Master of Science, Miami University, 2012, Chemistry and Biochemistry

The HIV genome is synthesized as a 9.5kb polycistronic transcript that undergoes alternative splicing to produce the complete viral protein compliment. Within the HIV genome both donor and acceptor sites are coupled together to determine which of the over 40 transcripts are made and thus which viral proteins are produced. Host proteins are responsible for both the up regulation and down regulation of these sites and play a key role in viral protein production. ASF, is a serine arginine rich protein that is capable of binding splice site A7 and upregulating splicing. The RNA recognition motif domains (RRMs) were studied both separately and together with the complete splice site as well as exonic splicing enhancer 3 (ESE3). A major goal of this study was to identify which domain has a greater responsibility in binding the splice site. In order to understand this, a series of electrophoretic mobility shift assays were run. It is seen that RRM-1 has a higher affinity for both the complete splice site as well as ESE3.

Committee: Christopher Makaroff PhD (Committee Chair); Carole Dabney-Smith PhD (Committee Member); Natosha Finley PhD (Committee Member); David Tierney PhD (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry

Doctor of Philosophy, Case Western Reserve University, 2022, Genetics

Balanced mRNA isoform diversity and abundance are spatially and temporally regulated throughout cellular differentiation. The proportion of expressed isoforms contribute to cell type specification and determine key properties of the differentiated cells. Neurons are unique cell types with intricate developmental programs, characteristic cellular morphologies, and electrophysiological potential. Neuron-specific gene expression programs establish these distinctive cellular characteristics and drive diversity among neuronal subtypes. Genes with neuron-specific alternative processing are enriched in key neuronal functions, including synaptic proteins, adhesion molecules, and scaffold proteins. Despite the similarity of neuronal gene expression programs, each neuronal subclass can be distinguished by unique alternative mRNA processing events. Alternative processing of developmentally-important transcripts alters coding and regulatory information, including interaction domains, transcript stability, subcellular localization, and targeting by RNA binding proteins. Here, we investigate the role of CLP1, a component of the 3' end processing complex, and THOC6, a component of the transcription/export complex, in mRNA transcription. We find CLP1 suppresses proximal polyadenylation and disease-associated variants exhibit toxic gain-of-function properties. We establish a regulatory pattern for CLP1 in motor neuron 3' end diversity and identify neuronal gene overexpression as a possible mechanism of disease. Furthermore, we find THOC6 is involved in regulation of splicing and is essential for neurodevelopment in the mammalian brain. Fine-tuning of mRNA processing is essential for neuronal activity and maintenance. Thus, the focus of neuronal RNA biology research is to dissect the transcriptomic mechanisms that underlie neuronal homeostasis, and consequently, pre-dispose neuronal subtypes to disease.

Committee: Ashleigh Schaffer (Advisor); Peter Scacheri (Committee Chair); Polyxeni Philippidou (Committee Member); Helen Miranda (Committee Member); Donny Licatalosi (Committee Member) Subjects: Biochemistry; Genetics; Neurosciences

Master of Science, Miami University, 2010, Botany

This thesis reports a study of the interaction between messenger RNA polyadenylation and splicing in the expression of an Arabidopsis gene OXT6. This gene encodes two proteins that may involve in both polyadenylation and splicing processes. Interestingly, alternative polyadenylation or splicing of intron-2 of the gene defines the expression ratio of the two transcripts. To reveal the relationship of these processing events, a set of mutations were introduced at the splice sites and polyadenylation signals within intron-2 and transformed into the oxt6 mutant plants. The splicing and polyadenylation events were monitored by quantitative RT-PCR, revealing a competition nature of alternative polyadenylation and splicing during the OXT6 transcript processing. ChIP (chromatin immunoprecipitation) analysis performed against RNA Polymerase II in splicing mutant lines suggests that Pol II binding may affect the switch between polyadenylation and splicing during transcript processing. This work is the first of its kind that exemplifies the interplay among transcription, splicing and polyadenylation that defines a gene's expression outcome in plants.

Committee: Qingshun Q. Li PhD (Advisor); Chun Liang PhD (Committee Member); Nicholas P. Money PhD (Committee Member) Subjects: Molecular Biology

