Bachelor of Arts, Wittenberg University, 2024, Biology

Obsessive Compulsive Disorder, or OCD, is a disorder characterized by intrusive thoughts and repetitive actions (Couto et al., 2018), and affects about 2-3% of the U.S. population (“What is obsessive-compulsive disorder”, 2024). Although there are no molecular concrete causes of OCD, it is thought that abnormalities in the glutamate and serotonin pathways may have large impacts on the symptoms that cause OCD. Furthermore, there are many genes that may have a linkage to OCD and its comorbidities. Currently, as a main treatment SSRIs may be combined with anti-psychotic drugs such as Risperdal, but they are only effective about 40%- 60% of the time (Allen et al., 2018). Cognitive Behavioral Therapy can also be used in combination as a treatment for OCD. There have been studies in both mice and humans that suggest N-Acetylcysteine, or NAC, may be useful in reducing OCD symptoms (CITATION). Mice were exposed to NAC for 1 or 3 weeks, and then injected with a saline or RU42969 (5HT1A/1B agonist) to induce OCD-like symptoms. They were then observed behaviorally, and their cDNA was harvested. The involvement of different genes in NAC and OCD pathways is not completely known. Therefore, qPCR was used to test the expression of seven different genes—DRD2, GRIK2, SLC1A2, HTR2A, MAOA, PRL, and OXTR—and their connection to OCD and NAC. Two of the seven genes (28.6%) that were studied were significantly affected by 1 Week NAC + OCD. Two of the seven genes (28.6%) were significantly affected by 3 Week NAC + OCD. Three of the seven genes (42.9%) were significantly affected by 3 Week H20 + OCD. Overall, the results suggest that many of the genes we looked at are not necessarily involved in the NAC pathways but perhaps are involved in the OCD pathway. More research is needed to determine the genes that are significantly affected by NAC on its own, and further research can be done to strengthen the literature on interactions between these genes and OCD.

Committee: Michelle McWhorter (Advisor); Gwynne Davis (Committee Member); Margaret Goodman (Committee Member) Subjects: Biology; Mental Health; Neurobiology; Neurology; Neurosciences; Psychobiology; Psychology

Master of Sciences, Case Western Reserve University, 2024, Pathology

Malaria caused by the Plasmodium vivax parasite is a leading cause of disease burden in many tropical and subtropical parts of the world. Infection leads to the generation of antibodies specific to parasite proteins, such as apical membrane antigen 1 (AMA1). This ligand plays a role in invasion of reticulocytes by binding of the rhoptry neck (RON) complex, which is composed of RON proteins. This complex is inserted by the parasite into the cell surface. In malaria infections, IgG1 and IgG3 isotype frequencies are increased. These isotypes can activate complement or bind Fc receptors to initiate effector functions. As part of a project that isolates cDNA from individual PvAMA1- specific IgG expressing B cells, we aim to develop a rapid method to identify the IgG subclass from these B cells to provide a more comprehensive view of immunoglobulin generation in these individuals.

Committee: Neena Singh (Committee Chair); Jürgen Bosch (Committee Member); Christopher King (Committee Member) Subjects: Immunology; Molecular Biology; Parasitology; Pathology

Bachelor of Science (BS), Ohio University, 2023, Biological Sciences

Fibrosis, a pathological process characterized by excess extracellular matrix (ECM) deposition, can occur in many internal organs and tissues in response to various stimuli. As fibrosis progresses, scarring occurs, which ultimately leads to tissue dysfunction and organ failure. Patients with acromegaly, a rare disease usually caused by a benign, GH-producing pituitary tumor, have been reported to have prominent ECM deposition and scarring in certain tissues, which is indicative of fibrosis. In bGH transgenic mice, which express high levels of bovine growth hormone, several tissues [white adipose tissue (WAT), heart, intestine, and kidney] demonstrate a fibrotic phenotype. However, there is no previous research that investigates various bGH tissues – particularly from mice derived from a single cohort – for fibrosis. Additionally, WAT fibrosis is associated with obesity and lipodystrophy, and seems to be particularly associated with excess GH. This study aims to investigate the role of different cell types and genes involved in the development and progression of WAT fibrosis and determine if fibrosis is increased in BAT, liver, quad, kidney, lung, and spleen of aged bGH mice. Results of this thesis included a striking observation of increased fibrosis in all bGH tissues examined. For WAT, decreases in fibrosis-associated RNA expression in 3-month-old bGH mice via qPCR analysis was only observed in the perigonadal depot and not the subcutaneous depot that has more prominent collagen deposition. Interestingly, we observed an intriguing increase in fibrosis-associated RNA expression in a population of adipose stem and progenitor cells in 6-month-old mice within subcutaneous bGH WAT. These results indicate a potential common GH-induced mechanism of fibrosis across bGH tissues and pave the way for future research into WAT fibrosis.

