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

Adenovirus infection mostly triggers cold-like symptoms, but in some cases, adenovirus infection may lead to severe symptoms and death. The primary receptor for many adenoviruses is the Coxsackievirus and Adenovirus receptor (CAR). There are two transmembrane isoforms of CAR; CAREx7 and CAREx8. CAREx7 localizes at the basolateral surface and CAREx8 localizes at the apical surface. I hypothesized that knockout of CAREx8 will prevent apical adenovirus transduction, while knockout of all CAR isoforms will prevent apical and basolateral adenovirus transduction. To test this, I utilized CRISPR/Cas9 to knockout endogenous CAREx8 or all CAR isoforms in MDCK-CAREx8 cells. The results indicate that knockdown of CAREx8 significantly decreased apical adenovirus transduction. Moreover, the knockdown of total CAR resulted in a decline in all adenovirus transduction. The data indicate that CAREx8 localization promotes adenovirus entry, which might provide potential targets for adenovirus gene therapy.

Committee: Katherine J. Excoffon Ph.D. (Advisor); Abimbola O. Kolawole Ph.D. (Advisor); Quan Zhong Ph.D. (Committee Member) Subjects: Biology; Molecular Biology; Virology

Doctor of Philosophy, Miami University, 2017, Cell, Molecular and Structural Biology (CMSB)

The first section of this dissertation describes the development of strategies for the treatment of pancreatic cancer using a novel synthetic DNA sequence that functions as decoy binding site for an oncogenic protein called high mobility group A (HMGA) and adenovirus mediated gene therapy. The second part of this dissertation describes a survey study for evaluating the antibacterial activity of mushroom samples collected from local areas. The Chapter 1 provides background information about the HMGA and its role in tumor development and cancer progression. The Chapter 1 also provides background information about the adenovirus and its use in cancer treatment. In addition, Chapter 1 discusses about the medicinal use and antibacterial potential of mushrooms. In Chapter 2, we have devised a strategy of using engineered replication-defective adenovirus (Ad) to deliver decoy hyper binding sites for HMGA to the nucleus of cancer cells with the goal of sequestering excess HMGA at the decoy hyper binding sites due to binding competition. Sequestration of excess HMGA at the decoy binding sites is intended to reduce HMGA binding at the naturally occurring genomic HMGA binding sites, which should result in normalized gene expression and restored sensitivity to chemotherapy. Infection of five different pancreatic and liver cancer cell lines with the Ad containing the HMGA decoy hyper binding sites resulted in significant reduction in cell viability and increased sensitivity to chemotherapy. The Chapter 3 discusses toxicity and biodistribution following injection of the Ad containing the HMGA decoy hyper binding sites into mice liver or pancreas. This study provides essential preclinical information regarding toxicity and biodistribution needed to determine the potential of our engineered virus for future treatment of cancer in humans. The Cahpter 4 demonstrated a strategy to develop a conditionally replicating Ad virus which will selectively replicate in cancer cells while being (open full item for complete abstract)

Committee: Michael Kennedy Dr. (Advisor); Carole Dabney-Smith Dr. (Committee Chair); Rachael Morgan-Kiss Dr. (Committee Member); Gary Lorigan Dr. (Committee Member); David Tierney Dr. (Committee Member) Subjects: Biochemistry; Molecular Biology

Master of Science (MS), Wright State University, 2023, Biochemistry and Molecular Biology

Current surveillance focuses on well characterized pathogens such as influenza. Since 2000, there have been multiple outbreaks of respiratory disease. These outbreaks have demonstrated the need for robust and collaborative global efforts to identify, monitor, and contain novel respiratory viruses. This study aims to improve the ability of public health agencies to monitor and respond to respiratory disease outbreaks. Using five respiratory pathogens, this study compares a molecular capture technology from Twist Biosciences to the shotgun sequencing approach of whole transcriptome amplification (WTA, Qiagen) with the goal of determining which method is most effective using cost, usability, and sequencing quality metrics for evaluation. Twist, though more expensive, had a 92.1% positive identification of targets on successful sequencing runs with greater depth and breadth of coverage. WTA failed to sequence and identify targets except Human Adenovirus 7, proving that Twist is more reliable and efficient in this study.

