Master of Science (MS), Ohio University, 2014, Computer Science (Engineering and Technology)

Within every cell is a copy of an organism's DNA. This copy of DNA has all of the information needed for the cell to express every gene in the organism's genome. Although each cell is capable, individual cells do not express every gene in their DNA. The genes expressed by a cell are regulated by transcription factors (TFs) that bind to a transcription factor binding site (TFBS) located in the promoter region of the gene. TFs must bind to TFBSs in order for a gene to be expressed. Tissues are groups of cells that perform a specific function; therefore, the cells of a specific tissue express genes that are not expressed in other cell types. Hydroxyproline-rich glycoprotein (HRGPs) are proteins that are found in the plant cell wall, and they can be further classified according to the degree they are glycosylated as arabinogalactan-proteins (AGPs), extensins (EXTs), and proline-rich proteins (PRPs). Currently, the TFBSs for EXTs, AGPs, and PRPs expressed in the pollen cells of Arabidopsis are unknown andtheir discovery will provide a better understanding of the regulatory and evolutionary processes of these genes. Motif discovery and other bioinformatics tools were used to search the promoter regions of EXT, AGP, and PRP genes expressed in the Arabidopsis pollen cells and select motifs that are putative TFBSs. The best set of motifs discovered as putative pollen-specific TFBSs are GCYAMGKA, ACTMGGAA, CATSAAAMGA, and ATTKGKTTCT. Of the 8 pollen-specific promoters, GCYAMGKA occurs in 5 promoters,ACTMGGAA occurs in 2 promoters, CATSAAAMGA occurs in 4 promoters, and ATTKGKTTCT occurs in 3 promoters. Also, all of the 8 HRGP pollen-specific promoters have anoccurrence of at least one of these four motifs and none of the four motifs occur in the 84 HRGP promoters of genes not expressed in pollen cells.

Committee: Lonnie Welch (Advisor) Subjects: Bioinformatics; Computer Science

Doctor of Philosophy, Case Western Reserve University, 2015, Molecular Medicine

The epidermal growth factor receptor (EGFR) drives downstream signaling pathways that promote cancer progression. EGFR is often constitutively active in tumors, e.g., non-small cell lung cancer (NSCLC) and glioblastoma (GBM), either by overexpression or mutations. Nuclear factor-κB (NF-κB) is a master transcription factor that controls inflammation and innate immunity. NF-κB, often constitutively activated in cancer, drives tumor development by activating antiapoptotic and prosurvival genes, and has been implicated in anticancer drug resistance. We and others have shown that EGFR activates NF-κB signaling in both noncancerous and cancer cells. In an attempt to overcome drug resistance to EGFR tyrosine kinase inhibitors (TKI): erlotinib and lapatinib in NSCLC and GBM respectively, we combined EGFR-TKI with a novel class of NF-κB inhibitors, including quinacrine and curaxins, which inhibit NF-κB-driven transcription by targeting the facilitates chromatin transcription (FACT) complex. Unexpectedly, we discovered preferential targeting of GBM stem cells (GSCs), another major player in cancer therapeutic resistance, by curaxins, uncovering a potential role of FACT in the maintenance of stem cell phenotypes. Beyond applications in anticancer therapeutics, we are interested in the basic biological question of how EGFR, compared to canonical NF-κB activators such as interleukin 1 (IL-1), regulates NF-κB distinctively. Specifically, we utilize genomic approaches including RNA-sequencing (RNA-sequencing) and chromatin immunoprecipitation (ChIP)-sequencing to investigate the signal-specific, genome-wide regulation of NF-κB-driven transcription by EGFR versus IL-1. One possible mechanism that underlies specificity in transcription factor regulation is differential phosphorylation. We speculate that phosphorylation of the serine 276 residue on the NF-κB subunit p65 (RELA) plays a role in EGFR-mediated NF-κB regulation. Using the gene editing approach CRISPR, we have introduced t (open full item for complete abstract)

Committee: George Stark PhD (Advisor); Mark Jackson PhD (Committee Chair); Charis Eng MD, PhD (Committee Member); Brian Rubin MD, PhD (Committee Member); Micheala Aldred PhD (Committee Member) Subjects: Cellular Biology; Genetics; Molecular Biology; Oncology

Doctor of Philosophy (PhD), Ohio University, 2013, Chemistry and Biochemistry (Arts and Sciences)