Committee: Darlene Berryman (Advisor) Subjects: Biology; Biomedical Research

Doctor of Philosophy, The Ohio State University, 2022, Environmental Science

We spend 90% of our time indoors where we are exposed to diverse microbial communities which can have profound effects on human health outcomes. Advances in DNA-based technologies offer greater ability to quantitatively measure indoor fungal communities and more accurately assess the influence of environmental conditions on fungal communities as well as the influence of fungal exposures on health. Trends are emerging in our understanding of pathways between built environments, indoor microbiomes, and ultimately human health, but much remains uncertain. Fungal diversity is consistently and inversely associated with development of asthma and allergic disease. Further, seasonal variation in asthma and allergy is well established. So too is the relationship between asthma development and sensitization to select allergenic fungi. However, the influence of fungal exposures—including diversity, total fungal concentration, and select fungal taxa—on measures of asthma morbidity is still not well understood. The influence of season and other indoor environmental conditions or occupant behaviors in shaping these measures of fungal exposure is also understudied. The goal of this work is to investigate novel associations between measures of childhood asthma morbidity and fungal exposures, and to examine the effect of comprehensive occupant behaviors and indoor environmental characteristics—including conditions mediated by season—on indoor fungal communities. Bedroom floor dust samples were collected from two cohorts: the New York City Neighborhood Asthma and Allergy Study (NAAS) and the Columbia Children's Center Environmental Health (CCCEH) birth cohort. DNA extracted from floor dust samples was analyzed using quantitative polymerase chain reaction and next-generation sequencing in order to obtain total fungal concentration, fungal diversity metrics, and concentration of individual fungal taxa. Fungal diversity was significantly and inversely associated with asthma symptom freq (open full item for complete abstract)

Committee: Karen Dannemiller PhD (Advisor); Jeffrey Bielicki PhD (Committee Member); Jiyoung Lee PhD (Committee Member); Virginia Rich PhD (Committee Member); Matthew Perzanowski PhD (Committee Member) Subjects: Environmental Health; Microbiology

Master of Science (MS), Bowling Green State University, 2021, Biological Sciences

Pharmaceutical companies have slowed the discovery and development of antibiotics due to low-profit margins. Therefore, antibiotic discovery is at an all-time low, and pathogens have evolved resistance to all currently available drugs. As a result, multi-drug resistant (MDR) bacterial infections are becoming more difficult to treat, especially in individuals at a high risk for infection such as cystic fibrosis (CF) patients. CF is a genetically inherited disease that inhibits or decreases chloride ion transport across epithelial cell membranes, resulting increased mucus viscosity, impairing normal clearance in the lungs. This environment is ideal for bacterial colonization and leads to a chronic lung infection. A major pathogen that colonizes the CF lung over time is Pseudomonas aeruginosa. A promising alternative treatment against MDR P. aeruginosa is bacteriophage therapy which has several advantages compared to antibiotics. First, phage therapy exhibits minimal side effects because phage are highly host-specific and do not inhibit other bacteria that are part of the human microbiome. Second, phage replicate itself exponentially when killing its host; and third, phage can be applied directly to the site of infection. However, like antibiotics, bacteria can evolve resistance to phage. To circumvent the problem of phage and drug resistance, trade-off effects may promote opportunities against both entities that may be exploited to treat MDR infections. I hypothesize that the effectiveness of antibiotics can be restored after selective pressure from bacteriophage. To test this hypothesis, MDR P. aeruginosa strains were exposed to phage in trade-off experiments, and results showed that the evolved phage resistant P. aeruginosa strain became antibiotic susceptible. In one trade-off experiment, a temperate phage recombined in the P. aeruginosa pathogen at a location downstream of a multidrug resistance efflux pump that may directly affect antibiotic susceptibility. In an (open full item for complete abstract)

Committee: Hans Wildschutte Ph.D (Advisor); George Bullerjahn Ph.D (Committee Member); Ray Larsen Ph.D (Committee Member) Subjects: Bioinformatics; Biology; Biomedical Research; Microbiology; Molecular Biology

Master of Science (MS), Bowling Green State University, 2021, Biological Sciences

