understanding of RVA pathogenesis taking into consideration RVA genotype-specific features, in our second study, we comparatively evaluated the effects of cholesterol and cholesterol-related additives [Methyl-β-cyclodextrin (MβCD), and diethylaminoethyl (DEAE), and bile acids (BAs)] on G9P[13] vs. OSU G5P[7] replication in vitro. Consistent with our previous findings, treatment with cholest (open full item for complete abstract)

Committee: Anastasia Vlasova (Advisor); Linda Saif (Committee Member); Qiuhong Wang (Committee Member) Subjects: Biology; Genetics; Virology

Doctor of Philosophy, The Ohio State University, 2022, Comparative and Veterinary Medicine

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus. It was initially reported in 1970s in Europe. However, massive porcine epidemic diarrhea (PED) epidemics did not occur in China until late 2010. PEDV was first introduced into the United States (US) and rapidly spread nationwide in 2013. PEDV infection of suckling piglets causes up to 100% mortality and the US pig industry lost 10% of domestic pig population and $0.9 to $1.8 billion during 2013-2014, posing a great threat to the pork industry sustainability. Live attenuated vaccines (LAVs) given orally to pregnant sows/gilts replicate in the intestines and induce lactogenic immunity in sows/gilts. They are the most effective approach to provide passive immunity to neonatal pigs against PED via colostrum and milk. However, safety concerns associated with potential virulence reversion hinder broad application of LAVs. The goal of this dissertation is to identify promising targets in two viral genes and to develop a recombination-resistant platform for the development of effective and safe PEDV LAVs. My first objective was to evaluate whether the exonuclease (ExoN) domain within PEDV nsp14 is a good target for LAV development. Based on the infectious cDNA clone of a highly virulent PEDV strain (icPC22A), eight mutants targeting nsp14 ExoN catalytic sites, zinc finger, or Mg2+-binding site were designed. Only one E191A mutant, carrying the mutation in Mg2+-binding site, was rescued that was characterized by poor growth in Vero or IPEC-DQ cells of the early passages no.1-3 (P1-3) with peak titers of 1.80 ± 0.12 and 1.42 ± 0.16 log10 TCID50/mL, respectively. However, the P4 of E191A was characterized by dramatically improved growth characteristics reaching a high infectious titer (5.55 ± 0.35 log10 TCID50/mL) in Vero cells, like icPC22A. Sequence analysis demonstrated that the introduced mutation site has reverted to wildtype in the P4 virus. To evaluate the pathogenesis of the E191A-P1, 4-5-day-old gnotobi (open full item for complete abstract)

Committee: Qiuhong Wang (Advisor); Zongdi Feng (Committee Member); Linda Saif (Committee Member); Anastasia Vlasova (Committee Member) Subjects: Virology

Master of Science, The Ohio State University, 2022, Anatomy

Under ideal circumstances, surgical skills training should emulate live surgical conditions as closely as possible. While new anatomically accurate models and virtual/augmented reality simulators make surgical anatomical knowledge acquisition more accessible, research shows that these resources are limited in their ability to fully reflect the live human anatomy. As such, the animal and human body donor models have remained the gold standards for surgical simulation. And while there have been studies comparing the porcine and human body donor models, very little is known about Imperial College of London – Soft Preservation (alcohol-preserved) human body donors in the context of surgical training and education. The objective of our study was to examine the alcohol-preservation technique and to further elucidate its utility in general surgery resident education. Utilizing questionnaire-based feedback and semi-structured interviews, we analyzed quantitative and qualitative data obtained from the accounts of general surgery residents and surgical faculty instructors who experienced both the porcine and alcohol-preserved models at The Ohio State University Wexner Medical Center. Results indicated that the porcine and alcohol-preserved human body donor models were preferred for different aspects of surgical training. While the porcine model was preferred for its ability to teach tissue-handling and to simulate live blood flow, the alcohol-preserved human body donor model was unanimously preferred for its anatomical relevance, colon suturing/stapling, and reflection of live operating conditions. Surgical faculty specifically noted that while there is anatomical overlap between the two models with the anatomy of the rectum, “the small bowel and colon are dramatically different, which does not give the full breadth of colon resection needed for the human model.” In addition, the alcohol-preserved model was regarded as superior to the formalin-fixed human body dono (open full item for complete abstract)