Committee: Richard Chapleau Ph.D. (Committee Co-Chair); Oleg Paliy Ph.D. (Committee Co-Chair); Michael Markey Ph.D. (Committee Member) Subjects: Biology; Molecular Biology; Public Health

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.

Committee: Abimbola O. Kolawole Ph.D. (Committee Co-Chair); Dawn P. Wooley Ph.D. (Committee Co-Chair); Nancy J. Bigley Ph.D. (Committee Member) Subjects: Biochemistry; Immunology; Microbiology; Virology

Master of Science (MS), Wright State University, 2014, Biological Sciences

Adenovirus's primary receptor, the Coxsackievirus and adenovirus receptor (CAR), has two transmembrane isoforms with differential localization within polarized epithelial cells (CAREx7 (basolateral), CAREx8 (apical)). I hypothesized that each isoform of CAR is degraded at different rates and is regulated by Src-family kinases. Increasing or decreasing the concentration of CAR is predicted to directly alter adenoviral entry. To test this hypothesis, inducible Madin Darby Canine Kidney (MDCK) cell lines either expressing CAREx7, CAREx8, or mCherry, under a doxycycline-inducible promoter were used in pulse-chase structured experiments to calculate the half-lives of these proteins. Alternatively, a polarized model lung epithelium (CaLu-3) treated with Src-kinase inhibitor, PP2, was used to test apical CAREx8 expression and viral transduction. Though CAREx7 and CAREx8 did have varying half-lives (5.1 h, 2.0 h respectively), inhibiting Src-family kinases did not decrease CAREx8 expression at early time points but did diminish adenovirus transduction. These data confirm that Src does not play a role in CAR turnover, but remains critical for adenoviral infection.

Committee: Katherine Excoffon Ph.D. (Advisor); Lynn Hartzler Ph.D. (Committee Member); Mill Miller Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology

Doctor of Philosophy (PhD), Wright State University, 2011, Biomedical Sciences PhD

A major factor in virus entry into cells is localization and abundance of the primary receptor. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for group B coxsackievirus and many serotypes of adenovirus. In most epithelia, a seven exon isoform of CAR (CAREx7) is exclusively localized at the basolateral surface where it behaves as a homophilic adhesion protein and is inaccessible for viral infection. However, in well-differentiated human airway epithelia, we recently discovered an alternatively spliced, low abundance isoform of CAR (CAREx8) that is apically localized where it may initiate apical viral infection. The two isoforms differ only in the last 26 (CAREx7) or 13 (CAREx8) amino acids of the cytoplasmic domain, which suggests that some intracellular interactions may differ. One such differential interaction involves MAGI-1, an essential PDZ-domain containing protein known to be involved in cell polarization and cancer. In non-polarized COS7 cells, the CAREx8 protein level is regulated by MAGI-1b. CAR-MAGI-1 interactions were investigated by MAGI-1 siRNA knockdown, in vitro translation, immunocytochemistry, co-immunoprecipitation-Western blot analysis, fluorescence resonance energy transfer, direct binding assays, and adenovirus infection. Data showed that both CAR isoforms were expressed in several cell lines with CAREx7 RNA consistently more highly expressed than CAREx8 and MAGI-1 siRNA knockdown improved adenovirus infection in polarized cells. Both isoforms strongly interacted with PDZ3. CAREx8 also interacted with PDZ1. Whereas co-expression of PDZ1 with CAREx8 did not affect adenovirus infection, it could compete with full length MAGI-1 to protect CAREx8 from loss. Co-expression of CAREx8 with PDZ3 significantly reduced CAREx8 cell surface expression and adenovirus infection but did not reduce total CAREx8 protein levels suggesting that this may be the MAGI-1 PDZ domain responsible for holding CAREx8 within the cell but it is like (open full item for complete abstract)