Aquaculture is one of the fastest growing food sectors of the world economy and reached a record high of 178.5 million tonnes in 2018. Saprolegnia sp. is a fish pathogen with a wide range of hosts including all salmonids. It is responsible for millions of dollars of damage annually to aquaculture. It is estimated that roughly ten percent of all hatched salmon succumb to Saprolegnia infections. The resulting economic losses range in the tens of millions of dollars. Early detection in large bodies of water can prove difficult with current capture methods. This study was conducted to determine if Daphnia magna, a filter feeder could be used as a biocontrol agent and as an indicator for the presence of small amounts of Saprolegnia zoospores in water. Time-lapse images of zoospores were documented at different time points (0-, 30-, 60-, and 90-minutes) after the introduction of Daphnia magna and plates were allowed 48 hours for encysted zoospores to germinate. By 90 minutes, number of visual zoospores had declined significantly compared to controls. After 48-hour incubation control had mycelial growth while Daphnia exposed plates did not, indicating that Daphnia magna did consume zoospores and could be used as a biocontrol agent to control Saprolegnia spp.. To determine if Daphnia magna can be used for detection of Saprolegnia spp., quantitative PCR (qPCR) with Saprolegnia specific primers targeting a cytochrome oxidase subunit I gene, was utilized. In Daphnia magna that had consumed zoospores at different times (5,10, 30, 40, and 50 minutes), qPCR failed to detect Saprolegnia spp.. This study indicates Daphnia magna does intake zoospores, indicating that it has potential to be used as a biocontrol agent against Saprolegnia infection. However, the DNA of Saprolegnia degrades too quickly once were inside Daphnia magna and cannot be used as an indicator for the presence of Saprolegnia spp.

Committee: Vipaporn Phuntumart PhD (Advisor); Raymond Larsen PhD (Committee Member); Paul Morris PhD (Committee Member) Subjects: Biology

Master of Science (MS), Bowling Green State University, 2021, Forensic Science

Microbial genetics is an emerging area of study within the field of forensic science. Topics of research include bioterrorism investigation and the relevance of microbes in human identification. In the area of forensic DNA analysis, a developing method includes the supplementation of human short tandem repeat profiles with microbiome profiles; however, studies concerning the collection of microbial DNA in a forensic context are scarce. Thus, the primary aim of this study was to evaluate the relative yield of microbial DNA from cotton and flocked swabs, both common tools in the discipline of forensic biology. To pursue this aim, the relative DNA yield from Proteus mirabilis samples pipetted onto the swabs was compared via qPCR. Compared to the DNA recovery of the non-manipulated positive control, Copan FLOQSwabs and Puritan cotton swabs yielded 17.52 % ± 2.47% (mean ± standard error) and 61.13% ± 13.39%, respectively. Furthermore, the manipulation control group, which demonstrated the effects of vortexing and centrifugation on DNA yield, resulted in a 61.56% ± 8.02% recovery compared to the positive control. The DNA recovery from the cotton swabs was not significantly different than the DNA recovery for the manipulation controls, but the recovery of microbial DNA using the flocked swabs was reduced significantly as compared to either the cotton swabs or the manipulation controls. Such results illustrate a greater DNA recovery ability of cotton swabs relative to Copan FLOQSwabs. However, further studies regarding the relative collection abilities of these swabs are required to fully assess their performance under field conditions.

Committee: Vipaporn Phuntumart PhD (Advisor); Travis Worst PhD (Committee Member); Crystal Oechsle PhD (Committee Member) Subjects: Biology; Genetics; Microbiology

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

Cauliflower mosaic virus (CaMV) is a member part of the Caulimoviridae, the virions of which, contain a circular, double-stranded DNA (dsDNA) genome. The 8kb CaMV genome encodes 7 different proteins (P1-P7). The non-enveloped, 50nm in diameter icosahedral (T=7) virions are mainly composed of the capsid protein (P4). The virion-associated protein, P3 acts as a linker joining virus particles to either the aphid transmission factor, P2 or the movement protein, P1. CaMV particles can be transmitted either by mechanical inoculation or by an insect vector (aphid). CaMV mainly infects members of the Brassicaceae (including: radish, canola, mustard, cauliflower, cabbage and broccoli). Two strains of CaMV (W260, D4) also have been found to infect species within the Solanaceae species such as Datura stramonium (devils's trumpet) and Nicotiana tabacum (tobacco). In agriculture, CaMV is a widely-distributed virus that is primarily found in temperate regions. CaMV-infected plants show yield reductions of 20-50% and can be detected in about >70% of crops. Unfortunately, tools for managing CaMV infections (isolation and aphid management) are of limited efficacy. However, to develop better ways to manage CaMV infection, we must first explore how the virus propagates within its host. A characteristic cytopathic effect of a CaMV infection is the formation of viroplasms or more commonly known as inclusion bodies (IBs). IBs are cytoplasmic structures, formed by CaMV where virus protein synthesis, genome replication, and virion assembly occur within an cell. CaMV P6 protein is the major constituent of IBs. Expression of P6 tagged on its C-terminus with GFP (P6-GFP) produces fluorescent green foci, visible by confocal laser-scanning microscopy, that were later demonstrated to be IBs. P6 contains 4 self-association domains (D1-D4). Here we show that deletion of any of these domains alters the formation of IBs. Surprisingly, deletion of D1, D2, or D3 individually did not abolish the (open full item for complete abstract)

