Doctor of Philosophy (PhD), Ohio University, 2024, Plant Biology (Arts and Sciences)

Plants encounter various biotic and abiotic stresses daily and have developed defense mechanisms to overcome these challenges. One key system involved in these defense mechanisms is the ubiquitin (Ub)-26S proteasome system (UPS), which targets malfunctioning proteins for degradation through Ub-tagged proteasomal pathways. The E3 ligases, specifically S-phase kinase-associated protein 1 (SKP1), Cullin 1 (CUL1), and F-box (SCF) complexes, play crucial roles in this process by recognizing and tagging specific protein substrates. Arabidopsis thaliana, with over 700 F-box proteins, has the largest group of E3 ligases, yet only 5% have been functionally characterized. Phylogenetic relationships among 111 plant species have identified four clusters of F-box genes, including a cluster with more conserved F-boxes, referred to as core Arabidopsis F-box (CAF) genes. Given that CAF genes have more known functions compared to other clusters, this dissertation hypothesizes significant potential for discovering new functions among the uncharacterized F-boxes within this group. Considering the evolutionary conservation of most CAFs, I adopted a genetic approach to investigate the roles of CAFs during seed germination and seed development. To address the challenges posed by functional redundancy of duplicated CAF genes and the lethality associated with constitutive F-box overexpression in transgenic plants, I created a library of inducible overexpression lines for 40 CAF genes, many of which lacked known biological functions. By systematically examining the effects of conditional overexpression of these 40 CAFs, I found that CAF overexpression during seed germination and seed development can positively or negatively regulate radicle rupture growth, thus controlling the germination process. Specifically, I identified 24 CAFs that enhance radicle rupture and two that inhibited it by interfering with abscisic acid (ABA)-mediated germination suppression. Induction of CAFs during seed (open full item for complete abstract)

Committee: Zhihua Hua (Advisor); Yang Li (Committee Member); John Schenk (Committee Member); Morgan Vis (Committee Member) Subjects: Biology; Genetics; Molecular Biology; Plant Biology; Plant Sciences

Doctor of Philosophy, Miami University, 2024, Biology

Fragile X syndrome (FXS) is a common form of inherited intellectual disability and the primary monogenic cause of autism spectrum disorder. FXS results from a mutation to the Fragile X Mental Retardation 1 (FMR1) gene in the form of extra CGG repeats; greater than 200 repeats silences the gene and leads to the loss of the gene product, Fragile X Mental Retardation Protein (FMRP). As a neurodegenerative disorder, FXS research has primarily focused on the role of FMRP during development. Moreover, the role of FMRP in the innate immune system has not been well studied and differences between males and females are underexplored. The goal of this study was to better understand sex differences in fmr1 expression and function in the innate immune system using the cricket Acheta domesticus. To accomplish this, we first examined Ad-fmr1 expression in male and female neural and immune tissues before and after an immune challenge at sexually immature and sexually mature time points using quantitative real-time PCR. We found significant sex-, time-, and tissue-specific differences in Ad-fmr1 expression. Secondly, to examine FMRP's role in the innate immune system before and after a Serratia marcescens immune challenge, we used RNA interference to knockdown (KD) Ad-fmr1 expression of male and female crickets and examined the following innate immune parameters: survivorship, nodulation, fat body lysozyme expression, hemolymph lytic activity, nitric oxide synthase (nos) expression, and nitrite concentration. We found both sexes had increased lysozyme expression and lytic activity; nos expression was decreased while nitrite concentration decreased only in female hemolymph following frm1 KD. We also identified sex-specific differences for Ad-fmr1 KD crickets following an immune challenge. KD males were less likely to survive an immune challenge and did not nodulate bacteria as well as females. KD crickets had a significant increase in lysozyme expression, though only males had incre (open full item for complete abstract)

Committee: Kathleen Killian (Committee Chair); Paul James (Committee Member); Donghyung Lee (Committee Member); Haifei Shi (Committee Member); Dawn Blitz (Committee Member) Subjects: Biology

