PhD, University of Cincinnati, 2024, Medicine: Molecular and Developmental Biology

Viruses are complex infectious agents that infect all living organisms and have been associated with both communicable and non-communicable diseases. Viruses encode proteins that alter the gene regulatory mechanisms of the host cell, some of which might explain these disease associations. Genetic differences in the genes encoding these proteins can affect the function of these viral transcriptional regulators (vTRs). Therefore, it is necessary to examine the genetics of vTRs to elucidate the impact of viral variation on the ability of these vTRs to interact with the human genome. Epstein Barr Virus (EBV) is a human gammaherpes virus that is categorized into two distinct phylogenetic groups: EBV-1 and EBV-2, with over 400 EBV genomes completely sequenced. EBV-1 and EBV-2 have been shown to infect individuals globally, with EBV-2 commonly observed in immunocompromised individuals, pointing to a potential role in disease processes. The largest amino acid variation between EBV types is found within Epstein Barr Nuclear Antigen 2 (EBNA2), with type 1 and type 2 EBNA2 sharing only 53% amino acid identity. Previous studies have shown that variation within EBNA2 affects its ability to infect and transform B and T cells. To date, our lab has shown that EBNA2 type 1 human genomic interactions significantly overlap with the genetic risk loci of seven distinct autoimmune diseases. Mechanistically, our lab has also shown that EBNA2 type 1 changes the chromatin landscape at known disease risk loci, altering human gene expression levels that might contribute to autoimmune disease processes. However, no work has shown if EBNA2 type 2 may also contribute to disease etiology by interacting with the same or additional disease risk loci. Therefore, we investigated the ways in which EBNA2 type 2 differentially interacts with the human genome and autoimmune disease risk variants, compared to EBNA2 type 1. Such information would reveal mechanistic insight into how different EBV typ (open full item for complete abstract)

Committee: Matthew Weirauch Ph.D. (Committee Chair); Leah Claire Kottyan Ph.D. (Committee Member); Brian Gebelein Ph.D. (Committee Member); William Miller Ph.D. (Committee Member); Raphael Kopan Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, Case Western Reserve University, 2024, Genetics

Pancreatic beta-cell loss and dysfunction is central to the development of diabetes. As the main function, beta-cells produce and secrete insulin to maintain blood glucose homeostasis. Insulin is converted from its precursor proinsulin in a tightly regulated process in the beta-cells, but the regulating mechanism remains largely unknown. Stem-cell derived beta-like cells serve as unlimiting source for cell replacement therapy, however the generated beta-like cells functionally cannot represent the bona fide human islet. Here in this thesis study, functional genomics study was performed to mapping proinsulin regulators and modifying the in vitro pancreatic beta-cell differentiation. We performed a genome-wide CRISPR screen and identified 84 proinsulin regulators, including well-known factors like Pcsk1 and Cpe, that alter intracellular proinsulin/insulin ratio in a mouse -cell line. We found our proinsulin regulators are distinct from the insulin regulators from a previous orthogonal CRISPR screen. Functional annotation of the proinsulin regulators highlights Golgi as the primary organelle for proinsulin storage and regulation: trafficking towards Golgi increases the intra-cellular proinsulin/insulin ratio, while trafficking away from Golgi, including exocytosis and Golgi-to-ER retrograde transport, decreases proinsulin levels. We also mapped mouse quantitative trait loci (QTLs) associated with plasma proinsulin levels and integrated with the CRISPR screen to pinpoint the causal genes within the QTL loci. The protein disulfide isomerase Pdia6 stands out as the strongest hit from both CRISPR screen and the in vivo QTL mapping. Interestingly, knocking down Pdia6 significantly reduced intracellular proinsulin content and exocytotic secretion. Intriguingly, Pdia6-depletion in both human and mouse -cells does not affect the folding status but disturbed proinsulin translation through a UPR-independent mechanism. Our genetic profiles provide mechanistic insights into the (open full item for complete abstract)

Committee: Yan Li (Advisor) Subjects: Biomedical Research; Cellular Biology; Genetics