Committee: Scott Leisner (Committee Chair); John Gray (Committee Member); Lirim Shemshedini (Committee Member); Qian Chen (Committee Member); Travis Taylor (Committee Member); Donald Ronning (Committee Member) Subjects: Molecular Biology; Virology

Master of Science (MS), Bowling Green State University, 2020, Biological Sciences

Cyanobacterial harmful algal blooms threaten freshwater quality and human health around the world. One specific threat is the ability of some cyanobacteria to produce multiple types of toxins, including a range of neurotoxins called saxitoxins. While it is not completely understood, the general consensus is environmental factors like phosphorus, nitrogen, and light availability, may be driving forces in saxitoxin production. Recent surveys have determined saxitoxin and potential saxitoxin producing cyanobacterial species in both lakes and rivers across the United States and Ohio. Research evaluating benthic cyanobacterial blooms determined benthic cyanobacteria as a source for saxitoxin production in systems, specifically rivers. Currently, little is known about when, where, why, or who is producing saxitoxin in Ohio, and even less is known about the role benthic cyanobacterial blooms play in Ohio waterways. With increased detections of saxitoxin, the saxitoxin biosynthesis gene sxtA, and saxitoxin producing species in both the Western Basin of Lake Erie and the lake's major tributary the Maumee River, seasonal sampling was conducted to monitor saxitoxin in both systems. The sampling took place from late spring to early autumn of 2018 and 2019. Monitoring including bi-/weekly water column sampling in the Maumee River and Lake Erie and Nutrient Diffusing Substrate (NDS) Experiments, were completed to evaluate saxitoxin, sxtA, potential environmental drivers, and benthic production. Overall, saxitoxin and sxtA was found throughout the entirety of the Ohio's portion of the Maumee River and east of the Lake Erie Islands during both years. Detections included sxtA peaks in July and saxitoxin detections as early as May and as late as October for planktonic samples. However, benthic experiments suggested higher saxitoxin production in September and October. In general, low correlations were found between qPCR detections, nutrients, and toxin detections, however; ELISA an (open full item for complete abstract)

Committee: Timothy Davis (Advisor); George Bullerjahn (Committee Member); Justin Chaffin (Committee Member) Subjects: Biology; Ecology; Freshwater Ecology; Microbiology

Master of Science, The Ohio State University, 2020, Animal Sciences

Necrotic enteritis (NE) is a multifactorial disease caused by Clostridium perfringens. A way to examine treatments on NE is through methods like body weight gain or lesion scores. Quantification of C. perfringens could be additional measurement. In experiment 3.1, four published extraction protocols, and a new protocol were compared to determine which would be ideal for extracting bacterial DNA. After extraction, DNA concentrations were measured with quality assessed, and a qPCR standard curve was established for each protocol. For Protocols 1 to 5, mean DNA yields and A260/280/A260/230 ratios were 459.02μg/g and 2.18/1.26, 989.66μg/g and 2.07/2.06, 110.71μg/g and 1.58/0.89, 437.03μg/g and 2.07/1.93 and 1,794.90μg/g and 1.91/1.42, respectively. Amplification efficiency percentages and standard curve regression coefficient values for Protocols 1 to 5 were 84.69%/1.00, 79.76%/0.99, 100.30%/1.00, 80.93%/0.99 and 95.76%/0.99, respectively. There was extensive variation in DNA quantity, quality, and PCR efficiency, highlighting challenges of DNA extraction from intestinal samples. Protocol 2 was identified as ideal based on DNA quantity and quality, and low standard deviations between qPCR samples. Experiments 4.1 and 4.2 compared qPCR and direct plating of fecal, ileal and cecal samples for C. perfringens quantification. In experiment 4.1, two groups were compared; an un-inoculated control and an inoculated group, while in experiment 4.2, the two groups compared were an un-inoculated control, fed a corn-based diet and an inoculated rye-based diet group. For quantifying C. perfringens, qPCR and two recently developed selective agar types were compared, with results analyzed by ANOVA factorials and Tukey's Honestly Significant Difference test. All methods were further evaluated using relative standard deviation of CFU/g percentage (RSD%) values. In experiment 4.1, quantification method was a significant variable (p<0.05), with significant differences between qPCR and C. p (open full item for complete abstract)

Committee: Lisa Bielke (Advisor); Thaddeus Ezeji (Committee Member); Zhongtang Yu (Committee Member) Subjects: Animal Diseases; Microbiology; Molecular Biology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2019, Biological Sciences