NF-κB is a transcription factor that controls expression of genes involved in the immune and inflammatory responses as well a being a key component in the onset of cancers. In this study we provided evidence that MSK1 is responsible for a non-canonical late-phase activation of NF-κB upon UVB irradiation. Our data demonstrated that following UV irradiation, MSK1 is activated via phosphorylation at the 24 h time point coinciding with translocation of NF-κB into the nucleus. Investigations into the signaling pathways upstream of MSK1 through the use of specific inhibitors for mitogen-activated protein kinase and p38 revealed that both kinases are required for full phosphorylation during the late-phase (24 h), while p38 is paramount for phosphorylation during the early-phase (6 h). Electromobilty shift assays (EMSA) showed that inhibition of MSK1 resulted in a marked reduction in NF-κB binding affinity without altering the nuclear translocation of NF-κB. Supershift EMSA implicate that the p65, but not p50, isoform of NF-κB is involved in late-phase activation in response to UVB irradiation. Together, the results of these studies shed light onto a novel pathway of MSK1 mediated late-phase activation of NF-κB in response to UV irradiation. Exposure to UV induces a prolonged expression of COX-2 via transcriptional activation that is due in part to an increase in NF-κB activity. While transcriptional regulation of COX-2 expression has been well studied, the role of translational regulation of COX-2 synthesis upon UV-irradiation is not yet clear. In this study, we show that the phosphorylation of the alpha subunit of eIF2α plays an important role in the regulation of COX-2 expression after UV-irradiation. Our data demonstrates that UV light induces COX-2 expression in wild-type mouse embryo fibroblasts (MEFS/S) and that the inducibility is reduced in MEFA/A cells in which the phosphorylation site, Ser-51 in eIF2α, is replaced with a nonphosphorylatable Ala (S51A). UV (open full item for complete abstract)

Committee: Shiyong Wu PhD (Advisor) Subjects: Biochemistry; Cellular Biology; Molecular Biology; Oncology

Doctor of Philosophy, The Ohio State University, 2007, Oral Biology

Transcription factor activation is a rapidly induced intermediary component of the innate immune response to lipopolysaccharide (LPS). Intracellular signaling cascades initiated by extracellular recognition of LPS, induce transient activation of a conserved set of constitutively expressed transcription factors. Factors such as NF-κB and AP-1 are able to rapidly localize to the nucleus and bind to specific DNA sequences associated with the gene promoters for proinflammatory cytokines and growth factors. Transcription of several proinflammatory genes such as IL-1β, IL-6 and TNF-α is influenced by transcription factors whose impact is to promote or suppress gene expression. Activated by glucocorticoids (GC) the glucocorticoid receptor (GR) has long been appreciated for its anti-inflammatory effects. The GR modulates gene expression of proinflammatory genes through both direct (DNA binding) and indirect (protein-protein) interactions with the gene promoter. Ultimately, the goal of inflammation is the elimination of the underlying stimulus while simultaneously preventing any unnecessary damage to the surrounding tissue that may be caused by prolonged cytokine activation. Over-expression of GC as observed in cases of long-term stress inhibits proinflammatory gene activation delaying the clearance of and increasing susceptibility to pathogens. Previous studies from our laboratory have indicated that androstenediol-induced activation of the androgen receptor (AR) is capable of altering the impact of activated GR on transcription factor activation and gene expression patterns following tissue injury. The AR and GR are structurally homologous proteins that are capable of interacting with identical DNA sequences in gene promoters. Their close structural relationship also imparts a high degree of overlap regarding the indirect protein-protein interactions that are essential in the assembly of factors that drive transcription. The studies detailed herein test the hypothesis that (open full item for complete abstract)

Committee: David Padgett (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2024, Molecular Genetics