Committee: Joy Balta (Advisor); Aslam Ejaz (Committee Member); Melissa Quinn (Committee Member) Subjects: Anatomy and Physiology

Doctor of Philosophy, The Ohio State University, 2021, Comparative and Veterinary Medicine

Rotaviruses (RVs) are a leading cause of acute viral gastroenteritis in young children and livestock worldwide. Cell glycans, such as histo-blood group antigens (HBGAs) and sialic acids (SA), are recognized by the RV surface protein VP4. However, a mechanistic understanding of these interactions and their effects on RV infection and pathogenesis is lacking. Here, we established porcine small intestinal enteroids (PIEs) expressing different HBGAs (A+, H+, and A+/H+) to study RV-glycan interactions. Differentiated PIEs were infected with human RV (HRV) G1P[8] Wa, porcine RV (PRV) G9P[13], PRV Gottfried G4P[6] and PRV OSU G5P[7] virulent and attenuated strains. Virulent Wa replicated to the highest titers in A+ PIEs, whereas virulent PRV G9P[13] and OSU strains preferentially replicated in H+ PIEs. The replication of all 4 attenuated strains was less affected by the PIE HBGA phenotypes. HBGA synthesis inhibitor 2-F-Peracetyl-Fucose (2F) treatment demonstrated that HBGAs are essential for Wa replication; however, they seemed dispensable for G9P[13] and OSU strains. Contrasting outcomes were observed following sialidase treatment on PIEs which significantly enhanced G9P[13] replication, but inhibited OSU G5P[7] growth. These observations suggest that additional receptors recognized by G9P[13] become unmasked after removal of terminal SA. To study the molecular mechanisms of RV attenuation and cell culture adaptation, we have sequenced and analyzed complete genomes of virulent Wa, (G1P[8]), M (G3P[8]), Gottfried (G4P[6]) and OSU (G5P[7]) strains and their attenuated counterparts. Most of the mutations clustered in the VP4 gene of the attenuated strains, with a high nonsynonymous substitution rate (81.2%). Two amino acid (aa) substitutions, aa385 and aa 471, found in the VP4 gene were conserved between two or more strain pairs. Of interest, the D393H and D385N substitutions identified within the VP4 hydrophobic domain important for virus cell entry. Collectively, these (open full item for complete abstract)

Committee: Vlasova Anastasia Dr. (Advisor); Scott P Kenney Dr. (Committee Member); Qiuhong Wang Dr. (Committee Member); Linda J Saif Dr. (Committee Member) Subjects: Biology; Virology

Master of Science, The Ohio State University, 2021, Comparative and Veterinary Medicine

Avian species often serve as transmission vectors and sources of recombination for viral infections due to their ability to travel vast distances and gregarious behaviors. Recently a novel deltacoronavirus (DCoV) was identified in sparrows. Sparrow deltacoronavirus (SpDCoV), coupled with close contact between sparrows and swine carrying porcine deltacoronavirus (PDCoV) may facilitate recombination of DCoVs resulting in novel CoV variants. We hypothesized that the spike (S) protein or receptor-binding domain (RBD) from sparrow coronaviruses (SpCoVs) may enhance infection in poultry. We used recombinant chimeric viruses, which express S protein or the RBD of SpCoV (icPDCoV-SHKU17, and icPDCoV-RBDISU) on the genomic backbone of an infectious clone of PDCoV (icPDCoV). Chimeric viruses were utilized to infect chicken derived DF-1 cells, turkey poults, and embryonated chicken eggs (ECEs) to examine permissiveness, viral replication kinetics, pathogenesis and pathology. We demonstrated that DF-1 cells in addition to the positive control LLC-PK1 cells are susceptible to SpCoV spike- and RBD- recombinant chimeric virus infections. However, the replication of chimeric viruses in DF-1 cells, but not LLC-PK1 cells, was inefficient. Inoculated 8-day-old turkey poults appeared resistant to icPDCoV-, icPDCoV-SHKU17- and icPDCoV-RBDISU virus infections. In 11-day-old ECEs, there was no evidence of viral replication, suggesting that differentiated cells are not susceptible to the virus infections. Collectively, we demonstrate that SpCoV chimeric viruses are not highly infectious in the turkey poults or the 11-day-old ECEs. Therefore, understanding the cell and host factors that contribute iii to resistance to PDCoV and avian swine chimeric virus infections may aid in the design of novel antiviral therapies against deltacoronaviruses.