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

Pediatric cancer is the leading cause of death by disease in children, and approximately 43 children are expected to receive a cancer diagnosis each day. The paucity of approved treatments, many of which induce lasting negative effects on the health of survivors, and the dismal prognosis for patients who present with metastases at the time of diagnosis or recurrent/relapsed disease warrant more focused efforts on understanding the biology of pediatric cancers and developing novel treatments. Unlike adult cancers, which are usually characterized by genomic alterations and/or contributing lifestyle/environmental factors, pediatric cancers are more likely to be caused by epigenetic dysregulation or post-transcriptional processes such as alternative splicing. All premature mRNAs in a cell undergo splicing in order to remove introns and ligate exons together to produce a mature mRNA transcript that can then be translated into protein. Differential inclusion or exclusion of exons or introns leads to alternative splicing and the generation of mature mRNA transcripts that differ from the parent premature mRNA, which contributes to protein diversity in normal conditions and disease. Cancers hijack this process to selectively regulate the production of oncogenic isoforms, such as the IR-A isoform of the insulin receptor, and create a favorable environment for tumor progression and eventual metastasis. The objectives of the work presented in this dissertation are to 1) critically review the literature to ascertain what is known about alternative splicing in pediatric cancer, 2) study the role of alternative mRNA splicing in pediatric bone sarcomas, 3) test splice-switching oligonucleotides (SSOs) against splicing defects, and 4) utilize the U7 snRNA system to engineer an AAV that expresses SSO sequences as antisense RNAs. First, we reviewed the current literature on the role of alternative splicing in adult cancer, cancer predisposition syndromes, and pediatric cancer (open full item for complete abstract)

Committee: Dawn Chandler (Advisor); Emily Theisen (Committee Member); Steven Sizemore (Committee Member); Timothy Cripe (Advisor) Subjects: Biomedical Research; Molecular Biology; Oncology; Therapy

Master of Science in Biological Sciences, Youngstown State University, 2023, Department of Biological Sciences and Chemistry

Burying beetles have served as a model to answer diverse questions about the evolution of social behavior in insects and to understand the physiological and evolutionary mechanisms involved in the manifestation of parental care behavior. More recently, an increasing number of genomic and transcriptomic resources have become available in Nicrophorus spp. This study utilizes this data to assay sources of variation in the Nicrophorus orbicollis transcriptome with the specific aims to 1) evaluate the extent of alternative splicing in this species and 2) assess differential expression and alternative splicing in genes that are associated with distinct parental behavioral phenotypes in male and female burying beetles during breeding. This was achieved by differential expression analysis of N. orbicollis transcriptomes for parents showing high and low care provisioning behavior. Two algorithms were used to increase predictive accuracy in detecting alternatively spliced genes. Genes that were alternatively spliced and differentially expressed between behavioral groups were subject to further functional characterization. An overall profile of alternative splicing was determined, indicating that many transcripts resulting from alternative splicing were the result of several types of alternative splicing events. There was also evidence for a higher occurrence of intron retention events than exon skipping events. Additionally, the occurrence of alternative splicing was not found to be more common in differentially expressed genes for any specific biological process associated with parental care behavior. Still, evidence for alternative splicing within genes relevant to variability of the parental care phenotype was presented and potential behavioral implications were discussed.

Committee: Stefania C. Panaitof PhD (Advisor); Xiangjia Min PhD (Committee Member); Jonathan Caguiat PhD (Committee Member) Subjects: Animals; Bioinformatics; Genetics; Molecular Biology

Master of Science in Biological Sciences, Youngstown State University, 2022, Department of Biological Sciences and Chemistry

Potato (Solanum tuberosum L.) is the third most important food crop in the world following rice and wheat. Due to its high calories, it is a good source of energy and nutrients such as proteins, vitamins and minerals. Thus, potato plays an important role in providing food, nutritional and economic security of the world. To further improve the quantity and quality of potato as a food crop, it is imperative to understand the transcriptome diversity, gene expression dynamics and associated developing methods. The aim of this study is to identify the alternative spliced events of genes and their differential expression in nitrogen-treated and drought-stressed potato plants. In nitrogen grown potato plants, alternative acceptor site was observed to be most dominant alternative spliced events while intron retention was dominant in drought-stressed potato plants. Additionally, differential gene and isoform expressions of four different varieties of drought-stressed leaf samples of potato plants were analyzed by cuffdiff module of cufflinks V2.2.1 package. Statistically significant (P ≤ 0.05) differential gene expressions with corresponding transcript isoforms were identified such as 10 (up-regulated) and 13 (down-regulated) in Algeria cultivars, 24 (up-regulated) and 21 (down-regulated) in Desiree cultivars with 39 transcript isoforms generated, 33 (up-regulated) and 51 (down-regulated) with 62 generated transcript isoforms in Saturna cultivars, 43 (up-regulated) , 32 (down-regulated) and 55 transcript isoforms generated in Milva cultivars. Overall, conserved alternative spliced genes and alternative spliced events were identified in both nitrogen-treated and drought-stressed potato plants. Furthermore, among the differentially expressed genes, PYR1-like (pyrabactin resistance 1) and heat shock protein families were most upregulated genes. These genes play a crucial role in enhancing development in potato plants under extreme conditions of drought.