In eukaryotes approximately 9% of genes encode proteins that utilize zinc as a cofactor. Zinc-dependent proteins can be found in the cytosol as well as other intracellular compartments including the nucleus, Golgi apparatus, endoplasmic reticulum, mitochondria, and vesicular bodies. Zinc transporters are utilized to ensure appropriate levels of zinc are maintained in compartments. While cells require zinc to function, excessive zinc can also be toxic. Because of this, organisms have evolved mechanisms for maintaining appropriate zinc levels. These mechanisms include altering the expression of genes involved in zinc homeostasis using zinc-responsive transcription factors. Schizosaccharomyces pombe is a model organism that was recently discovered to have its own zinc-responsive transcription factor, Loss of Zinc-sensing 1 (Loz1). Currently, it is not known how Loz1 activity is regulated by zinc. It is also unclear how zinc levels are regulated within intracellular compartments to changes in intracellular availability. The work in this dissertation had two approaches to better understand how S. pombe senses zinc and how changes in zinc status affect zinc levels in intracellular compartments. The first part of the study focuses on Loz1 and a potential mechanism that affects its ability to sense zinc. Previous in vitro work in a collaborating lab has shown that an X-Pro bond (with X referring to any amino acid) within the linker region of the two zinc fingers from Loz1 can be found in a cis or trans conformation. Conformational changes from cis to trans in vivo are slow but can be used as switches for changing protein activity. Here, I investigated whether the cis/trans isomerization of the X-Pro bond of Loz1 has a regulatory function related to Loz1 activity by using knockouts of genes encoding proteins involved in catalyzing the switch of cis/trans X-Pro bonds. I discovered that expression of reporter constructs of Loz1 targets (adh4, vel1, pho8) was decreased in a (open full item for complete abstract)

Committee: Amanda Bird (Advisor); Harold Fisk (Committee Member); Susan Cole (Committee Member); Anita Hopper (Committee Member) Subjects: Genetics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2024, Medical Science

Ewing sarcoma is a highly aggressive tumor of bone and muscle tissues, primar- ily affecting children, adolescent, and young adults. While the clinical outcome for patients with localized Ewing sarcoma have improved due to a combination of ap- proaches including surgery, radiation, and a high-dose chemotherapies, the same can not be said for patients with metastatic and relapsed diseases. The standard treatment regimen has seen little innovation over the past decades. This stagnation in therapeutic advancement reflects a critical gap in our knowledge, which, if addressed, could pave the way for more targeted interventions for Ewing sarcoma patients. Central to Ewing sarcoma pathogenesis is the EWS::FLI fusion oncoprotein. This fusion results from a translocation between chromosomes 11 and 22, merging the EWSR1 and FLI1 genes. EWS::FLI is an aberant transcription factor that dysregu- lates the gene expression profile of normal cells, leading to their malignant transfor- mation. The fusion of the EWS domain with the FLI domain endows the final protein a novel ability to bind at GGAA microsatellites, which are scattered across the hu- man genome in great numbers and with varied characteristics. EWS::FLI reprograms the epigenetic landscape by binding at GGAA repeats, opening nucleosome-wrapped DNA, recruiting chromatin modifiers, and altering chromatin architecture and the enhancer landscape. We investigated the role of the DBD-α4 helix in the FLI domain of the fusion protein in genome regulation. Employing a mutli-omics approach, we delineated the mechanisms by which this alpha-helix participates in the transcriptional activation capacity of EWS::FLI. Our studies unveiled that the DBD-α4 helix is required for col- lective binding at long and dense GGAA microsatellites, a process integral to various forms of genome regulation by EWS::FLI including formation of topologically asso- ciated domains (TADs), short-range loops, Ewing-specific enhancers, and pr (open full item for complete abstract)

Committee: Emily Theisen (Advisor) Subjects: Medicine; Oncology

Master of Science (MS), Ohio University, 2023, Molecular and Cellular Biology (Arts and Sciences)

Gravity is among the most critical of environmental cues in shaping plant growth; however, its ubiquity on Earth limits available options in the study of the plant gravity response. NASA has circumvented this obstacle by hosting experiments in the microgravity environment of the International Space Station (ISS). Gene expression data gleaned from RNA-seq and microarray analyses of Arabidopsis seedlings grown aboard the ISS has provided a wealth of information regarding how plants respond to this unique gravity condition. A meta-analysis of these datasets intersected with one of terrestrial plants exposed to an alternate gravity stimulus - a new gravity vector – has identified several novel components in the gravity signaling pathway. Of particular interest in the analysis were transcription factors, for their role in regulating downstream expression patterns. Two transcription factors were identified at this fundamental level of the Arabidopsis gravity response: ERF104, which is upregulated on earth in response to a new gravity vector and downregulated in microgravity, and IQD21, which displays the inverse expression pattern. A third gene, CIB1, was shown to be upregulated in both scenarios. Phenotypic characterizations of mutant lines of each of these genes show significant gravity sensitivity in the root tip and/or inflorescence stem, confirming their gravitropic role. Fusion constructs of these transcription factors joining each with a Human Influenza Hemagglutinin (HA) tag were generated, placed under the control of a constitutive promoter, and expressed in each gene's respective Arabidopsis T-DNA mutant line. These lines are enabling chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify binding sites of each transcription factor in the Arabidopsis genome, and consequently to infer their downstream targets. This work was done in tandem with qPCR analyses targeting the expression patterns of each protein in response to (open full item for complete abstract)