Committee: Scott Kenney (Advisor); Qiuhong Wang (Committee Member); Linda Saif (Committee Member) Subjects: Comparative; Food Science; Veterinary Services; Virology

PHD, Kent State University, 2020, College of Arts and Sciences / School of Biomedical Sciences

Background: Animal agriculture is an industrialized, globalized system of meat production that will continue to increase in demand through the 21st century. This is a greatly resource intensive process that produces greenhouse gases and zoonotic disease development that contribute to climate change and public health pandemics. Since the demand for meat shows no sign of slowing in the coming years, alternative methods of meat production are required to feed a growing human population. Cell-based meat is one possible solution, which is meat grown from cell culture technology. Cell-based meat can be grown from developing edible cell lines and expanding them in bioreactors using biomedical techniques and equipment. The field in in its infancy, however, and many questions remain about how to develop useful cell lines and which are the most effective ways to grow them. This work demonstrates a research framework from which to characterize and compare two of the main cell types in meat: skeletal muscle cells and intramuscular fat cells. Methods were developed to analyze the difference between porcine and bovine cells, what characterizes their sensory and pigment properties, and how to efficiently grow them skeletal muscle tissue form. Methods: Bovine and porcine myoblasts and intramuscular fibroblast were isolated from the hind leg of 2 month old pigs and cows. Myoblasts were used for myogenesis assays, and intramuscular fibroblasts were used for adipogenesis assays. Gene and protein expression and volumetric hypertrophy data was obtained for myoblasts and lipid staining was quantified for fibroblasts. Meat color and pigment was determined for pork, beef, and cultured cells using colorimetry. Myoglobin gene and protein expression was assessed during myogenesis assays with and without electrical stimulation, mimicking an exercise regimen. Conditioned media from polarized porcine macrophages was used to characterize the potential for developing serum free media for myobl (open full item for complete abstract)

Committee: Min-Ho Kim Dr. (Advisor); Kristy Welshhans Dr. (Committee Member); Oleg Lavrentovich Dr. (Committee Member); Feng Dong Dr. (Committee Member); Songping Huang Dr. (Committee Member) Subjects: Biomedical Engineering; Biomedical Research; Food Science

PhD, University of Cincinnati, 2020, Engineering and Applied Science: Biomedical Engineering

Recent advances in point of care clinical diagnostic ultrasound scanners and image-guided procedures have been accompanied by an exponential expansion of therapeutic ultrasound technologies. Treatment strategies for stroke, a leading cause of morbidity and mortality worldwide, have been limited using lytic agents and interventional approaches with poor clinical outcomes. Sonothrombolysis, the adjuvant use of ultrasound exposure to enhance and accelerate thrombus break down with a thrombolytic agent, has been successful in vitro but has lacked demonstration of clinical success in large randomized controlled stroke treatment trials to date. This dissertation describes the development of unfocused low frequency (= 220 kHz) therapeutic ultrasound to enhance lysis through mechanically agitation and improved lytic diffusion.

Committee: Christy Holland Ph.D. (Committee Chair); Kevin Haworth Ph.D. (Committee Member); T. Douglas Mast Ph.D. (Committee Member); Marepalli Rao Ph.D. (Committee Member); Mario Zuccarello MD (Committee Member) Subjects: Biomedical Research