Committee: Xiangjia Min PhD (Advisor); David Asch PhD (Committee Member); Jonathan Caguiat PhD (Committee Member) Subjects: Agriculture; Bioinformatics; Biology

Doctor of Philosophy, Case Western Reserve University, 2022, Molecular Medicine

Myeloid neoplasms consist of a large group of hematological malignancies frequently characterized by genetic abnormalities in splicing factors. We investigated the role of several mammalian splicing factors, DDX41 and members of the LUC7-like family, in splicing and disease pathogenesis. DDX41 encodes a DEAD-box RNA helicase with functions implicated in innate immunity, snoRNA processing, and pre-mRNA splicing. Germline DDX41 mutations predispose to late-onset myeloid neoplasms with somatic mutations commonly occurring on the second allele. We designed a system to deplete DDX41 in human and mouse cell lines for in vitro mechanistic studies. However, technical problems arose, preventing utilization of this approach. Vertebrates have evolved three paralogs, termed LUC7L, LUC7L2 and LUC7L3, of the essential yeast U1 snRNA-associated splicing factor Luc7p. Only LUC7L2 is mutated or deleted in myeloid neoplasms. Notably, these paralogs share two conserved zinc finger motifs with Luc7p while adding divergent arginine-serine rich domains. The highly conserved second zinc finger domain of Luc7p stabilizes the binding of weak 5′ splice sites (5′SS) by directly contacting U1 snRNA at the 5′SS-U1 snRNA duplex. We investigated the mechanistic and regulatory functions of the LUC7-like family of splicing factors using a multi-omic approach to characterize protein-protein interactions, RNA binding profiles, regulated gene expression and alternative splicing events. Protein interaction data showed that all three proteins bound similar core but distinct regulatory splicing factors, likely mediated through their divergent arginine-serine rich domains. RNA binding studies revealed that LUC7L2 and LUC7L3 interacted with SR proteins and crosslinked to weak 5′SSs and to the 5′ end of U1 snRNA, establishing an evolutionarily conserved role in 5′SS selection. In contrast, LUC7L bound within introns and interacted primarily with hnRNP proteins. Knockdown of eac (open full item for complete abstract)

Committee: Richard Padgett Ph.D. (Advisor); Donal Luse Ph.D. (Committee Chair); Angela Ting Ph.D. (Committee Member); Yogen Saunthararajah M.D. (Committee Member); George Stark Ph.D. (Committee Member) Subjects: Biochemistry; Bioinformatics; Biology; Biomedical Research; Cellular Biology; Genetics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

Multiple sclerosis (MS) is a chronic inflammatory disease that affects the brain and spinal cord leading to demyelination. Symptoms in patients can present in a broad range. Mild symptoms may manifest as peripheral tingling or numbness, while severe symptoms may present as severe motor disabilities or sudden blindness. A number of disease modifying therapies are available to help improve quality of life, but there is presently no cure available. Per the current numbers, there are nearly 1 million adults in the US who suffer from MS. Proinflammatory T helper (Th) cells that infiltrate the central nervous system (CNS) are thought to be the initial mediators of disease. Findings from the mouse model of MS, experimental autoimmune encephalomyelitis (EAE) lead to the understanding that MS is an autoimmune disease where T cells are reactive to myelin antigens. Controlling the biology of these T cell responses could bring therapeutic benefits to patients. Previous work from our lab has helped establish the role of the type II arginine methyltransferase, PRMT5 (protein arginine methyltransferase 5) in Th cell expansion and EAE autoimmunity using selective inhibitors. However, the role and mechanism by which PRMT5 modulates T cell responses and EAE progression haven't been determined. During my time in the lab, we developed two conditional knockout mouse models to evaluate PRMT5's function in T cells. In my thesis work I used our mouse models, exploratory RNA sequencing (RNA-Seq) and mass spectrometry techniques to uncover the mechanisms of action that drive T cell differentiation, expansion and the PRMT5 mediated symmetric dimethylation (SDM) targets of T cells. We identified a link between PRMT5 and cholesterol metabolism in Th17 cell development. We found that cholesterol pathway intermediates act as agonists for ROR-γt, the signature transcription factor encoding Th17 cells. PRMT5 expression was necessary for proper activity of the enzymes driving cholester (open full item for complete abstract)

Committee: Mireia Guerau-de-Arellano (Advisor); Stephen Kolb (Committee Member); Federica Accornero (Committee Member); Robert Baiocchi (Committee Member) Subjects: Biomedical Research; Immunology