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

Osteosarcoma, the most prevalent primary bone cancer, frequently metastasizes to the lungs. Lung metastasis and the downstream sequelae are the major contributor to mortality, with current patient survival rates mirroring those from four decades ago. Among all the stages of the metastasis-invasion cascade, the colonization of secondary organs is arguably the most critical yet remains poorly understood. This gap in understanding arises from the unclear processes occurring between a cancer cell's arrival in the secondary organ and its evolution into a full-blown metastatic lesion. In Chapter 2 of this thesis, we employ in vivo epigenomic and transcriptomic profiling to discover that osteosarcoma cells, while colonizing the lungs, transition through specific reproducible epigenetic states. Through in vivo and in vitro CRISPR screens, we demonstrate that the transcription factors regulating these epigenetic changes are metastasis-specific dependency genes. Intriguingly, small molecule targeting of these genes inhibits metastasis, emphasizing the functional relevance of the intermediate transcriptional states. Our findings show that the colonization stage of metastasis actually comprises a spectrum of epigenetic transitions, offering novel avenues for metastasis-targeted therapies. Further, this work underscores the importance of exploring the dynamic nature of metastasis in diverse cancers, urging a shift from static to dynamic studies of disease progression.

Committee: Peter Scacheri (Advisor); Michelle Longworth (Committee Chair); Yogen Saunthararajah (Committee Member); Jacob Scott (Committee Member); Yan Li (Committee Member) Subjects: Genetics; Molecular Biology; Oncology

Doctor of Philosophy (PhD), Ohio University, 2023, Plant Biology (Arts and Sciences)

Glycosyltransferases (GTs) are important proteins that are widely distributed in both prokaryotes and eukaryotes and play a crucial role in the biosynthesis of carbohydrates and glycoconjugates. They catalyze the formation of specific glycosidic linkages by transferring sugar moieties from activated sugars to a variety of biomolecules such as carbohydrates, lipids, proteins, or water. Progress in functional genomics technologies, such as DNA sequencing and proteomics, has allowed the identification of a large number of GT genes in several species. In general, functional genomics approaches, which include genomics, genetics, proteomics, and biochemistry, are used to determine the function of a gene (i.e., GTs). In biochemical approaches, the most direct way to assign a function to a gene is through direct testing of the enzyme activity of its product (carbohydrate) in vitro. However, in contrast to genomics/proteomics approaches, the biochemical approaches are the most difficult to adapt to high-throughput screening. The reason is that biochemical approaches require the use of isolated/purified proteins, which is labor-intensive and prone to the formation of undesired products resulting from background enzyme activity. Therefore, there is a need for the development of protein-based in vitro high-throughput platforms for determination of biochemical functions of proteins, including GTs, and their interactions with other proteins. To be advantageous, a protein-based high-throughput platform should have the following characteristics: i) the platform can be adapted to all GTs and synthases, ii) the detection method should be sensitive enough to demonstrate the formation of GT products, and iii) the platform should be simple and easy to implement or accessible to any laboratory. This work describes the development of a novel platform for screening of enzyme activities of GTs in vitro. It is called the in vitro GT-array (i-GTray) platform. This platform uses an in vitro ce (open full item for complete abstract)

Committee: Ahmed Faik (Advisor) Subjects: Biochemistry; Cellular Biology; Molecular Biology; Plant Biology

Doctor of Philosophy, The Ohio State University, 2018, Evolution, Ecology and Organismal Biology

Most broadly, this study aimed to develop a better understanding of how organisms evolve novel functions and traits, and examine how seemingly complex adaptive trait syndromes can convergently evolve. As an ideal example of this, the carnivorous plants were chosen. This polyphyletic grouping contains taxa derived from multiple independent evolutionary origins, in at least five plant orders, and has resulted in striking convergence of niche and morphology. First, a database study was performed, with the goal of understanding the evolutionary trends that impact carnivorous plants as a whole. Using carnivorous and non-carnivorous plant genomes available from GenBank. An a priori list of Gene Ontology-coded functions implicated in plant carnivory by earlier studies was constructed via literature review. Experimental and control samples were tested for statistical overrepresentation of these functions. It was found that, while some functions were significant in some taxa, there was no overall shared signal of plant carnivory, with each taxon presumably having selected for a different subset of these functions. Next, analyses were performed that targeted Sarracenia alata specifically. A reference genome for S. alata was assembled using PacBio, Illumina, and BioNano data and annotated using MAKER-P with additional preliminary database filtration. From these, it was found that Sarracenia alata possesses significant and substantial overrepresentation of genes with functions associated with plant carnivory, at odds with the hypothesis that the plant primarily relies on symbioses. Finally, pitcher fluid was collected from S. alata in the field. RNA was extracted from the fluid, sequenced via Illumina, and assembled with Trinity. Sequences were sorted into host plant and microbiome based on BLAST match to the S. alata reference genome. It was found that, while S. alata contributes two-thirds of the transcripts, these encode no digestive enzymes and a very limited set o (open full item for complete abstract)