MS, University of Cincinnati, 2019, Pharmacy: Pharmaceutical Sciences

Permeation studies are an essential tool in ensuring the efficacy of topically applied drugs and in studying the absorption of toxic compounds. There are many factors that can affect permeation studies such as the choice of solvent, the physiochemical properties of the drug, and skin donor conditions. In this study, we aimed to investigate two of these aspects in permeation studies: dose effect and impact of skin furrows. In skin permeation studies, the rate of skin penetration of a compound is not necessarily proportional to the applied dose. In this thesis research, the dose dependence effect on skin permeation was determined in Franz diffusion cell under finite dose conditions. Radiolabeled solutions of urea, dexamethasone, estradiol, caffeine, and ethylene glycol were prepared in ethanol at doses ranging from 0.1 -200 μg. A 72-h study was conducted, and the receptor solution was sampled at various time points. The percent absorbed was calculated and analyzed in terms of the applied dose. The result showed a dose dependence effect on caffeine, dexamethasone, and estradiol up to a dose of 100 μg. No dose dependence was observed in the permeation profiles of urea and glycerol. Another factor investigated was the presence of furrows on the skin surface. These furrows might lead to erroneous interpretation of the results in skin permeation studies using tape stripping, in which the material trapped in the furrows removed by the tapes representing the deeper layers of the SC might be interpreted as the material permeating within these layers. The objective of this study was to investigate the effect of skin furrows on tape stripping results. Non-penetrating fluorescent materials were topically applied to split-thickness human and full-thickness porcine skin samples. Tape stripping was applied, and the tapes were assessed by fluorescence microscopy and quantitative analyses. The microscopic images were assessed visually to determine the presence of the applied mat (open full item for complete abstract)

Committee: Kevin Li Ph.D. (Committee Chair); Kavssery Ananthapadmanabhan Ph.D. (Committee Member); Gerald Kasting Ph.D. (Committee Member) Subjects: Pharmaceuticals

Doctor of Philosophy, The Ohio State University, 2019, Comparative and Veterinary Medicine

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, weight loss, dehydration, and up to 100% mortality in neonates. Vaccination of sows with a live attenuated vaccine (LAV) stimulates protective immunity in sows that can provide passive protection to piglets against PEDV via colostrum and milk. The goal of this dissertation is to determine attenuation mechanisms of deletions in two viral genes and to develop an effective and safe PEDV LAV candidates using reverse genetics technology. Our first objective was to study the pathogenesis of a PEDV variant TC-PC177 which contains a 197-aa deletion in the spike protein. In 4-day-old conventional piglets, TC-PC177 caused mild diarrhea, lower titers of fecal viral RNA shedding and no mortality, compared with a virulent strain PC21A. To investigate the role of this deletion in the attenuation of TC-PC177, we generated a mutant (icPC22A-S1Δ197) bearing this deletion from an infectious cDNA clone of the highly virulent PEDV PC22A strain (icPC22A). In 4-day-old gnotobiotic pigs, we found that the icPC22A-S1Δ197 virus caused mild to moderate diarrhea, lower titers of viral shedding and no mortality, compared with the virulent icPC22A. Our second objective was to elucidate the functions of motifs YxxΦ; and KVHVQ at the cytoplasmic tail of the S protein in intracellular sorting and in viral pathogenesis. By transiently expressing PEDV S proteins with mutations in these motifs, we confirmed that the motif KVHVQ is involved in the retention of the S protein in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and the YxxΦ; motif triggers endocytosis of the S protein. Next, we generated mutants by introducing deletions or a mutation into the two motifs of the icPC22A. Infection of Vero cells with icΔ10aa (ΔYxxΦEKVHVQ) resulted in larger syncytia and reduced numbers of S protein projections on the surface compared with icPC22A. In 5-day-old gnotobiotic piglets, mutant icΔ10aa (open full item for complete abstract)

Committee: Qiuhong Wang (Advisor); Linda Saif (Advisor); Anastasia Vlasova (Committee Member); Chang-Won Lee (Committee Member); Scott Kenney (Committee Member) Subjects: Virology

Doctor of Philosophy, The Ohio State University, 2018, Comparative and Veterinary Medicine