Doctor of Philosophy, The Ohio State University, 2021, Pharmaceutical Sciences

The human DNA topoisomerase IIα (170 kDa, TOP2α/170) enzyme is essential in proliferating cells by functioning as a homodimer resolving DNA topological entanglements that form during chromosome condensation, replication, and segregation. The TOP2α/170 homodimer disentangles DNA by introducing transient double strand breaks in a DNA helix via a transesterification reaction between the active site Tyr805 from each TOP2α/170 subunit and the phosphodiester DNA backbone, creating the intermediate state known as the TOP2α/170-DNA cleavage complex. An intact DNA duplex passes through the cleaved DNA. Subsequently, the DNA strands are religated restoring the integrity of the DNA and preparing the TOP2α/170 dimer for another catalytic cycle. TOP2α/170 enzymatic activity is indispensable for the survival of highly proliferating cells including cancer cells. This has made TOP2α a prominent target for anticancer therapies. Some of the most widely used topoisomerase II targeting drugs such as etoposide, mitoxantrone, amsacrine and doxorubicin, stabilize the TOP2α/170-DNA cleavage complex preventing the religation of the DNA strands. As a result, these agents exert their cytotoxic effects by the accumulation of double strand DNA breaks which ultimately lead to the initiation of apoptotic pathways. Acquired chemoresistance to topoisomerase II targeting drugs continues to be a major obstacle in cancer treatment in the clinic. In order to characterize the mechanisms of resistance to etoposide, our laboratory developed etoposide resistant human leukemia K562 cells, designated K/VP.5 in which levels of TOP2α/170 were decreased along with identification of a novel C-terminal truncated isoform of TOP2α, TOP2α/90. This 90 kDa protein is present in both in K562 and etoposide resistant K/VP.5 cells with expression levels increased ~3-fold in K/VP.5 cells. TOP2α/90 is the translation product of novel alternatively spliced mRNA via intron 19 (I19) retention and processing, confirmed by 3 (open full item for complete abstract)

Committee: Christopher Coss (Committee Member); Jack Yalowich (Advisor); Dawn Chandler (Committee Member) Subjects: Pharmaceuticals; Pharmacology; Pharmacy Sciences

Doctor of Philosophy, The Ohio State University, 2021, Molecular, Cellular and Developmental Biology

Alternative splicing of the pre-mRNA is an essential post-transcriptional process in eukaryotes that generates multiple protein isoforms from a single gene. However, this process can be disrupted by mutations leading to several diseases including cancer. One such example is the oncogene and negative regulator of p53 Mouse Double Minute 2 homolog (MDM2), which undergoes alternative splicing to produce a splice isoform that has novel implications in cancer development. MDM2-Alt1 comprised of coding exons 3 and 12, is highly expressed in several cancers including those of the breast, liposarcomas, high-grade gliomas and rhabdomyosarcomas (RMS), and can be induced by genotoxic stress. Another good example of cancer-related aberrant alternative splicing, is the insulin receptor gene (INSR), comprised of 22 exons, and that skipping of the exon 11 allows the production of the IR-A splice isoform. Overexpression of the IR-A isoform compared to the full-length IR-B isoform, has been reported in several cancers including RMS, osteosarcoma, breast cancer, prostate cancer and hepatocellular carcinoma. Understanding of the splicing mechanisms of these two genes is crucial to improve current cancer therapies. In this work we report that SRSF2 positively regulates the alternative splicing of MDM2 and that its binding to the exon 11 can be modulated to generate novel mouse model for cancer studies. Furthermore, we show a novel and unique regulatory mechanism of splicing wherein a nuclear microRNA, miR-29b, influences the alternative splicing of MDM2 by directly binding to the MDM2 pre-mRNA. We show that miR-29b is downregulated by genotoxic stress, a similar characteristic of cancer cells. Finally, we study the usage of antisense oligonucleotides (ASOs) as cancer therapies, by blocking the binding of the CUG-BP1 (CUG Binding Protein 1) splicing factor with an ASO, we promote the alternative splicing of the IR-B isoform which decreases cell proliferation and angiogenesis. Moreover, (open full item for complete abstract)

Committee: Dawn Chandler (Advisor); Guramrit Singh (Committee Member); Kotaro Nakanishi (Committee Member); Anita Hopper (Committee Member) Subjects: Biochemistry; Cellular Biology; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2020, Pharmacology

Despite extensive advances in early breast cancer detection, diagnosis and treatment, effective cancer therapies to prevent tumor cell dissemination and overt metastasis remain an area of unmet clinical need. Many factors underlying metastatic processes have been identified; however, the central nodes that govern these elements are poorly understood. Our studies elucidate two nodal pathways that drive metastatic phenotypes in cancer cells: the activin/follistatin signaling pathway and the BCL11A-governed transcriptome. We found that breast cancers display decreased follistatin expression, a selective inhibitor of activin with high-affinity binding. Simultaneously, breast cancers also have increased expression of the INHBA subunit of activin, suggesting unabated activin signaling in this disease. Restoration of follistatin expression in mammary tumors arising in HER2/Neu transgenic mice completely abrogated metastasis to the lungs, indicating that follistatin is capable of suppressing metastatic progression. Follistatin is also prognostic of extended recurrence-free and overall patient survival. These studies suggest that therapeutics that inhibit activin signaling may be useful for the prevention of metastatic disease. We also present data demonstrating that the transcription factor, BCL11A drives optimal invasion and metastatic outgrowth in cell line and mouse models of triple-negative breast cancer (TNBC). We identified two pathways governed by BCL11A that facilitate its metastasis promoting properties. First, we found that BCL11A sustained the invasive capacity of TNBC cells by suppressing the expression of muscleblind-like splicing regulator 1, a splicing regulator that suppresses metastasis. This ultimately increased the levels of an alternatively spliced isoform of integrin-α6 that is associated with worse patient outcomes. We also uncovered that the BCL11A-controlled transcriptome was enriched in genes encoding matrisome proteins. Notably, matrix m (open full item for complete abstract)