Saprolegniasis, caused by oomycete pathogens of the Saprolegnia genus is a serious emergent disease of fish causing losses worth approximately $40 million annually in USA. This study aims to analyze the efficacy of copper sulfate and peracetic acid as chemical agents to combat saprolegniasis; and to develop a molecular strategy for the rapid, sensitive and specific detection of Saprolegnia spp., suitable for on-site applications. The study indicates that copper sulfate and peracetic acid effectively reduced the various parameters of Saprolegnia spp. growth. Peracetic acid was effective against other oomycete pathogens to varying degrees, and its persistence in environmental water samples depends on the organic matter content of the water samples. Peracetic acid has been proposed as an effective, non-toxic, and eco-friendly approach to combat saprolegniasis. This study reports the isolation Saprolegnia spp. from various sources. Using cytochrome c oxidase subunit I (COI) and Internal Transcribed Spacer of rDNA (ITS) as molecular markers, these have been identified phylogenetically. Based on these markers, qPCR primers have been developed specific to the Saprolegnia genus and could detect as low as 2pg of Saprolegnia spp. genomic DNA. Also, qPCR based absolute quantification could be used as an approach to quantify the Saprolegnia spp. levels in environmental samples. Additionally, a LAMP assay was developed using the ITS marker. The established LAMP assay was specific to the Saprolegnia genus and could detect as low as 10 fg of Saprolegnia spp. genomic DNA within 30 min, thus making it significantly more sensitive compared to qPCR. Both qPCR and LAMP could also detect as low as 1 zoospore directly. The LAMP reactions could be performed using a simple equipment such as heat block, and results could be detected visually. Further, LAMP has the potential for direct on-field applications for detecting Saprolegnia spp. from water samples collected from Recirculating Aqu (open full item for complete abstract)

Committee: Vipaporn Phuntumart Dr. (Advisor); Lubomir Popov Dr. (Other); Raymond Larsen Dr. (Committee Member); Paul Morris Dr. (Committee Member); Scott Rogers Dr. (Committee Member); David Straus Dr. (Committee Member) Subjects: Biology

Master of Science, The Ohio State University, 2019, Environmental Science

Asthma represents both a significant health and economic burden worldwide and is expected to increase in prevalence in the future, especially among high prevalence regions in high income countries (HIC) and in some low to middle income countries (LMIC). This is partly due to growing inclusiveness in our definition of asthma and is likely related to increasing rates of sensitization and allergy, which in turn may be a result of changing environmental factors, including microbial and pollutant exposures. Asthma is a complex respiratory disease with multiple subtypes, and severity is affected by both genetics and environmental exposures. One exposure, fungal diversity, is known to be inversely associated with asthma prevalence. Exposure to mold in housing is also associated with negative respiratory effects. Fungal exposures can be assessed from indoor environmental samples via relative abundance data obtained through next-generation DNA sequencing. However, relative abundances do not provide information about total fungal load. This factor is crucial for symptom severity among non-allergic asthmatics especially. Additionally, excluding this quantitative information may mask other associations. Combining sequencing data with absolute quantification of spore equivalents—obtained using qualitative polymerase chain reaction (qPCR)—offers an improved characterization of exposure. The goal of this study was to compare differences in analysis results between relative abundance and absolute quantification, and explore associations between quantitative fungal exposures and asthma outcomes in children. Dust samples were collected from bedroom floors in homes of asthmatic children classified as living in neighborhoods with either higher or lower asthma prevalence in the Neighborhood Asthma and Allergy Study in New York City. We homogenized dust through sieving and performed DNA extractions on samples of known mass. We analyzed DNA samples using qPCR to measure total fungal spore (open full item for complete abstract)

Committee: Karen Dannemiller PhD (Advisor); Jeffrey Bielicki PhD (Committee Member); Matthew Perzanowski PhD (Committee Member); Christopher Weghorst PhD (Committee Member) Subjects: Environmental Health; Microbiology