Neonates are immune competent but immunologically immature at birth and are highly susceptible to enteric pathogens. For example, porcine epidemic diarrhea virus (PEDV) is a highly virulent re-emerging enteric coronavirus that causes acute diarrhea, dehydration and 80-100% mortality in neonatal piglets. Since its emergence in the United States (US) in 2013, PEDV caused over 8.5 million piglet deaths and $900 million to $1.8 billion in losses to US swine producers in 2014. Lactogenic immunity remains the most promising and effective way to protect neonatal suckling piglets from enteric diseases like PEDV. This is particularly true for domesticated animals like swine and cattle whose epitheliochorial placenta inhibits immunoglobulin (Ig) transfer in utero. Therefore, colostrum [first secretions from the mammary gland (MG)] and milk-derived antibodies (Abs) and other immune factors are the sole source for immune protection after birth. Passive lactogenic immunity is achieved through high titers of IgG Abs in colostrum and a continuous supply of secretory IgA (sIgA) Abs in milk. Specifically, because of their persistence in milk at high titers, sIgA Abs play a major role in conferring passive lactogenic protection against enteric pathogens in suckling neonates. Therefore, our goal was to improve the understanding of maternal and lactogenic immunity induced via the gut-MG-sIgA axis and its role in protection against PEDV in neonatal suckling piglets. Our first objective was to study the impact of stage of gestation at time of PEDV infection in pregnant gilts and its implications for generation of lactogenic immunity and protection of PEDV-challenged piglets. To understand how stage of gestation affects maternal immune responses to PEDV, three groups of gilts were orally infected with PEDV in the first, second or third trimester. Control (mock) gilts were inoculated with medium in the third trimester. To determine if lactogenic immunity correlated with protection, all (open full item for complete abstract)

Committee: Linda Saif (Advisor); Anastasia Vlasova (Committee Member); Qiuhong Wang (Committee Member); Prosper Boyaka (Committee Member) Subjects: Agriculture; Animal Diseases; Immunology; Veterinary Services; Virology

PhD, University of Cincinnati, 2017, Engineering and Applied Science: Biomedical Engineering

Ischemic stroke affects nearly 700,000 patients in the United States each year and is the fifth most common cause of death. In less than 6% of ischemic stroke patients, lysis of the occlusive clot is attempted with recombinant tissue-plasminogen activator (rt-PA). The addition of Definity® microbubbles and 120-kHz ultrasound to rt-PA treatment has been shown to enhance lytic activity in vitro and ex vivo. However, preclinical trials must be completed in an animal model such as pigs prior to human clinical trials. One porcine thrombosis model is the intracerebral hemorrhage model, in which an intracerebral hemorrhage is treated with a lytic and exposed to ultrasound. An assay for a biochemical marker of clot breakdown, D-dimer, was evaluated for quantification of thrombolysis in this model. A porcine D-dimer purification protocol was developed and the identity of the purified D-dimer was confirmed by immunoblotting and MALDI TOF-TOF analysis. We evaluated a commercially available D-dimer ELISA kit and 5 commercially available D-dimer antibodies for development of an in-house ELISA protocol. In porcine samples produced in an in vitro thrombolysis system, D-dimer concentration was shown to correlate with mass loss. However, no current assay is known to be able to quantitate D-dimer with adequate sensitivity (10 ng/mL). To create an arterial thromboembolism model of ischemic stroke, porcine ascending pharyngeal arteries (APA) were occluded bilaterally. Most arteries were occluded with a single clot chosen to be about 1 mm larger than the inner diameter of the target artery. However, intraarterial treatment of the occluded arteries with rt-PA was ineffective and did not recanalize any of the occluded arteries. A protocol for post-mortem APA excision from swine was also developed. The lack of rt-PA efficacy in the porcine arterial thromboembolism model suggested that porcine clots were resistant to rt-PA thrombolysis. In vitro evaluation of the lytic response o (open full item for complete abstract)

Committee: Christy Holland Ph.D. (Committee Chair); Todd Abruzzo Ph.D. (Committee Member); Kevin Haworth Ph.D. (Committee Member); Andrew Herr Ph.D. (Committee Member); Daria Narmoneva Ph.D. (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2017, Veterinary Biosciences