Committee: Katherine Excoffon PhD (Advisor); Nancy Bigley PhD (Committee Member); Michael Leffak PhD (Committee Chair); Mill Miller PhD (Committee Member); Robert Putnam PhD (Committee Member) Subjects: Biomedical Research

Master of Science (MS), Wright State University, 2023, Biological Sciences

The study was conducted to investigate the prevalence of four enteric viruses in mouse research facilities that may not be captured by routine pathogen screening. Fecal samples were collected from contact-sentinel mouse cages between April 2022 and January 2023, and screened for the presence of viral genome by RT-qPCR. Out of a total of 50 samples, murine norovirus and murine astrovirus were detected in two (4%) and one (2%) sample, respectively, while murine adenovirus and murine rotavirus were not detected. Furthermore, all samples were screened for the presence of infectious murine noroviruses and astroviruses using the RAW264.7 and Vero cells respectively. Interestingly, infectious murine norovirus was observed in ten (26.31%) samples and no infectious murine astrovirus. Using Sanger sequencing, the species of the detected norovirus was determined as murine norovirus type-1 (MNV-1). These findings highlight the need to update health monitoring practices in mouse research facilities and develop tools to monitor emerging viruses.

Committee: Abimbola O. Kolawole Ph.D. (Advisor); Athanasios Bubulya Ph.D. (Committee Member); Quan Zhong Ph.D. (Committee Member) Subjects: Biology

Doctor of Philosophy, Miami University, 2022, Microbiology

The eukaryotic cell cycle is a highly regulated and orderly process comprising DNA replication and mitotic cell division. Progression of the cell cycle is tightly monitored by cell cycle checkpoints, in which DNA damage and genomic integrity are assessed. In the event of exogenous or endogenous DNA damage, cells orchestrate highly sophisticated DNA damage response (DDR) signaling cascades that can initiate DNA repair mechanisms, or induce cell cycle arrest or apoptosis. Adenovirus (Ad) infected cells perceive viral genomes as cellular DNA breaks and activate DDR cascades by via signal transducing kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), and DNA protein kinase (DNA-PK). These kinases activate a myriad of effector kinases some of which can induce G2/M cell cycle arrest. The contribution of Ad-activation of the DDR to the host cell cycle in the context of Ad infection is not clearly understood. The goal of this study is to investigate the role of Ad-induced DDR in the cell cycle profile of Ad infected cells. We show that cells infected with an E4 deletion mutant (1007) activates the G2/M checkpoint in response to virus-induced activation of ATM. 1007 infections accumulate phosphorylated Cdk1 in ATM-dependent manner which prevents mitosis. 1007 infection also results in reduced expression of the mitotic marker, phosphorylated (Ser10) histone 3 (pH3). Ad early proteins E1A and E1B induce the S phase of the cell cycle by promoting cellular DNA synthesis, which can result in the production of aneuploid cells with >4N DNA content. However, viral infection eventually inhibits cellular DNA synthesis during the late phase. We investigated the role of Ad E4 34 kDa in cellular DNA synthesis. Our study shows that an Ad mutant (1010) with a defective E4 34 kDa gene, produces an increased number of aneuploid cells; this suggests a potential role of E4 34 kDa in shutdown of cellular DNA synthesis. 1010 infected cells produced a higher amount of newly (open full item for complete abstract)

Committee: Eileen Bridge (Advisor); Joseph Carlin (Committee Member); Yoshinori Tomoyasu (Committee Member); Timothy Wilson (Committee Member); Xiao-Wen Cheng (Committee Member) Subjects: Microbiology; Virology