Committee: Ruth Keri Ph.D. (Advisor); Marvin Nieman Ph.D. (Committee Chair); Donny Licatalosi Ph.D. (Committee Member); Mark Jackson Ph.D. (Committee Chair); Hung-Ying Kao Ph.D. (Committee Chair) Subjects: Molecular Biology; Pharmacology

PhD, University of Cincinnati, 2019, Medicine: Systems Biology and Physiology

Disruption of circadian rhythm can lead to serious sleeping disorders and predispose to a number of life-threatening diseases, including cancer. Circadian splicing adds an additional regulatory layer to the time keeping mechanism in plants, flies and mammals. The circadian regulation of alternative splicing, including that of core clock genes themselves, are speculated to be a central mediator of clock function, however, no comprehensive analyses of this mechanism exist to date. To develop an improved understanding of circadian splicing in mammals I describe a series of comprehensive analyses of circadian splicing within and across diverse healthy mouse, baboon and human tissues, spanning thousands of samples. These analyses confirm that conserved tissue-specific and tissue-shared circadian splicing events (CSEs) are frequent and can be identified from multiple study designs and recommend workflows for accurate circadian splicing analysis. Our analysis demonstrates a higher number of tissue-specific CSEs compared to circadian gene expression. Transcriptionally, temperature sensitive and other circadian splicing factors (SFs) are also found rhythmic in majority of the tissues. Cross-tissue CSEs frequently contain binding sites for these circadian SFs likely targeting specific CSEs and regulating splicing at the peripheral tissue level. Notably, these evolutionarily conserved CSEs and pan-tissue CSEs frequently impact prior defined cancer regulators, RNA binding proteins previously implicated in thermoregulation and splicing auto-regulation. I demonstrate the importance of these circadian findings specific in Lung cancer, which suggest the existence of novel putative chronotherapeutic targets. To enable the broad research community, we have developed an easy-to-use online web-portal to explore and compare these results across species. As such, these data have the potential to highlight intriguing new roles for splicing regulation in normal circadian biology.

Committee: Nathan Salomonis M.D. (Committee Chair); Christian Hong Ph.D. (Committee Member); Jaroslaw Meller Ph.D. (Committee Member); Yana Zavros Ph.D. (Committee Member); Tongli Zhang Ph.D. (Committee Member) Subjects: Bioinformatics

PhD, University of Cincinnati, 2019, Engineering and Applied Science: Computer Science and Engineering

Over the past decade, numerous clustering approaches have been developed and applied to gene expression studies for the unsupervised detection of sub-populations that inform disease prognosis, treatment and mechanism. For example, in diverse cancers, the identification of novel patient subtypes from gene expression can highlight novel therapeutic pathways and cooperating mutations. In addition to the measurement of transcriptional activity from genes, modern high-throughput sequencing technologies enable the sensitive detection of higher-resolution features including alternative splicing, RNA-editing and chromatin modifications. The detection of such features presents a number of computational challenges, due in large part to the sparse nature of that data, high dimensionality (hundreds of thousands of features) and presence of both broad and exceedingly rare molecular/genetic subtypes that are overlapping. In this dissertation, I describe the development of a series of novel methodologies to address these computational challenges that aim to uncover the hidden heterogeneity within complex molecular datasets. The first of these algorithms, splice-ICGS, provides an automated and accurate solution for the detection of complex overlapping splicing-defined subtypes, from large bulk RNA-sequencing datasets. Our solution required the introduction of several key innovations including new methods for sparse matrix filtering, correlation-based feature prioritization, iterative sparse-NMF analysis and a new strategy for multi-label classification. I demonstrate the improved performance of this approach in multiple clinical cancer datasets with an emphasis on Leukemia. To improve our understanding of the causal nature of such known and novel splicing subtypes, I further have developed several downstream analysis tools that can predict causal regulators from splicing subtypes in an automated manner (Bridger, RBP-Finder). These unsupervised approaches were further adapted to so (open full item for complete abstract)