Master of Science (M.S.), University of Dayton, 2024, Biology

This thesis sought to advance the understanding of a gene regulatory network (GRN) that is responsible for the development of a male-specific (dimorphic) pattern of pigmentation on the 5th and 6th (A5 & A6) abdomen segments of the fruit fly species Drosophila (D.) melanogaster. The A5 and A6 protective cuticle tergites are fully black in color from the melanin pigment deposited from the underlying epidermal cells. These black tergites differ from the non-melanic A1-A4 tergites, and the A5 and A6 tergites of females. Previous studies identified 18 transcription factor genes whose functions are needed for this dimorphic pattern of tergite pigmentation. They also found the cis-regulatory elements (CREs) that control the epidermis expression of four “realizator” genes that encode enzymes for pigment metabolism (Ddc, tan, yellow, and ebony), and one CRE that controls expression of the bab paralog (bab1 and bab2) genes which encode key transcription factors that shape sex-specific patterns of pigmentation gene expression. Here, the activity of these CREs were assessed in genetic backgrounds in which RNA-interference (RNA-i) turned down the expression of these 18 genes one by one, and were imaged utilizing confocal microscopy. On the low end, the activity of the Ddc gene's MEE1 CRE and the bab dimorphic element CRE were impacted by eight of the 18 transcription factors. On the high end, the e_AMS CRE of ebony and the t_MSE2 CRE for tan were impacted by 12 transcription factors. Among the nuances of these numerous CRE interactions were transcription factors like bab (bab1 and bab2) and grh whose expressions were required for the proper activity of the CREs for all five of the analyzed genes. In contrast, the transcription factor pdm3 was found to regulate the expression of a single gene, ebony. A second objective of this thesis was to see whether any of the regulatory relationships between transcription factors and CREs might involve direct physical interactions. To this (open full item for complete abstract)

Committee: Thomas Williams (Advisor); Yvonne Sun (Committee Chair); Pothitos Pitychoutis (Committee Chair) Subjects: Biology; Developmental Biology; Evolution and Development; Genetics

Doctor of Philosophy, Miami University, 2019, Biology

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is the primary monogenetic cause of autism spectrum disorders. FXS is caused by a mutation in the regulatory region of the Fragile X Mental Retardation 1 (FMR1) gene, that ultimately leads to loss of the gene product Fragile X Mental Retardation Protein (FMRP). As FXS is a neurodevelopmental disorder, most studies have focused on the role of FMRP during development. However, a few studies have shown that the loss of FMRP during adulthood can impact learning, cognition, and behavior. The primary goal of this study was to decrease FMRP during adulthood and examine the impacts on immune function and social behavior using the cricket Acheta domesticus as a novel insect model. We examined immune function in males and females by assessing several important immune parameters including: the total number of circulating hemocytes in the hemolymph, the total hemolymph protein content, total phenoloxidase enzyme activity, and fat body lysozyme expression. We found that males and females exhibited similar changes in these immune parameters as a result of decreased Fmr1, but males were less likely to survive an immune challenge with an injection of lipopolysaccharide (LPS) from Serratia marcescens. This is the first study to demonstrate that decreased FMRP during adulthood causes sex-specific effects on the immune system. We also examined the effects of decreased Fmr1 on the agonistic behavior of adult male crickets. We observed that the average time to first physical contact during an agonistic interaction was significantly increased in fights between a control male and Fmr1 knockdown male and that Fmr1 knockdown males were less likely to initiate an aggressive interaction than control males. Despite initiating interactions less often, these males fought just as aggressively and won as often as controls. In this study, we also evaluated the parameters required to generate a successful parental R (open full item for complete abstract)

Committee: Kathleen Killian Dr. (Advisor) Subjects: Biology; Entomology; Genetics