Doctor of Philosophy, Miami University, 2022, Microbiology

The stability and integrity of the genetic information inscribed in DNA molecules are essential to all living organisms and are under constitutive surveillance by sensor proteins that will elicit the DNA damage response (DDR) upon detecting DNA lesions. The presence of input and replicating viral genomes in infected cells may also trigger the cellular DDR, as viral genomes and replication intermediates can have structures that mimic features of damaged DNA. Double-stranded DNA breaks (DSBs) can be catastrophic for cell viability and are predominately repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is canonically regulated by the Mre11-Rad50-Nbs1 (MRN) sensor complex. In contrast, NHEJ is mediated by the DNA-dependent protein kinase (DNA-PK) complex, consisting of the Ku70/Ku86 (Ku) sensor proteins and the DNA-PK catalytic subunit. DSBs induced by chemotherapeutics such as etoposide result in DNA ends with covalently-attached proteins. The resolution of these DSBs requires nucleolytic processing by the MRN complex to remove these proteins and complete NHEJ. The molecular mechanisms governing the collaboration of the MRN and DNA-PK complexes in resolving these lesions are not fully understood. The purpose of this study is to investigate the cellular and viral factors that govern DNA-PK activation during adenovirus (Ad) infection. Ad contains a double-stranded DNA genome with a covalently-attached terminal protein (TP) at each 5' end. Since Ad infection elicits DDRs that can obstruct viral propagation, Ad encodes several proteins from early region 4 (E4) that can limit the function of DDR factors. Both E4-11 and -34 kDa interact with DNA-PK and inhibit some of its activities. Infection with an Ad mutant lacking E4 genes (E4-) results in the concatenation of viral genomes via NHEJ. This process requires the removal of the 5' covalently-attached TP by the MRN complex and DNA-PK for NHEJ-mediated ligation. The data indicates viral (open full item for complete abstract)

Committee: Eileen Bridge (Advisor) Subjects: Microbiology

Doctor of Philosophy, The Ohio State University, 2022, Ohio State University Nutrition

Genome editing is one of the most important techniques to investigate gene function of organisms. The advancement of genome editing techniques and the emergence of the CRIPSR/Cas9 system enable scientists to conduct genome editing conveniently and rapidly. In mammals, pronuclear injection of CRISPR/Cas9 has been accepted as a standard genome editing method due to the advantage of using the one-cell stage embryo. In avian species, pronuclear injection is not applicable because of the different reproductive system, including the development of extraembryonic components, egg white, membrane, and eggshell. Instead, genome editing in the avian species has been conducted using primordial germ cells (PGC). However, the conventional PGC-mediated method requires technically demanding in vitro steps including isolation, culture, genome editing, proliferating, and injecting back into recipient embryos. In addition, the germline competence of PGCs is extremely difficult to maintain throughout the complex and long in vitro steps. Therefore, a great need exists for the development of a novel avian genome editing method that can be easily utilized and applied to various avian species. To develop a novel method without the in vitro PGC culture step, PGCs located within avian embryos were targeted directly using an adenovirus containing the CRIPSR/Cas9 system. In the freshly laid eggs, embryos are at the blastodermal stage and PGCs are located in the central region of the blastoderm. Thus, adenovirus was injected into the central region of the quail blastoderm to target the blastodermal PGCs and produce potential germline mutant founders and genome edited offspring. For distinct phenotypic change caused by mutation, the melanophilin (MLPH) gene was targeted and MLPH homozygous mutant quail showed a feather color change from brown to gray. Therefore, production of MLPH mutant offspring demonstrated that the adenovirus-mediated method can induce targeted mutation in the blastoderm (open full item for complete abstract)

Committee: Kichoon Lee (Advisor); Alejandro Relling (Committee Member); Ouliana Ziouzenkova (Committee Member); Michael Davis (Committee Member) Subjects: Animal Sciences; Genetics

Master of Science (MS), Wright State University, 2021, Microbiology and Immunology