Committee: Nathan Salomonis M.D. (Committee Chair); Gowtham Atluri Ph.D. (Committee Member); Raj Bhatnagar Ph.D. (Committee Member); Kakajan Komurov Ph.D. (Committee Member); Ali Minai Ph.D. (Committee Member) Subjects: Computer Science

PhD, University of Cincinnati, 2019, Medicine: Cancer and Cell Biology

The innate immune system is a complex network that recognizes and responds to foreign particles. Innate immune signaling is fundamentally involved in inflammation and increasing evidence implicates chronic innate and inflammatory signaling as a risk factor in cancer and hematologic malignancies. Recent studies have implicated the dysregulation of innate immune signaling in the pathogenesis of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). However, the precise genetic alterations that cause innate immune signaling activation in hematologic malignancies are not fully defined. Hematologic malignancies have a particularly high frequency of mutations in RNA splicing factors. How these mutations contribute to disease is not fully understood. A global analysis of exon usage in AML samples revealed a subset of genes is regulated exclusively at the isoform level in leukemia, resulting in anticorrelated expression of individual RNA isoforms. Many of these genes regulated by RNA isoform changes are associated with inflammatory and immune pathways. IRAK4 was the most significant immune pathway gene that undergoes isoform switching. Increased expression of the long isoform of IRAK4 (IRAK4-L), which includes exon 4, was found in MDS and AML cell lines and primary patient samples and results in maximal activation of NF-kB. Elevated IRAK4-L isoform expression is associated with poor prognosis in MDS and AML and is significantly associated with mutations in splicing factor U2AF1. Further, U2AF1 directly regulates the splicing of IRAK4 to increase the expression of IRAK4-L. Inhibition of IRAK4 abrogates leukemic growth in vitro and in vivo and is more efficacious in AML cells with U2AF1 mutations and/or higher expression of the IRAK4-L isoform. Thus, mutations in U2AF1 splicing factor induce expression of therapeutically targetable "active" IRAK4 isoforms and provide the first genetic link to activation of chronic innate immune signaling in MDS and AML. The conseq (open full item for complete abstract)

Committee: Daniel Starczynowski Ph.D. (Committee Chair); Jose Cancelas-Perez M.D. (Committee Member); Matthew Flick Ph.D. (Committee Member); Kakajan Komurov Ph.D. (Committee Member); Nathan Salomonis M.D. (Committee Member) Subjects: Oncology

Doctor of Philosophy, Case Western Reserve University, 2019, Biochemistry

Alternative RNA processing is a critical factor in increasing transcriptome complexity during cell differentiation and tissue development. Remarkably, spermatogenic cells express more alternatively processed transcripts compared to most whole tissues. While alternative mRNA processing is known to be prevalent in the testis, the full extent of its regulation and biological impact remain unclear. Here, we describe distinct stage-specific programs of pre-mRNA alternative splicing in male germ cells, revealing that the greatest number of splicing changes occur between mitosis and meiosis. Further, we identify an essential function for the RNA binding protein, Ptbp2, in regulating stage-specific alternative splicing. Using high-throughput sequencing technologies, we demonstrate that Ptbp2 regulates the alternative splicing of over 200 genes and inhibits splicing of alternative exons by directly binding near 3' splice sites. Strikingly, Ptbp2 regulates the alternative splicing of a network of genes important for germ-Sertoli cell communication, protein transport, and mitochondria dynamics. Indeed, using ultrastructural approaches, we observed cellular protein trafficking defects and increased mitochondria fission in the absence of Ptbp2. Using polyA-seq, we revealed stage-specific regulation of alternative polyadenylation during male germ cell development. In contrast to the regulation of alternative splicing during spermatogenesis, the majority of differences in 3'-end formation occur in post-meiotic germ cells and are accompanied by significant 3'UTR shortening. Collectively, the data reveal that alternative pre-mRNA processing is highly regulated during spermatogenesis and offers important insights into the physiological functions of alternative splicing in the mammalian germline. 

Committee: Donny Licatalosi (Advisor) Subjects: Biochemistry; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2017, Molecular, Cellular and Developmental Biology