Doctor of Philosophy, The Ohio State University, 2017, Plant Pathology

In Ohio, soybean yield can be affected by several diseases caused by soil borne pathogens. In the past decade, there have been consistent annual outbreaks of Sclerotinia stem rot (SSR), caused by the fungal pathogen Sclerotinia sclerotiorum, in Northeast and Northcentral regions of Ohio. Management guidelines for SSR were more than 10 years old, and were developed based on older fungicides products and planting of highly susceptible cultivars. The objective of the first study was to evaluate the effect of soybean cultivars (MR, moderately resistant; MS, moderately susceptible) and chemical treatment programs on disease development and soybean yield in field trials conducted over three years. Out of the 12 environments (field/year), SSR was observed in 6 of them. Three environments had disease incidence in the untreated check higher than 20%, and therefore, were used for further statistical analysis. Significantly lower levels of SSR were consistently observed in MR cultivars. Both boscalid fungicide and lactofen herbicide reduced SSR on the MS cultivars. Pyraclostrobin fungicide increased SSR compared to the untreated check. In the fields where disease did not develop, chemical treatment did not increase yield, nor was the yield from the MR cultivar significantly different from the MS cultivar. Thus, MR cultivars alone were effective for management of SSR in Ohio soybean fields. An unexpected increase in incidence and severity of SSR was observed following application of pyraclostrobin, a strobilurin fungicide, compared to the untreated check in several field trials. There are numerous studies indicating that strobilurin fungicides can affect plant physiology, and one of the most common effects reported is delay in senescence, which is attributed to reduction in ethylene synthesis. Ethylene can either be directly or indirectly involved in plant defense response to necrotrophic pathogens. Therefore, the patterns of expression of defense-related genes were studied i (open full item for complete abstract)

Committee: Anne Dorrance Dr. (Advisor); Christopher Taylor Dr. (Committee Member); Laurence Madden Dr. (Committee Member); Pierce Paul Dr. (Committee Member); Sally Miller Dr. (Committee Member) Subjects: Agriculture; Agronomy; Biology; Plant Biology; Plant Pathology; Plant Sciences

Master of Science, The Ohio State University, 2017, Comparative and Veterinary Medicine

Enteric coronaviruses (CoVs) of pigs include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine delta coronavirus (PDCoV). They cause significant economic losses in pig industry worldwide. In the US, PEDV emerged in 2013 and caused the death of over 7 million piglets within the first year. Feral pigs have contributed to the transmission of different viral, bacterial and parasitic infections to domestic pigs, other domestic animals and even to humans. However, their role in maintenance and transmission of porcine enteric CoVs is not well understood. In this study, fecal samples of 44 feral pigs were collected from California, USA in early-mid 2016. RNA was extracted from the fecal content supernatant and subjected to conventional reverse transcription (RT)-PCR using two different panCoV primers to detect the presence or absence of porcine enteric CoVs. Quantitative RT-PCR (RT-qPCR) using PEDV N gene and PDCoV M gene specific primers and probes were also performed for amplification of specific viral genes. DNA sequencing of PCR products from likely positive sample was also performed for further confirmation. None of the samples were clearly positive using conventional RT-PCR assays. Few samples were doubtful for PEDV or PDCoV genes and showed Ct values slightly above the detection limit. Repeated testing and/or DNA sequencing confirmed that those samples were negative for PEDV or PDCoV presence. Our results indicated that no porcine enteric CoVs were circulating in the feral pigs in California in early-mid 2016. Similar studies from other states of USA with larger sample sizes will be helpful to ascertain whether wild pigs in USA are playing a role in the maintenance and transmission of porcine enteric CoV or not.

Committee: Qiuhong Wang (Advisor); Renukaradhya Jayamurthy Gourapura (Committee Member); Anastasia Nickolaevna Vlasova (Committee Member) Subjects: Epidemiology; Virology

Doctor of Philosophy, The Ohio State University, 2017, Entomology

Mosquitoes are the most dangerous animals on the planet due to the pathogens they transmit to humans. The yellow fever mosquito, Aedes aegypti, is the study organism of this dissertation, as well as the primary vector for the viruses that cause yellow, dengue, chikungunya, and Zika fevers in humans. Unfortunately, many of these diseases lack effective vaccinations and/or therapeutics and instead must be prevented through control of the mosquito vector. Our current approach to mosquito control relies primarily on the use of insecticides to suppress mosquito populations. While these chemicals are incredibly effective at killing mosquitoes, they also exert a strong selective pressure driving the evolution of resistance. In order to combat this resistance, current compounds must be modified and new targets need to be identified. In this dissertation I focus on the latter with mosquito gap junctions as my potential targets of interest. Gap junctions are intercellular channels that mediate direct communication between adjacent cells via the transfer of small molecules and/or ions. Gap junctions are formed by protein subunits known as connexins in vertebrates and innexins in invertebrates, which are evolutionarily distinct sharing no significant amino acid homology. Despite their distinct evolutionary origins, both connexin and innexin formed gap junctions play integral roles in processes from embryogenesis to reproduction. Here I expand the knowledge base of mosquito gap junctions through examination of gene expression, iii protein localization, pharmacological inhibition, and RNAi based gene knockdown. I find that gap junction inhibitors kill and incapacitate larval and adult female mosquitoes, and inhibit diuresis in adult females. Moreover, RNAi to knockdown the mRNA expression of innexin genes decreases survival. Additionally, I show splice variation for 2 innexin mRNAs, describe innexin gene expression throughout the animal, localize the inx3 protein, and character (open full item for complete abstract)