Co-infection is common among viruses and bacteria in the human respiratory system. Adenovirus (AdV) and Streptococcus pneumoniae are clinically relevant respiratory pathogens that cause morbidity and mortality in a variety of patient populations with the highest morbidity occurring among immunocompromised individuals, but also prevalent in infants and the elderly. Acute respiratory distress syndrome may become severe in healthy individuals when co-infection with S. pneumoniae and AdV occurs due to synergistic effects of the pathogens on the host. I hypothesized that S. pneumoniae infection decreases AdV transduction of airway epithelia. To test this hypothesis, we utilized the polarized immortalized airway epithelial cell line Calu-3. Calu-3 cells were inoculated with S. pneumoniae followed by the addition of recombinant adenovirus, AdVLacZ. Then, the effect of bacteria on AdV transduction into Calu-3 cells was determined by quantifying β-galactosidase. Cell-associated viral genomes were measured by quantitative polymerase chain reaction. These results showed that S. pneumoniae infection decreased significantly AdV transduction in Calu-3 cells, but there was no significant difference in quantity of cell-associated AdV genomes.

Committee: Dawn P. Wooley Ph.D. (Committee Chair); Nancy J. Bigley Ph.D. (Committee Member); Marjorie M. Markopoulos Ph.D. (Committee Member); Lynn K. Hartzler Ph.D. (Committee Member) Subjects: Microbiology; Virology

Master of Science, Miami University, 2021, Microbiology

Adenovirus type 5 (Ad5) is used as a model system to study the DNA damage response. Ad5 mutants that lack the early region 4 (E4-) activate the DNA damage response, which is detrimental to a productive life cycle. Ataxia-telangiectasia mutated kinase (ATM) is a DDR signal transducer which interferes with viral DNA replication. We found recruitment of phosphorylated ATM to replication centers was important for inhibition of E4- viral replication. Recruitment of DDR factors was ATM-dependent suggesting that ATM alters viral chromatin at replication centers. We analyzed viral DNA configuration using micrococcal nuclease digestion and observed wild-type Ad5 DNA was resistant to digestion while E4- DNA was sensitive to digestion. Treatment with an ATM inhibitor increased the fraction of E4- micrococcal nuclease-resistant fraction. The deacetylase SIRT7 is a proposed antiviral factor during Ad5 infection. We found SIRT7 was downregulated during Ad5 infection and E4orf6 is important for SIRT7 degradation. We observed when SIRT7 was knocked down prior to infection, an increase in viral DNA replication and late gene expression, but not early gene expression was observed in Ad5 and E4-. These findings suggest that a function of SIRT7 is inhibitory to the life cycle of wild-type Ad5 and E4- mutant.

Committee: Eileen Bridge (Advisor); Joseph Carlin (Committee Member); Mitchell Balish (Committee Member) Subjects: Microbiology; Molecular Biology; Virology

Master of Science, Miami University, 2020, Microbiology

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are epigenetic and non-epigenetic modifiers that are important for regulating cellular processes. Viruses are able to exploit their activities to benefit the viral life cycle at the expense of the host. Both HATs and HDACs have important roles in cellular proliferation and manipulating these activities has been shown to increase the development of diseases such as cancer. The HAT p300/CBP and the HDAC SIRT7 both target histone 3 lysine 18 (H3K18) and are manipulated by early adenovirus proteins, suggesting that p300/CBP and sirtuin activities are important for viral growth and cellular proliferation. Inhibition of p300/CBP activity did not affect cellular proliferation but delayed the onset of viral DNA replication, while inhibition of the class III HDACs, the sirtuins, had little effect on either viral or cellular DNA replication. We also determined that H3K18 hypoacetylation begins at the onset of viral DNA replication and the HDAC SIRT7 contributes to the H3K18 hypoacetylation. Knowledge gained from this study has the potential to identify possible therapeutic targets to reduce viral infection or limit the development of disease such as cancer.