Doctor of Philosophy, Case Western Reserve University, 2016, Pathology

Brain cancer is a devastating disease and a dreaded diagnosis for patients. Cancer within the brain, more than any other organ in the body, more than skin or colon or breast, is a difficult diagnosis to treat and live with because the brain is not just a part of the person, it is the person. Memories, personality, emotions, intelligence, speech and movement all originate in the brain. Therefore, when cancer, or the treatments used to fight the cancer, affect the brain by disrupting or damaging an area of the brain that controls these functions, it not only changes the body of the person, but changes the person. It is a particularly cruel disease. Glioblastoma is the most common and aggressive form of malignant brain cancer, which we currently have no way to effectively treat. The majority of patients die within 16 months of receiving a diagnosis, many times following aggressive surgery, radiation and chemotherapy, and then a sequelae of declining physical and cognitive function. Effective therapies are urgently needed for these patients. Many studies have identified potential therapeutic targets through in vitro screens or genomic sequencing, but this has not led to effective clinical therapies. Here we present a novel method of identifying potential drug targets in glioblastoma using advanced RNA interference screening in an in vivo orthotopic microenvironment. Using this system, we discover that screening within a functional microenvironment reveals novel targets that have been missed by traditional in silico or in vitro screening. We also demonstrate that our targets identified in vivo are more clinically relevant than targets we were able to identify in vitro using the same system. We focused our screening on epigenetic modifier genes, as many of these enzymes are sensitive to microenvironmental conditions and are potentially druggable. Within epigenetic modifier genes, we revealed that factors involved in transcription pause-release and elongation ar (open full item for complete abstract)

Committee: Jeremy Rich MD, MHSc, MBA (Advisor); Paul Tesar PhD (Advisor); Alan Levine PhD (Committee Chair); Peter Scacheri PhD (Committee Member); Bruce Trapp PhD (Committee Member); Steven Rosenfeld MD, PhD (Committee Member); Mark Jackson PhD (Committee Co-Chair); Clive Hamlin PhD (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Genetics; Medicine; Molecular Biology; Neurology; Pathology

Master of Science (MS), Wright State University, 2016, Biological Sciences

Evolutionarily conserved molecular processes involved in construction of the germline and embryonic development are essential for the procreation of many species. Infertility affects 15% of couples in the world and can be caused by dysfunctions during egg and sperm development, anatomic defects, as well as faulty embryonic development. Although there are some infertility disorders that are genetically defined, such as Turner and Klinefelter syndromes, many clinical infertility cases are diagnosed as idiopathic due to the lack of understanding of basic fertility mechanisms. Schmidtea mediterranea is a freshwater planarian species that has the ability to regenerate complete organisms, including germ cells and reproductive structures, from small tissue fragments containing pluripotent somatic stem cells. The developmental plasticity of planarians provides a wonderful opportunity to investigate the molecular mechanisms behind the differentiation and development of specialized cells, including gametes. Smed-boule encodes for an RNA-binding protein and is the most ancestral member of the Deleted in AZoospermia (DAZ) gene family. DAZ family genes function in different aspects of germ cell development and fertility in species ranging from sea anemone to humans. Whole-mount in situ hybridization experiments revealed Smed-boule expression is enriched in the testes and ovaries of planarian flatworms. Interestingly, Smed-boule RNA-interference (RNAi) planarians lost the ability to produce gametes, yet still were able to deposit sterile egg capsules. Virgin Smed-boule(RNAi) and control planarians maintained in isolation also continuously produced sterile egg capsules. Altogether these results demonstrate that egg capsule production in S. mediterranea occurs independently of ovulation, fertilization, and mating events. In addition, detailed analysis of gametogenesis defects revealed that Smed-boule functions at different stages during male and female germline development. These (open full item for complete abstract)

Committee: Labib Rouhana Ph.D. (Advisor); David Goldstein Ph.D. (Committee Chair); Scott Baird Ph.D. (Committee Member); Mill Miller Ph.D. (Committee Member) Subjects: Biology; Developmental Biology