Alternative splicing of the oncogene MDM2 is induced in response to genotoxic stress. MDM2-ALT1, the major splice variant generated, is known to activate the p53 pathway and impede full-length MDM2's negative regulation of p53. Despite this perceptible tumor-suppressive role, MDM2-ALT1 is also associated with several cancers including soft tissue sarcomas, breast, and ovarian cancers. We have shown that expression of MDM2-ALT1 is capable of initiating anchorage-independent growth, which is a hallmark of cancer cells. Furthermore, MDM2-ALT1 is the most common genetic perturbation characterized in pediatric rhabdomyosarcoma (RMS) irrespective of histological subtype. Despite its pervasiveness in tumors, there is little known about the regulators of that govern this alternative splicing event as well as its functions in vivo. In order to study the alternative splicing of MDM2 we have developed a damage-inducible minigene system. The MDM2 3-11-12s minigene recapitulates the splicing of the endogenous gene by excluding its intervening exon under genotoxic stress. Using SELEX-based bioinformatics prediction algorhythms in ESEfinder 3.0 we identified conserved consensus sequences for splicing regulators SRSF1 and SRSF2 in exon 11 of MDM2. We report that SRSF1 and SRSF2 promote MDM2 exon exclusion and inclusion, respectively. Additionally, by targeting these sites we can modulate MDM2 alternative splicing using splice switching oligonucleotides (SSOs). To understand the role of this splice variant in tumorigenesis we generated a conditional mouse model of MDM2-ALT1 expression. We show MDM2-ALT1 accelerated tumor onset and increased incidence of RMS on p53 null and hemizygous backgrounds. However, when expressed in B cells on a p53 wild-type background, MDM2-ALT1 leads to an initial decrease in B cell numbers despite driving lymphomagenesis in aged mice. Here, we have shown for the first time a co-occurrence of phenotypes that reflect the dichotomous nature of MDM2-A (open full item for complete abstract)

Committee: Dawn Chandler (Advisor); Denis Guttridge (Committee Member); Anita Hopper (Committee Member); Daniel Schoenberg (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 2016, Comparative and Veterinary Medicine

Feline oral squamous cell carcinoma (FOSCC) is the most common oral cancer in cats. This cancer is very aggressive and has the ability to invade into the adjacent bone resulting in poor quality of life and a grave prognosis in affected cats. FOSCC has been shown to be comparable to human OSCC in many aspects. However, the pathogenesis of FOSCC remains unclear. Similar to human OSCC, FOSCCs respond poorly to the available standard therapy. Therefore, novel molecular targets as well as effective therapies are required for this cancer. The usefulness of p16, p53, and pRb immunohistochemistry (IHC) for human OSCC classification has been reported. Low p16 frequently associates with non-viral-associated OSCCs, while high p16 with low p53 and pRb usually occurs in HPV-associated OSCC. In addition, a favorable prognosis was found in patients with HPV-associated OSCC and a grave prognosis was observed in patients with non-viral-associated OSCC. In this study, the IHC pattern of p16, p53, and pRb in FOSCC was investigated. We found that low p16 IHC is common in FOSCCs and high p16 IHC is present in a subset of patients with this cancer. However feline papillomavirus was not detected in these samples. These results indicate that FOSCCs have more than one type of classification and a variation in pathogenesis may occur in FOSCC. Increased osteoprotegerin (OPG) has been found to reduce tumor size and bone invasion in many human bone-invasive cancers. Low levels of feline OPG (fOPG) mRNA expression were previously observed in a bone-invasive FOSCC cell line (SCCF2). The role of fOPG in bone-invasive FOSCC in vitro and in vivo was investigated using a novel bone-invasive FOSCC cell line with high fOPG mRNA expression (SCCF2-OPGv.1).We found that overexpression of fOPG reduced tumor growth and inhibited bone invasion in bone-invasive FOSCC in vivo. Thus, fOPG could be effective adjuvant therapy for FOSCC-associated bone lysis. Telomerase, a protein complex with cell immortaliz (open full item for complete abstract)

Committee: Thomas Rosol J. (Advisor) Subjects: Animal Diseases; Experiments; Molecular Biology; Veterinary Services

Doctor of Philosophy, Miami University, 2016, Cell, Molecular and Structural Biology (CMSB)

In the first chapter I have discussed the advantages of Drosophila as a model organism, the ΦC31 transgenesis system, various aspects of splicing and splice factors and role of RNA in regulating gene expression. Chapter 2 is a detailed study of the truncated dADAR protein during Drosophila embryogenesis. 3'-RACE has shown that during D. melanogaster embryogenesis truncated dADAR mRNA levels are developmentally controlled. Western analysis showed that both full-length and truncated dADAR isoform classes are translated into protein in all Drosophila species used in the study. RNAi to specifically knock down the full-length dADAR transcript caused no changes to the long rnp-4f mRNA levels, suggesting an evolutionarily-conserved function of the truncated dADAR in regulating alternative splicing of rnp-4f during Drosophila embryogenesis. Chapter 3 includes a description of the ΦC31 based rnp-4f – GFP reporter gene expression vectors. Using the UAS-Gal4 system, we showed that presence of the regulatory rnp-4f stem-loop enhances reporter expression, which is further enhanced by overexpression of the RNP-4F transgene. We have proposed a model which suggests that RNP-4F protein acts as an enhancement factor and binds to its own mRNA 5'-UTR to enhance protein synthesis in the developing fly CNS by positive autoregulation. Chapter 4 is a study of an affinity-based approach to isolate RNA probe UV-crosslinked to embryo protein, to investigate the composition of the dsRNA binding protein complex which regulates rnp-4f gene expression. The RNA-protein complex was analyzed by MALDI-TOF MS and several protein candidates were identified. In Chapter 5, I have discussed the research objectives accomplished in my dissertation. I have mentioned future goals designed to further our understanding of rnp-4f and dADAR regulation during Drosophila embryogenesis. The last section includes the appendices divided into three parts. The first part is a REMSA based st (open full item for complete abstract)