Committee: Sarah Wyatt (Advisor); Zhihua Hua (Committee Member); Michael Held (Committee Member); Allan Showalter (Committee Member) Subjects: Molecular Biology; Plant Biology

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Sciences

CD4+ T cells are a subset of the adaptive immune response that are classically defined as “helper” type cells, responsible for aiding in the effective recruitment and function of other immune cells. In addition to these traditional helper type roles, however, an additional subset of CD4+ T cells characterized by the ability to perform cytotoxic functions classically attributed to Nature Killer (NK) and CD8+ T cells, has emerged. This additional subset, deemed CD4+ cytotoxic T lymphocytes (CD4-CTLs), produce cytolytic effector molecules and directly kill compromised cells in a major histocompatibility complex class-II (MHC-II) restricted fashion. While initially thought to be a bi-product of in vitro culturing, CD4-CTLs have now been identified in a broad range of both human and murine immune responses. Specifically, CD4-CTLs have been shown to play largely protective roles in numerous antitumor and antiviral responses, including those to influenza and SARS-CoV-2. Conversely, dysregulated CD4-CTLs have been implicated in the pathogenesis of autoimmune diseases, including multiple sclerosis and ulcerative colitis. Despite their documented importance in a wide range of immunological contexts, the mechanisms that underly their differentiation and function remain enigmatic. Here, we first identify the Ikaros Zinc Finger transcription factor Aiolos (encodedby Ikzf3) as a reciprocal regulator of CD4-CTL and T-follicular helper (TFH) cell populations. TFH cells represent a classical CD4+ T helper cell populations that, upon activation and subset specific differentiation, interact with and provide aid to B-cells in the production of high-affinity neutralizing antibodies. In this work, we utilized a murine model of influenza infection, an immunologic context in which both TFH and CD4-CTLs have been shown to play important protective roles. We find that the absence of Aiolos resulted in global disruptions in TFH cell programming, including decreases in key transcription fa (open full item for complete abstract)

Committee: Ken Oestreich (Advisor); Hazem Ghoneim (Committee Chair); Thomas Mace (Committee Member); Murugesan Rajaram (Committee Member); Purnima Dubey (Committee Member) Subjects: Biomedical Research; Immunology

Doctor of Philosophy, Case Western Reserve University, 2023, Systems Biology and Bioinformatics

Gene expression from bulk RNA-seq studies is an average measurement between two chromosomes and across cell populations. Signifcant heterogeneity in gene expression between alleles and among cells can not be accurately characterized by a single averaged measurement. Transcription burst, where the promoter repeatedly alternates between an activated and inactivated state, is a major contributor to gene expression heterogeneity. Increased cell-to-cell heterogeneity in gene expression is a hallmark of aging, potentially induced by the change of transcription burst patterns. Due to technical restrictions in measuring multiple transcript levels over time, studies on transcription burst have been conducted only in a limited number of genes within selected model organisms. In this dissertation, we develop an approach to construct a transcriptomic kinetics map with scRNA-seq data and phased genotype profles. We apply our method to two clonal cell lines and systematically investigate the regulatory effect of genetic variants and TF binding on transcriptional kinetics. We found that the transcription initiation rate and burst frequency correlate most with eQTL effect sizes from bulk RNA-seq studies, suggesting that eQTLs affect average gene expression mainly through altering the transcription initiation rate and burst frequency. Roughly 90% of the variance of burst frequency can be explained by TF occupancy within the core promoter, and allele-specifc binding events of individual TFs are associated with the change of transcription initiation rate and burst frequency. Microglia are the resident macrophages of the brain parenchyma and play critical roles in maintaining tissue homeostasis and are responsible for innate immune response. AD-associated microglia sub-cell types are not consistently replicated in human samples. We hypothesize that aging increases the transcriptional variability of microglia and introduces cell-to-cell heterogeneity. By applying our approach to micro (open full item for complete abstract)