Doctor of Philosophy, The Ohio State University, 2014, Entomology

The northern house mosquito, Culex pipiens L., is a vector of several diseases including West Nile virus, St. Louis encephalitis, and filariasis. Adult females of this species enter an overwintering dormancy known as diapause. Like most temperate insects, diapause in Cx. pipiens is programmed by the short daylengths of late summer and early fall although the molecular mechanism by which mosquitoes and other insects measure day length is unknown. Like other insects, Cx. pipiens, have circadian clocks which are composed of several genes whose mRNA transcripts and proteins oscillate throughout the day and, in doing so, regulate daily behaviors. In this thesis, I explore the possibility that circadian clock genes, which provide Cx. pipiens with information on the time of day, are also involved in measuring day length and hence initiating the adult overwintering diapause in this species. The clock genes in Cx. pipiens are highly similar to the circadian clock genes in the other mosquitoes and more distantly related insects. This suggests that the circadian clock in Cx. pipiens is similar in structure to other, non-drosophilid insects. To determine whether clock genes are differentially expressed during diapause, their expression was measured over a 24-hr period in nondiapausing mosquitoes as well as in females of Cx. pipiens throughout the diapause program and after diapause termination. I found that clock genes continue to cycle robustly throughout the diapause program, although early in diapause the expression of several core circadian clock genes show an altered expression profile with peak mRNA levels occurring later in the night than anticipated. This shift in clock gene expression may explain the shift in flight and feeding activity that has been previously observed in diapausing females of Cx. pipiens early in diapause. Using RNA interference (RNAi), the expression of several circadian clock genes was suppressed and the ability of females of Cx. pipiens to ente (open full item for complete abstract)

Committee: David Denlinger (Advisor); Woodbridge Foster (Committee Member); Andrew Michel (Committee Member); David Sommers (Committee Member) Subjects: Biology; Entomology; Molecular Biology

Master of Science, The Ohio State University, 2014, Plant Pathology

Oomycete and nematode pathogens cause major damage to soybean worldwide. In response to an oomycete pathogen invasion, plants activate one or both of their qualitative and quantitative resistance pathways. Qualitative resistance involves the activation of R---genes and is known as the gene---for---gene resistance pathway. Although R---genes provide a strong level of expression for resistance towards individual pathogen strains or populations, pathogens readily adapt due to the selection pressure. Quantitative resistance, or partial resistance, is mediated by many genes, each of which contributes to a reduction in the level of disease, and is thought to be more durable in some host---pathogen systems than qualitative resistance. This study was conducted to gain insight into soybean interactions with three different root pathogens. A Conrad x Sloan F9:11 recombinant inbred population was evaluated to determine the location of quantitative trait loci (QTL) that confer partial resistance to Pythium irregulare. Two QTL on chromosomes 14 and 19 contributing to partial resistance against P. irregulare were identified in a greenhouse cup assay. In this second study, the potential for suppressing gene silencing was evaluated in transgenic soybean cultivars. Williams, Williams82, Conrad, and Sloan soybean lines with transgenic hairy roots were inoculated with root---knot nematode (RKN) juveniles. The feeding sites of the RKN were observed for the presence of a normally suppressed marker two weeks post inoculation. Suppression of gene silencing within the RKN feeding site was observed. A QTL on chromosome 18 was previously identified to contribute to partial resistance towards Phytophthora soaje. An analysis of eight genes within this QTL identified SNPs and deletions in promoter sequences of the genes from the resistant and susceptible soybean parent lines. These genes will serve as excellent targets for functional analysis to s (open full item for complete abstract)

Committee: Christopher Taylor Dr. (Advisor); Anne Dorrance Dr. (Advisor); Leah McHale Dr. (Committee Member) Subjects: Molecular Biology; Plant Biology; Plant Pathology

PhD, University of Cincinnati, 2008, Medicine : Toxicology (Environmental Health)