Committee: Peter Piermarini Ph.D. (Advisor); Denlinger David Ph.D. (Committee Member); Anelli Carol Ph.D. (Committee Member); Reed Johnson Ph.D. (Committee Member) Subjects: Entomology

Master of Science in Biological Sciences, Youngstown State University, 2016, Department of Biological Sciences and Chemistry

As a filamentous fungus, Neurospora crassa serves as an ideal model for eukaryotic organisms. Like many fungi, Neurospora is able to utilize many different carbon sources for energy. This however, requires the presence of genes that code for a variety of metabolic pathways that are not always needed. An example of such a group of genes would be the genes involved in utilizing quinic acid. When Neurospora grows in the presence of a less preferred carbon source, such as quinic acid, gene expression of the quinic acid (qa) gene cluster is up-regulated. This allows the organism to metabolize quinic acid and survive in the less favorable conditions. In contrast, when in the presence of a preferred carbon source, such as dextrose or sucrose, the qa genes are repressed. This study examines how changing the carbon source effects gene expression in wild-type N.crassa. N.crassa was first grown in presence of either quinic acid or sucrose and harvested for tissue. Then, protein was extracted from this tissue and analyzed by 1- Dimensional Gel Electrophoresis (1-DGE). Differences in protein expression was compared using the Quantity One® 1-D analysis software. Proteins unique to growth on quinic acid were identified after being submitted for mass spectrometry. Finally, gene transcription was quantitated in order to determine which genes coded for proteins expressed only in the presence of quinic acid by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

Committee: David K. Asch PhD (Advisor); Gary R. Walker PhD (Committee Member); Jonathan J. Caguiat PhD (Committee Member) Subjects: Bioinformatics; Biology; Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2017, Comparative and Veterinary Medicine

Mycoplasma bovigenitalium (Mbvg) is a common inhabitant of the reproductive tract of diseased and apparently normal cattle. The bovine mycoplasma has been associated with multiple reproductive tract disorders in both male and female cattle. Additionally, Mbvg can colonize the prepuce and distal urethra of bulls, often without producing disease, resulting in Mbvg contaminated semen. The use of Mbvg-contaminated semen for AI has been reported as a potential source of infection for the bovine female genital tract. Although the pathogenic nature of Mbvg has been reproduced experimentally, it is not evident what triggers certain animals to exhibit disease while others remain asymptomatic carriers. Therefore, it is critical to increase the sensitivity of Mbvg detection as well as improve our knowledge regarding the pathogenicity and epidemiology of Mbvg. The first objective of this study was to develop and validate a TaqMan qPCR for the rapid detection of Mbvg in bovine semen. The assay exclusively amplified 81 field and laboratory strains of Mbvg when tested against a panel 15 bovine mycoplasma species. Fresh bovine semen samples (n=77) were assessed for Mbvg by qPCR and culture. M. bovigenitalium was recovered in 57 (74%) semen cultures and detected by qPCR in 62 (80.5%) samples. The rapid nature of the assay (results obtained in 5 h) may provide an alternative diagnostic and research tool for the detection and quantification of Mbvg in bovine semen samples as opposed to traditional culture, which requires up to 10 days. The second objective of this study was to develop a Multilocus Sequence Typing (MLST) assay for Mbvg strain differentiation. The sequences of three targeted loci (ppa, tpiA, rpoB) were analyzed from a collection of 86 Mbvg isolates from around the world. A high degree of nucleotide variability was observed and the isolates were organized into 61 strain types (ST). A phylogenetic tree constructed from the concatenated sequences displayed coherent (open full item for complete abstract)

Committee: Thomas Wittum (Advisor); Gustavo Schuenemann (Advisor); Gregory Habing (Committee Member); Christopher Premanandan (Committee Member); James M. DeJarnette (Committee Member) Subjects: Animal Sciences; Epidemiology; Microbiology; Molecular Biology

Master of Science in Biological Sciences, Youngstown State University, 2016, Department of Biological Sciences and Chemistry