Committee: Jack Vaughn (Advisor); Susan Hoffman (Committee Member); David Pennock (Committee Member); Haifei Shi (Committee Member); Xiao-Wen Cheng (Committee Member) Subjects: Biology; Molecular Biology

Master of Science, The Ohio State University, 2016, Animal Sciences

Increasing muscle mass is one of the primary goals of the United States poultry industry; however, skeletal muscle development at the molecular level is not fully understood. Myostatin (MSTN) is a well-known negative regulator of muscle growth by inhibiting both myoblast proliferation and differentiation into muscle fibers. Drastic increases in muscle mass have been observed by inhibiting MSTN activity in several species, including mice, cattle, and even humans. Recently, five alternative splicing isoforms of MSTN were discovered in domestic avians. Two of these isoforms, MSTN-A, which encodes the full-length peptide, and MSTN-B, which encodes a truncated peptide devoid of the active MSTN domain, are highly expressed in skeletal muscle. MSTN-B is able to bind and inhibit proteolytic processing of MSTN-A to the mature form, and overexpression of MSTN-B increases myoblast proliferation and myotube differentiation in vitro. Thus, MSTN-B may modulate muscle growth in avians, but further investigation is required to confirm its role in myogenesis. Japanese quail overexpressing MSTN-B in skeletal muscle were generated to elucidate the activity of this isoform in vivo. A 1.2 kb promoter of chicken skeletal muscle alpha actin was used to drive expression of the MSTN-B transgene specifically in skeletal muscle; however, MSTN-B protein was present in thigh muscle but not the pectoralis major muscle in three independent lines. The two lines with the highest MSTN-B expression (A1 and A2) were selected for further study. The growth curves between non-transgenic and transgenic groups in A1 and A2 were not different during a 6-wk growth period beginning at hatch. The pectoralis major muscle weight and heart weight as percentages of body weight were not different between any groups. On the other hand, the right gastrocnemius was a greater percentage of body weight in all transgenic male and female groups in both lines at 21 d posthatch (D21) and 42 d posthatch (D42; p < 0.05) (open full item for complete abstract)

Committee: Kichoon Lee (Advisor); Macdonald Wick (Committee Member); Pasha Lyvers-Peffer (Committee Member) Subjects: Animal Sciences; Developmental Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2015, Integrated Biomedical Science Graduate Program

Coronary artery disease (CAD) accounts for more deaths in America than any other disease, and places a considerable economic burden on the healthcare system. Statins have successfully reduced the risk of cardiac mortality; however, a residual risk of approximately 20-26% has been observed. Reduced activity of cholesteryl ester transfer protein has been shown to increase the risk of atherosclerotic death in males on statins, supporting a possible genetic basis for a portion of the observed residual risk. SNPs in CETP have been associated with a non-active splice form, increased HDL cholesterol, and allelic expression imbalance in CETP. The goal of the first portion of this study is to identify the functional SNPs responsible for the differential regulation of CETP splicing and expression through the use of molecular genetics. I use mini-genes and a qRT-PCR based assay to examine the effect of 2 SNPs on alternative splicing. I also investigate the interactions between SNPs residing in an upstream haplotype block with transcription factor binding sites. I demonstrate the effect of 3 candidate SNPs on expression of a luciferase reporter. Additionally, I interrogate RNA-sequencing data to uncover expression and alternative splicing of ABC and SLC transporter proteins in the blood brain barrier using computational tools. The effect of each candidate SNP (rs5883, and rs9930761) on splicing was apparent in the in vitro system that I assayed. The minor allele of rs5883 significantly increased the amount of alternatively spliced mRNA in HepG2 cells by 1.25 fold (p-value=0.0001), while rs9930761 had no effect. Thus, demonstrating the measureable effect of only rs5883 in regulating the amount of alternative splicing in liver. In the upstream haplotype block, I found that 3 SNPs interact with putative transcription factor binding sites for factors that are highly expressed in liver. rs17231506 had no effect in HepG2 cells, however, rs247616 caused a signific (open full item for complete abstract)

Committee: Wolfgang Sadee Der. Ret. Nat. (Advisor); Amanda Toland PhD (Committee Member); Kalpana Ghoshal PhD (Committee Member); Joseph Kitzmiller MD, PhD (Committee Member) Subjects: Biomedical Research; Genetics; Molecular Biology; Pharmacology