Any breakdown of epidermal barrier function leaves the host susceptible to infection. The innate immune system is tasked with the ability to clear these infections and provide an environment that can progress through the remaining stages of wound healing. There is a growing population of both immune competent and immunocompromised individuals that develop non-healing soft tissue injuries. Consistent identification of opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds has focused our attention on these bacterial species. Specifically, these opportunistic pathogens often exist as sessile, aggregated communities within these wounds and are profoundly resistant to exogenous and host derived antimicrobials. Therefore, we hypothesized that P. aeruginosa and S. aureus are able to subvert the innate immune system leading to persistent inflammation and delayed wound healing. The first part of this thesis investigates the host response to P. aeruginosa and S. aureus in a chronic porcine burn wound model. Previous work in our lab, established this model in collaboration with Drs. Shahwati Roy and Chandan Sen and other members of the Ohio State University's Comprehensive Wound Center. In the establishment of this wound it was apparent that although epidermal wound healing was achieved after poly-microbial infection, there remained incompetence of the barrier function. Mono-species infections with P. aeruginosa and S. aureus or co-infections with both of these species had previously not been conducted in a chronic wound model using a clinically relevant species. We discovered that bacterial infection results in a host response unique to the infective bacterial species and additive pathologic effects were expressed when present together. Specifically, we were able to identify mono-species infection induced responses by the epidermis that recapitulate defining features of chronic wounds in humans. Ultimately, this work generated a stan (open full item for complete abstract)

Committee: Daniel Wozniak (Advisor); Paul Stromberg (Committee Member); Luanne Hall-Stoodley (Committee Member); Sashwati Roy (Committee Member) Subjects: Microbiology; Veterinary Services

Doctor of Philosophy (PhD), Ohio University, 2016, Biological Sciences (Arts and Sciences)

Low back pain (LBP) is the leading cause of disability worldwide with more than eight hundred billion dollars of direct and indirect costs associated with LBP being incurred annually in the US alone. About 80-90% of all LBP patients do not have a definitive diagnosis of the etiology of pain, and are grouped under the non-specific LBP cohort. A group of such patients with unspecified etiology for their back pain are believed to have their LBP due to un-controlled and often, more than normal segmental motion involving one or more of their lumbar vertebrae. As such, many surgical treatments for LBP are directed at reducing inter-vertebral motion at and around an affected segment. The most common approaches for quantifying vertebral motion are based on radiographic assessments, which in many cases preclude scientific inquiry (e.g., use of high radiation equipment is not permitted for scientific investigations in some states, and, even when permitted, presents a major roadblock for serial assessments). This project was undertaken to develop and examine the feasibility, reliability and accuracy of a technique that used magnetic resonance (MR) images, custom built 3-D models and animations of spine-segment motion to quantify displacements in a calibrated imaging space. Experiment 1 explored the feasibility of developing an MRI-based spine kinematics quantification technique that involved (i) creating MRI compatible solid-body objects, (ii) scanning axial images of the objects in the MRI machine to create static 3-D virtual models of the solid-bodies, (iii) scanning the solid bodies as they were displaced within the MR imaging space through pre-determined magnitudes, and (iv) using images from the displacement trials to create background 'scenes' in an animation software where the user performed an image-to-model matching. This process called `rotoscoping', resulted in reliable quantification of the displacements achieved with the technique developed in this aim (see Cha (open full item for complete abstract)

Committee: Brian Clark Ph.D. (Committee Chair); Susan Williams Ph.D. (Committee Member); Robert Staron Ph.D (Committee Member) Subjects: Anatomy and Physiology; Biomedical Research; Medical Imaging

PhD, University of Cincinnati, 2016, Medicine: Molecular and Developmental Biology

Calcific Aortic Valve Disease (CAVD) is the most prevalent type of heart valve disease, affecting ~2% of the US population. The risk for developing CAVD increases with age, and CAVD is becoming more common with increased average lifespan. Currently, the standard of care involves aortic valve (AoV) replacement surgery, which is often contraindicated for the elderly. Importantly, the pathogenic mechanisms driving CAVD, which could serve as potential therapeutic targets, are not well understood. Studies of human diseased AoV provide initial evidence that the Bone Morphogenetic Proteins (BMPs), essential for normal bone formation, play a critical role during CAVD. However, the function of BMP signaling during osteochondrogenic gene induction and AoV calcification, both CAVD features, remain unknown. Here, studies are directed towards the understanding of molecular pathways involved in CAVD using a combination of a murine model of premature aging in vivo and directed mechanistic studies of porcine aortic valve interstitial cells (VICs) in vitro.