The white rot fungus Phanerochaete chrysosporiumis primarily known for its ability to degrade a wide range of xenobiotic compounds including the highly recalcitrant polycyclic aromatic hydrocarbons. The natural substrate of this basidiomycete fungus is however, lignin, the most abundant aromatic polymer on earth. The versatililty of this fungus in breaking down a wide array of compounds arises from the presence of a highly nonspecific enzyme system (peroxidase enzyme system) in its repertoire. Most of the research involving degradation of toxic chemicals has focused on this biodegrading enzyme machinery. Cytochrome P450 monooxygenases (P450s) on the other hand, are heme-thiolate proteins that are known to be involved in metabolism of endogenous compounds as well as xenobiotic compounds in higher eukaryotes. Nearly 150 P450s are present in this organism, which is the highest number known till date among fungal species. Based on the sequence similarity criteria and our phylogenetic analysis, these P450s have been classified under 12 families and 23 sub-families. Despite indirect evidences suggesting the role of P450s in oxidation of xenobiotics, there have been hardly any reports on characterization and role of individual P450s either in regulation of physiological processes or in direct metabolism of xenobiotics in this organism. Here we characterized and investigated the role of P450 enzymes in two different mechanisms in this fungus. One, indirect involvement of P450s in peroxidase–mediated oxidation of xenobiotics, and two, direct involvement of P450s in metabolism of xenobiotics. In order to achieve the first objective, we investigated the role of PC-bphgene, the only member of the P450 CYP53 in synthesis of a secondary metabolite, veratryl alcohol, which regulates the activity of the peroxidase enzyme system of this fungus. In order to achieve the second objective, we used the functional genomic approach based on a custom-designed microarray and heterologous exp (open full item for complete abstract)

Committee: Dr. Jagjit Yadav (Advisor) Subjects:

MS, University of Cincinnati, 2006, Arts and Sciences : Biological Sciences

RNA interference (RNAi) is a cellular process whereby gene expression is controlled via microRNA (miRNA) or small-interfering RNA (siRNA) recognition and cleavage of target messenger RNA (mRNA). RNAi also functions in the inhibition of viral propagation in a host cell, thus providing the cell a primitive immune system. The purpose of this study was to examine the mRNA levels of dicer, the gene which encodes for the Dicer protein, an RNAi component, in response to natural viral infection. Poliovirus and astrovirus were used to infect Caco-2 cells to determine the effect that each has on the expression of dicer mRNA. Using real-time RT-PCR, dicer expression was measured over the time course of eight hours of infection. It was determined that poliovirus caused dicer expression to be upregulated over one and a half fold by eight hours of infection. Astrovirus caused dicer expression to be downregulated over one-half fold by eight hours of infection. Both results were statistically significant, indicating that poliovirus and astrovirus induce changes in dicer mRNA expression levels during natural viral infection.

Committee: Dr. Stephen Keller (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2011, Veterinary Biosciences

Osteoarthritis (OA) is a debilitating disease associated with pain and dysfunction that remains an unresolved and widespread source of symptomatic health problems for many individuals, particularly those 40 years and older. Interleukin-1β (IL-1β) has been cited as a major cytokine involved in OA-related joint degeneration. Recognized as one of the most important mediators of inflammation and host response to infection, increased production of this cytokine has been linked to a wide variety of autoimmune conditions and autoinflammatory disorders. Characterization of its role in primary OA, however, remains elusive and potentially contradictory. Thus, the molecular interactions to explain the relationship between IL-1β and maintenance of healthy articular cartilage have been proposed but are not yet definitively established, and characterization of the function of IL-1β in a spontaneous, in vivo model remains elusive. As such, a primary aim of this study was to provide a comprehensive analysis of the temporal expression and tissue distribution of IL-1β using immunohistochemistry (IHC) throughout initiation and progression of OA in a naturally-occurring animal model. We subsequently elucidated that OA-prone Hartley guinea pig did not demonstrate reduced IL-1β in weight-bearing articular cartilage, synovium, meniscus, or subchondral bone during achievement of adult maturity as in the control guinea pig strain, and that this aberrant expression may correlate to early incidence of OA. As this temporal study provided evidence that IL-1β is a biomarker relevant to the development and progression of OA, and we then performed in vitro and in vivo studies to block and/or reduce the dysregulation of this cytokine's expression such that we could provide evidence of its contribution to premature onset of spontaneous OA. Successful reduction of the IL-1β transcript was achieved via RNA interference (RNAi) techniques in vitro using a novel adeno-associated viral vector serotype 5 ( (open full item for complete abstract)