Committee: Eileen Bridge Dr. (Advisor); Mitchell Balish Dr. (Committee Member); Rebecca Balish Dr. (Committee Member); Joseph Carlin Dr. (Committee Member) Subjects: Molecular Biology; Virology

Doctor of Philosophy (PhD), Wright State University, 2019, Biomedical Sciences PhD

Human adenoviruses (AdV) are double-stranded DNA viruses that can cause a range of diseases. While AdV respiratory infections are often mild and self-limited, severe manifestations such as fulminant pneumonia and acute respiratory distress syndrome (ARDS) are not uncommon. In order to identify potential modalities to treat AdV infections and potentially enhance the effectiveness of AdV vector delivery, a thorough mechanistic understanding of how AdV enters host cells is needed. Previous studies have revealed polarized epithelia, one of the primary targets for AdV respiratory infections, are highly resistant to AdV entry from the apical (luminal) surface. However, upon exposure to interleukin 8 (IL-8), the 8-exon encoded isoform of the Coxsackie and Adenovirus receptor (CAREx8) localizes to the apical surface of airway epithelial cells where it is able to mediate AdV entry. Furthermore, neutrophils, or polymorphonuclear leukocytes (PMN), that migrate to the epithelium as a result of the IL-8 stimulus have been shown to further enhance AdV infection of the epithelium through an uncharacterized mechanism. I hypothesized that neutrophilic factors alter epithelial physiology in such a way that renders them more susceptible to AdV infection and that a combination of enhanced apical CAREx8 expression and PMN factor signaling drastically enhances epithelial susceptibility to AdV5 infection. In order to address this hypothesis, a variety of pharmacological inhibitors were tested for their ability to influence AdV entry in epithelial cells that had been exposed to PMN or various PMN factors. Furthermore, CRISPR/Cas9 techniques were used to develop a CAREx8-knockdown epithelial model system to address the importance of CAREx8 in AdV entry in the presence and absence of neutrophilic factors. Using these methods, I demonstrate that neutrophil elastase (NE), a PMN serine protease with diverse functions, is a major neutrophilic factor that drives PMN-mediated enhancement of epit (open full item for complete abstract)

Committee: Katherine J.D.A. Excoffon Ph.D. (Advisor); Jeffrey B. Travers M.D., Ph.D. (Committee Member); Dawn P. Wooley Ph.D. (Committee Member); Dan R. Halm Ph.D. (Committee Member); Hongmei Ren Ph.D. (Committee Member) Subjects: Biomedical Research; Cellular Biology; Virology

Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences

The ability of pathogenic viruses to alter host metabolism has been recently characterized. Infection by the human adenovirus serotype 36 (AD36) promotes obesity in animal models and correlates to increased adiposity in humans, yet improves glycemic control. Based on in vitro studies, the E4ORF1 protein is responsible for both the adipogenic and insulin sparing properties of AD36 infection via insulin independent Akt activation. We generated a recombinant adeno-associated viral (rAAV) vector to express AD36E4ORF1. Through intravenous delivery we expressed AD36E4ORF1 in the livers of diabetic, insulin resistant, and wild-type mice. Hepatic AD36E4ORF1 improved glucose tolerance and attenuated hyperglycemia in obese diabetic Db -/- mice without improving insulin sensitivity. AD36E4ORF1 also reduced hyperinsulinemia and improved glucose tolerance in insulin resistant mice with diet induced obesity (DIO). Liver specific glucose uptake was increased without improving insulin sensitivity. Confirming the findings of previous in vitro studies, Akt activity was not only increased but also required for AD36E4ORF1 mediated glucose uptake. AD36E4ORF1 expression is a model of insulin independent AKT activation and provides a novel therapeutic mechanism to improve glycemic control in cases of insulin resistance. Next, we looked at how environmental factors might contribute to healthy aging and prevention of metabolic syndrome. Animals housed in a larger, more complex enriched environment (EE) are provided with increased somatosensory stimulation, physical exercise, and enhanced social interactions. Together, these stimuli increase expression of brain derived neurotrophic factor (BDNF) in the hypothalamus, activating a hypothalamic sympathoneural-adipocyte axis (HSA). In young animals, HSA activation has been shown to improve glycemic control and overall health but its impact in older animals has not been previously characterized. Middle-aged 10 month old female mice were h (open full item for complete abstract)