Committee: Katherine Yutzey Ph.D. (Committee Chair); Burns Blaxall Ph.D. (Committee Member); Rolf Stottman PhD (Committee Member); James Wells Ph.D. (Committee Member); Yana Zavros Ph.D. (Committee Member) Subjects: Molecular Biology

Master of Science (MS), Wright State University, 2016, Pharmacology and Toxicology

Introduction: (Oasis-ultra) is an extra cellular collagen rich matrix derived from porcine intestinal sub-mucosa. A prospective, multi-centered, randomized, single-blinded clinical trial was conducted to study the effects of Oasis-ultra combined with negative pressure wound therapy (NPWT) on the healing rate of stage IV pressure wounds versus NPWT alone. Materials and Methods: Twelve subjects were involved in the study: six patients in the study group and six in the control group. NPWT was changed twice a week for all subjects, and Oasis-ultra was applied weekly. The wounds were measured weekly, and the healing rate was calculated for each subject for 12 weeks. The canisters were collected monthly for three months. For cytokine and growth factors analysis, 100 µl 1XPBS were added to the sample, and protein concentration was determined using the Bradford assay. A Bio-Rad BioPlex 96 well plate was set up with 50 µl of the sample and duplicated for cytokine analysis using Bio-Plex. Results: In the study group, the healing rate calculated at 12 weeks was found to be ~87% when compared to the control group, which was ~55%. Analysis of different growth factors, normally present in stage IV pressure wounds, revealed higher concentrations in the oasis-ultra treated group when compared with the control group. Additionally, the other proinflammatory cytokines that accused of wound chronicity were down regulated as a result of treating the subjects in the study group with oasis-ultra. Conclusion: Our study demonstrates that the use of Oasis-ultra accelerates the healing rate of stage IV pressure wounds when combined with NPWT. Also, in the Oasis-ultra treated group, the proinflammatory cytokines were successfully inhibited. At the same time, Oasis-ultra promoted and upregulated the beneficial growth factors that had positive impact on the healing rate.

Committee: Richard Simman M.D, FACS, FACCWS. (Advisor); David Cool Ph.D. (Committee Member); Yanfang Chen M.D., Ph.D. (Committee Member); Ji Bihl MD., Ph.D. (Committee Member) Subjects: Medicine; Pharmacology

Doctor of Philosophy, The Ohio State University, 2015, Comparative and Veterinary Medicine

Human enteric caliciviruses are a leading cause of viral gastroenteritis in humans of all ages worldwide. However, most attempts to grow human enteric caliciviruses in routine cell cultures have failed. This has hampered research on pathogenesis, immunity, virus inactivation, and the development of antivirals and vaccines. Porcine sapovirus (PoSaV) Cowden strain causes diarrhea in pigs, and is one of only a few culturable enteric caliciviruses. The PoSaV Cowden strain was adapted to a porcine kidney cell line, LLC-PK, after serially passaging in gnotobiotic pigs and in primary porcine kidney cells. Compared with the wild-type (WT) PoSaV Cowden strain, the tissue culture-adapted (TC) PoSaV has eight conserved amino acid substitutions: two in the RNA-dependent RNA polymerase (RdRp) region and six in the capsid protein (VP1) region. By using a reverse genetics system for the TC Cowden strain, pCV4A, four (178, 289, 324, and 328) amino acid substitutions in the VP1, but not the substitutions in the RdRp region, were identified to be critical for the cell culture adaptation of PoSaV Cowden strain. Two (291 and 295) substitutions in the VP1 enhanced virus replication in vitro, but reduced virus replication in vivo. In addition, the 291 and 295 revertants induced higher serum and mucosal antibody responses than TC PoSaV Cowden strain. Three dimensional (3D) structural analysis of the VP1 showed that residue 178 was located in the shell domain near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at the protruding subdomain 2 (P2), which may influence virus binding to the cellular receptors; residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Next, these findings were tested using other Cowden-like genogroup III PoSaVs. The complete genomes of two Cowden-like PoSaV strains, WT JJ259 and WT LL14 were sequenced and compared with those of Cowden (open full item for complete abstract)

Committee: Linda Saif Ph.D. (Advisor); Qiuhong Wang Ph.D. (Advisor); Gireesh Rajashekara Ph.D. (Committee Member); Armando Hoet Ph.D. (Committee Member) Subjects: Molecular Biology; Pathology; Virology