Neurospora crassa, a model filamentous fungus, contains the quinic acid (qa) gene cluster, allowing the organism to effectively metabolize quinic acid as an alternative carbon source. In the presence of quinic acid, the qa gene cluster is significantly unregulated, showing an increased expression of both regulatory proteins as well as the structural proteins required for the utilization of quinic acid as an energy source. Of these structural genes, qa-2, qa-3, and qa-4 encode for the enzymes required for the conversion of quinic acid to protocathechuic acid while qa-y encodes for a quinate permease and the function of qa-x remains unknown.The present study aims to elucidate the expression pattern of qa-y, the quinate permease-producing gene required for efficient uptake and utilization of quinic acid in N. crassa, as well as qa-1F, the activator protein of the qa gene cluster, at differing times of quinic acid exposure. Quantitative real-time PCR (RT-qPCR) using reverse transcriptase was first used to determine Histone-3 as the optimal housekeeping reference gene. This gene was then used to accurately quantify the expression of both qa-y and qa-1F at differing times of exposure to quinic acid (0min, 15min, 30min, 1hr, 2hr, and 3hr). The expression of qa-y was observed to steadily increase for the first 30 minutes, showing up to a 17-fold change in expression after only 30 minutes following introduction of quinic acid. A prompt response of the qa-1F gene was also observed. At the 15-minute time point, a 3-fold increase in expression of the qa-1F occurred, presumably to initiate activation of transcription of the genes of the qa gene cluster, including itself. When combined, these data allow for a comprehensive representation of the timing and method of regulation of the qa gene cluster in N. crassa.

Committee: David Asch PhD (Advisor); Jonathan Caguiat PhD (Committee Member); Gary Walker PhD (Committee Member) Subjects: Biochemistry; Bioinformatics; Biology; Cellular Biology; Molecular Biology

Master of Science, The Ohio State University, 2014, Animal Sciences

A fermentation extract of the fungus Aspergillus oryzae can be utilized as a direct fed microbial. The objective was to determine if dietary inclusion of an extract of A. oryzae would alter growth parameters including rumen development of Holstein bull calves from birth thru 1 wk post weaning; it was hypothesized that it would. Bull calves (n=52) were used in this experiment. Calves were randomly assigned to a slaughter age, 4 wk (n = 16) or 8 wk (n = 36) and treatment, control (CON; n = 27) or direct fed microbial (AMF; n = 25). Calves averaged 43.2 ± 1.0 kg BW and 2.8 ± 0.3 days of age at the beginning of the experiment. Calves were housed and fed individually; no bedding was used. Calves assigned to AMF were fed 2 g of AMF daily. Liquid DFM was delivered in milk replacer for the first 4 wk of the trial; solid AMF was top-dressed on texturized starter thereafter. Calves were fed non-medicated milk replacer twice daily (22.0% CP, 20.0% fat DM basis; 680 g/d) and were weaned upon consumption of 0.91 kg of starter (20% CP, 2.0% fat; medicated with decoquinate) for 3 consecutive days or on d 45 of the study, whichever came first. Calves had ad libitum access to starter and water throughout the trial. Feed intake was recorded daily. Body weight (BW), withers height (WH), and hip height (HH) were recorded weekly. At 4 wk of age calves from each treatment (n = 8) were slaughtered and their rumen tissue/contents were weighed and sampled for gross, histological, and molecular measurements, pH, and lactate concentrations to assess rumen development. The remaining calves from each treatment (n = 16) were slaughtered at 8 wk of age and the same procedure for sample collection was followed. Masson's Trichrome stain to measure the different tissue layers of the rumen epithelium and quantitative PCR (qPCR) to measure rumen epithelial genes as well as microbial populations in the rumen contents. There was no effect of treatment on BW, WH, HH, average daily gain (ADG), feed effici (open full item for complete abstract)

Committee: Kristy Daniels (Advisor); Francis Fluharty (Committee Member); Zhongtang Yu (Committee Member) Subjects: Animal Sciences

MS, Kent State University, 2014, College of Arts and Sciences / Department of Biological Sciences

Recent studies suggest that the neuropeptide oxytocin (Oxt) may be important for organizing the neural circuitry that regulates adult male aggressive behavior. Specifically, male Oxt knockout (Oxt -/-) and male Oxt receptor knockout (Oxtr -/-) mice have heightened aggressive behavior in adulthood, which is thought to be due to an absence of Oxt signaling during development. These data contrast with that from male forebrain specific Oxtr knockout mice, in which the Oxtr gene is excised 21-28 days after birth and have normal male aggressive behavior. Based on these data it has been hypothesized that fetal exposure to Oxt is necessary for normal displays of aggressive behavior in adulthood. To investigate this, initial expression of mouse Oxt and Oxtr mRNA as well as Oxtr protein was examined. Then, using the time point for Oxtr protein initial expression, a transuterine injection of an oxytocin antagonist (OTA) was administered to block fetal Oxtr signaling to examine if it modulates adult intermale aggression. Results indicate Oxtr mRNA is present before the Oxtr peptide’s expression on embryonic (E) day 16 and a sex difference in Oxt mRNA initial expression. Preliminary data from the OTA administration suggests increased adult intermale aggressive behavior in E14 injected mice.

Committee: Heather Caldwell (Advisor); Colleen Novak (Committee Member); Wilson Chung (Committee Member) Subjects: Behavioral Sciences; Biology; Developmental Biology; Endocrinology