Committee: Lei Cao Ph.D. (Advisor); Denis Guttridge Ph.D. (Committee Member); Courtney DeVries Ph.D. (Committee Member); Kay Huebner Ph.D. (Committee Member) Subjects: Biomedical Research

Bachelor of Science (BS), Ohio University, 2018, Biological Sciences

Coxsackievirus (CV) infection has been associated with acute kidney damage, a problem that can be life-threatening. An observed lack of kidney damage following CV infection of the non-obese diabetic (NOD) mouse strain suggested a relationship between virus infection and expression of its receptor, Coxsackievirus and Adenovirus receptor (CAR), which could influence the effect of the virus on the kidney. Very little is known about the mechanism of virus-induced kidney injury in humans or in mice, but it appeared that NOD mice may be utilizing a protection mechanism. This project analyzed the influence of CV infection on CAR expression in the kidneys of NOD mice as a first step toward defining the mechanism of protection from viral damage. This study showed that virus can gain access to kidney cells and that receptor expression is affected by viral presence, indicating that decreased access to receptor is limiting viral spread. Because viral infection itself can cause kidney damage in humans and other animal models, defining the mechanism of how NOD mice protect themselves could someday help humans who have been infected with a virus and are at risk for kidney damage.

Committee: Karen Coschigano PhD. (Advisor); Debra Walter MS. (Other) Subjects: Biology; Biomedical Research

Doctor of Philosophy, The Ohio State University, 1970, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

Doctor of Philosophy (PhD), Wright State University, 2014, Biomedical Sciences PhD

The airway epithelium poses a formidable barrier for the entry of pathogenic viruses due to the formation of tight junctions between adjacent epithelial cells. The coxsackievirus and adenovirus receptor (CAR), a member of the Ig superfamily of cell junction adhesion proteins, is the primary receptor for adenovirus entry and infection. As a result of alternative splicing, two transmembrane isoforms of CAR are generated. While the seven-exon isoform of CAR (CAREX7) is hidden on the basolateral surface of polarized epithelia, the eight-exon isoform of CAR (CAREX8) localizes within the sub-apical region and at the air-exposed apical surface. Apical localization of CAREX8 makes it accessible to invading adenovirus entering the lumen of the airway and able to facilitate viral entry into the epithelium. Previous studies have shown that Interleukin-8 (IL-8), a proinflammatory cytokine and a neutrophil chemoattractant, increases the susceptibility of the airway epithelium to adenoviral infection. I hypothesized that the apical CAREX8 protein expression level and localization are responsible for the susceptibility of a polarized epithelium to viral infection. Moreover, I hypothesized that CAREX8expression is tightly regulated by mediators of IL-8 signaling and the endogenous function CAREX8is to tether neutrophils at the apical surface of the polarized epithelium. Finally, I hypothesized that adenovirus has co-opted CAREX8 and neutrophil transmigration to enhance infection of the polarized epithelium. Consistent with these hypotheses, I demonstrate that IL-8 increases the expression and the apical localization of CAREX8 in polarized airway epithelial cells. In addition, IL-8 differentially activates AKT/S6K and inactivates GSK3ß to augment the protein synthesis of CAREX8. Increased CAREX8 is able to mediate increased neutrophil binding at the apical surface of the epithelium that is completely abolished by competition with CAR-binding adenovirus fiber-knob. Finally, I a (open full item for complete abstract)

Committee: Katherine Excoffon Ph.D. (Advisor); David Goldstein Ph.D. (Committee Member); Robert Putnam Ph.D. (Committee Member); Dawn Wooley Ph.D. (Committee Member); Julian Gomez-Cambronero Ph.D. (Committee Member) Subjects: Cellular Biology; Virology

Master of Science, The Ohio State University, 2014, Comparative and Veterinary Medicine