Committee: Bryan Carstens Ph.D. (Advisor); Marymegan Daly Ph.D. (Committee Member); Zakee Sabree Ph.D. (Committee Member); Andrea Wolfe Ph.D. (Committee Member) Subjects: Bioinformatics; Biology; Botany

Doctor of Philosophy, The Ohio State University, 2002, Plant Pathology

Genomics offers new perspectives for the discovery of novel genes in the oomycetes, a unique group of eukaryotic microbes comprising saprophytes as well as pathogens of plants and animals. Among the oomycetes, the most devastating plant pathogen is the potato and tomato late blight pathogen Phytophthora infestans. Currently tens of thousands of expressed sequence tags (ESTs) are available for P. infestans. The challenge of the post-genomic era is to link a sequence to a phenotype (functional genomics) with as little experimental effort as possible. In the first study, we combined EST data mining with functional assays to identify extracellular effector proteins from P. infestans, which can manipulate plant molecular and cellular processes to induce defense or disease like responses. We developed the PexFinder algorithm for automated identification of Phytophthora extracellular proteins (Pex). Using a virus based high throughput functional assay to express Pex cDNAs in Nicotiana benthamiana and tomato, we identified two novel necrosis inducing effectors crn1 and crn2, which are members of large gene family. crn1 and crn2 were demonstrated to be expressed during P. infestans infection on tomato. In addition, crn2 induced defense related genes in tomato. In a second study, we characterized a Pex cDNA, pipg1 which represents the first endopolygalacturonase (endoPG) described from P. infestans. Generally, endopolygalacturonases are plant cell wall degrading enzymes primarily found and characterized extensively in plants and fungi. We showed that pipg1 is a member of a gene family and is expressed in both preinfection and infection stages. Phylogenetic analyses showed an affinity of pipg1 to fungal endoPGs, a feature that contrasts with phylogenies obtained with ribosomal sequences or compiled mitochondrial and chromosomal genes. In a third study, We generated ESTs from a cDNA library constructed from mRNA of Saprolegnia parasitica, an oomycete pathogen that causes sapr (open full item for complete abstract)

Committee: Sophien Kamoun (Advisor) Subjects:

Master of Science (MS), Ohio University, 2008, Computer Science (Engineering and Technology)

More organisms are having their genomes sequenced recently than in the past, thus creating a greater demand from the biological community to better understand the exact biological mechanisms which are encoded within the genomic blueprint of each organism. While biologists continue to analyze genomes and to identify new functional elements within organisms, there remain several regions of the genomes which are often overlooked, such as non-protein encoding regions, introns, and intergenic regions. Several bioinformatics algorithms exist to discover functional elements (which are also referenced within as words) in these regions.In this thesis, a functional genomics toolkit for finding functional words of genomes (vocabularies) is presented and described. With currently available vocabulary based tools, limitations arise when analyzing large input sequences. To overcome this limitation, a scalable word searching approach is presented and tested with genomic sequences with file sizes up to 2 Gigabytes (GB). In addition, the toolkit is utilized to provide a genome-wide characterization of the Arabidopsis thaliana genome in terms of over- and under-represented repeats within specific genome regions and to search for similarities between putative functional elements in the human genome and Arabidopsis thaliana thereby producing a putative vocabulary. The difficulties encountered during the research process and suggestions for future work are also further discussed.

Committee: Lonnie R. Welch PhD (Advisor); Frank Drews PhD (Committee Member); Klaus Ecker PhD (Committee Member); Sarah Wyatt PhD (Committee Member) Subjects: Bioinformatics; Computer Science