Committee: Alicia Bertone DVM PhD (Advisor); Jeffrey Bartlett PhD (Committee Member); Gerard Nuovo MD (Committee Member); Steven Weisbrode VMD PhD (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, 2008, College of Graduate Studies

RNA viruses have a high mutation rate due to the absence of post replicative error correction machinery. This, coupled with high yield and a short replication interval leads to the generation of extensive genetic diversity that promotes rapid adaptation to novel environments. The fitness of a population determines the frequency of neutral, deleterious and beneficial mutations that arise during replication, with the latter increasing in frequency as viral fitness increases. This work shows that there is a direct correlation between fitness and log CBS with population size ranging over five orders of magnitude, and this correlation explains limits to fitness gains and drift in populations of very high fitness.Two theories of asexual evolution were tested. Solitary wave theory was shown to explain accurately the fitness changes of viral populations evolved under a variety of conditions. Furthermore, fit of experimental data to solitary wave allowed the determination of biologically-meaningful, important parameters relevant to the evolution of VSV strains, including number of sites under moderate selection, number of sites under positive selection with their average selective value, and diversity within the population. Clonal interference theory is a good predictor of DNA-based microbes, but is proved unsatisfactory as a descriptor of RNA virus evolution because of their high mutation rates and assumptions of the model that do not apply to these pathogens (e.g. populations are at mutation-selection balance). In particular, the results of determining the order of mutation accumulation clearly showed the ability of RNA viruses to generate secondary beneficial mutations before the primary beneficial mutation is fixed, or even dominant. This result clearly demonstrates that clonal interference theory is a poor descriptor of the evolutionary behavior of RNA viruses.

Committee: Isabel Novella PhD (Committee Chair); James Trempe PhD (Committee Member); Sonia Najjar PhD (Committee Member); Joana Chakraborty PhD (Committee Member); Dorothea Sawicki PhD (Committee Member) Subjects: Biology; Genetics; Virology

Doctor of Philosophy, Case Western Reserve University, 2011, Macromolecular Science and Engineering

Low delivery efficiency and poor biocompatibility of nonviral gene delivery vectors remain the major obstacle for their clinical use. Herein, molecularly tailored acid-responsive polymers were developed in order to achieve efficient and biocompatible nucleic acid delivery systems by addressing key extra- and intracellular challenges, including high stability in serum, receptor-mediated targeted delivery, endosomal escape, and intracellular target-specific release of nucleic acid. Branched polyethylenimine (PEI) or linear PEI with low and high molecular weights was modified with acid-cleavable primary amine-bearing ketals. Physicochemical properties and biological activities of ketalized PEI were highly correlated with molecular weights, ketalization ratios, and topology (branched vs. linear) of the PEI. Regardless of types of nucleic acid, ketalized high molecular weight branched PEI polyplexes with inhibited nucleic acid complexation efficiency were mainly localized in the cytoplasm, resulting in efficient RNA interference. On the contrary, a significant number of ketalized low molecular weight branched PEI polyplexes were distributed in the nucleus, leading to efficient DNA transfection. Moreover, ketalized linear PEI mediated efficient, serum-resistant, and sequence-specific gene silencing via efficient cytosolic release of intact siRNA. Ketalized linear PEI was also utilized as a tumor stimuli-responsive nanotheragnostic agent by conjugating small gold nanoparticles on the surface of its siRNA-encapsulating polyplexes via acid-degradable ketal linkages. Under a tumor pH-mimicking condition, this acid-transforming nanoparticle resulted in various optical signal changes (blue-shifted UV absorbance, diminished scattering intensity, and increased variance of Doppler frequency) with simultaneously enhanced cellular internalization and gene silencing. PEG-conjugated acid-transforming poly(ketalized serine) [PEG-poly(kSer)] was also developed as a biocompatible polypep (open full item for complete abstract)

Committee: Young Jik Kwon (Advisor); Stuart Rowan (Committee Chair); David Schiraldi (Committee Member); Horst von Recum (Committee Member) Subjects: Biomedical Engineering; Chemical Engineering; Polymer Chemistry