Fetal mesenchymal stem cells (fMSC) have been shown to be superior to adult MSCs (aMSC) for bone tissue engineering applications due to greater proliferation capacity, osteogenic potential, and lower immunogenicity. However, gene delivery to fMSCs may differ from aMSC due to immaturity of cell surface receptors and gene expression pathways. We compared fMSCs to aMSCs for permissiveness to selected chemical transfection reagents and adenoviral (Ad) vectors, gene expression, and osteoinduction using Ad-bone morphogenetic protein-2 (BMP2) in a mouse model. We hypothesized that gene transduction would be efficient in fMSCs without affecting measures of cell stemness in vitro and function as a cell therapy in vivo. Fetal MSCs and aMSCs were compared for efficiency of transduction using green fluorescent protein (GFP) and BMP2, cell proliferation, viability, differentiation capacity, and in vivo osteoinduction as assessed by microradiography and histochemistry. Polyethylenimine (PEI) provided greater transfection and viability in fMSCs than other commercial chemical reagents. Ad transduction efficiency was superior to PEI-mediated transfection of GFP in fMSCs (81.3±1.3% vs 35.0±1.6%; P<0.05) and was similar in aMSCs (78.1±1.9%). Ad transduction had a shorter duration but greater intensity of both GFP and BMP2 expression in fMSCs than in aMSCs. Cell proliferation and osteogenic differentiation in vitro were unimpaired in both fMSCs and aMSCs, but fMSC had reduced chondrogenic differentiation in vitro. Ad-BMP2 tranduced fMSCs produced less ossification in vivo than Ad-BMP2 transduced aMSCs. These results suggested that fetal MSCs may be genetically manipulated ex vivo with Ad vectors without major effects on stemness. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications. Fetal MSC-mediated BMP2 gene delivery and osteogenic induction in vivo was less efficient than adult MSCs.

Committee: Alicia Bertone DVM, PhD (Advisor); Marco Coutinho da Silva DVM, PhD (Committee Member); Tracey Papenfuss DVM, PhD (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Comparative; Health; Medicine; Therapy

Doctor of Philosophy, Miami University, 2014, Microbiology

Cellular genomic integrity is constantly attacked by a variety of exogenous and endogenous agents. In response to damaged DNA, the cell activates a DNA damage response (DDR) pathway to maintain genomic integrity. Cells can also activate DDRs in response to infection with several types of viruses. The cellular DDR pathway involves sensing DNA damage by the Mre11, Rad50, Nbs1 (MRN) sensor complex, which activates downstream ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) kinases. These kinases phosphorylate downstream effector proteins implicated in cell cycle arrest, DNA repair, and, if the damage is irreparable, apoptosis. The induction of DDRs includes focal accumulation and phosphorylation of several DDR proteins. Adenovirus (Ad) mutants that lack early region 4 (E4) activate a cellular DDR. E4 proteins normally inactivate the MRN sensor complex and prevent downstream DDR signaling involved in DNA repair and cell cycle checkpoint arrest in wild-type Ad5 infections. The characteristics of Ad infection that activate the cellular DDR are not well understood. We have investigated the ability of replication defective and replication competent Ad mutants to activate cellular DDRs and G2/M cell cycle arrest. Ad infection induced early focal accumulation of DDR proteins such as Mre11, Mdc1, phosphorylated ATM (pATM), phosphorylated Chk2 (pChk2), and 53BPI, independent of the replication status of the mutants studied. However, Mre11 and pATM foci were transient in replication defective infections and were only maintained in infections with replication competent mutants. Viral DNA replication was correlated with amplification of pATM levels as well as its substrates, pChk2 and pNbs1. Furthermore, we found that G2/M cell cycle arrest was not activated by a replication defective mutant or a mutant expressing the E4orf3 encoded 11kDa protein. Our results suggest that the initial induction of DDR foci does not require viral DNA replication. In contrast, viral DNA (open full item for complete abstract)

Committee: Eileen Bridge (Committee Chair); Gary Janssen (Committee Member); Joseph Carlin (Committee Member); Xiao-Wen Cheng (Committee Member); David Pennock (Committee Member) Subjects: Microbiology