Committee: William Bush (Advisor); Mark Cameron (Committee Chair); Xiaofeng Zhu (Committee Member); Gurkan Bebek (Committee Member); Hao Feng (Committee Member); David Lodowski (Committee Member) Subjects: Bioinformatics; Genetics

Doctor of Philosophy, The Ohio State University, 2022, Molecular, Cellular and Developmental Biology

Ewing sarcoma is the second most common pediatric malignancy of bone and soft tissue. It is treated with highly aggressive therapy that involves surgery, radiation, and multiple chemo agents. However, the long-term survival rates are less than ideal (70% for localized disease, and less than 30%for metastatic disease) and the quality of life in survivors are diminished after intense therapy. A better understanding of the molecular underpinnings of this disease is therefore necessary to improve patient outcome. Genetically, Ewing sarcoma is characterized by a single chromosomal translocation event, t(11;22)(q24;q12) in ~85% of cases, that results in the expression of an oncogenic fusion transcription factor EWS/FLI. In fact, all cases of Ewing sarcoma express either EWS/FLI or a highly related FET/ETS fusion transcription factor as the sole recurring genomic aberration. Massively parallel sequencing have established Ewing sarcoma as a cancer with low mutational burden, and oncogenesis is thought to be driven by EWS/FLI mediated epigenetic reprogramming. The FLI portion of EWS/FLI contains an ETS type DNA binding domain (DBD) that allows EWS/FLI to bind consensus ETS sequences containing a single GGAA core motif (ACCGGAAGTG). The EWS portion is a low complexity domain (LCD), when fused to FLI allows EWS/FLI to bind DNA sequences containing repeats of GGAA motifs (GGAA-microsatellites) that otherwise remain inaccessible. EWS/FLI orchestrates widespread transcriptional dysregulation, including both activation and repression of thousands of genes, to promote oncogenic transformation in Ewing sarcoma. Upregulation of target genes correlate highly with EWS/FLI bound GGAA-microsatellite response elements that show length polymorphisms between cell lines and patient ethnicities. The EWS LCD has been shown to interact with and recruit histone modifying enzymes and chromatin remodelers to GGAA-microsatellites to induce epigenetic changes characteristic of enhancer elements. Thi (open full item for complete abstract)

Committee: Stephen Lessnick Dr. (Advisor) Subjects: Bioinformatics; Biology; Biomedical Research; Molecular Biology

Doctor of Philosophy, University of Akron, 2022, Integrated Bioscience

Cadherins are cell-adhesion molecules that play important roles in animal development, maintenance and/or regeneration of adult animal tissues. In order to understand cadherins' functions in adult vertebrate visual structures, one must study their distribution in those structures. First, I examined expression of cadherin-6, cadherin-7, protocadherin-17 and protocadherin-19 in the visual structures of normal adult zebrafish using RNA in situ hybridization, followed by studying expression of two Kruppel-like transcription factors (klf6a and klf7), that are known markers for regenerating adult zebrafish retinas and optic nerves, in normal adult zebrafish brain, normal and regenerating adult zebrafish retinas. Then, I investigated expression of these cadherins in regenerating adult zebrafish retinas using both RNA in situ hybridization and quantitative PCR. Finally, as the first step in elucidating molecular mechanisms underlying protocadherin-17 (one of the cadherins that I studied) function in zebrafish visual system development, I used DNA microarray analysis to study gene expression of zebrafish embryos with their protocadherin-17 expression blocked by morpholino antisense oligonucleotides (these embryos are called protocadherin- 17 morphants). The major findings include: 1) cadherin-6, cadherin-7, protocadherin-17 and protocadherin-19 were differently expressed in the retina and major visual structures of normal adult zebrafish brain. 2) klf6a and klf7 showed similar expression patterns in most visual structures in the adult fish brain, and in regenerating retinas, but klf6a appeared to be a superior regeneration marker based on RNA in situ hybridization. 3) These four cadherin molecules showed distinct expression patterns in the regenerating zebrafish retinas. 4) Several genes involved in vision and/or visual development were significantly downregulated in the protocadherin-17 morphants compared to control embryos. My results suggest that (open full item for complete abstract)

Committee: Qin Liu (Advisor); Richard Londraville (Committee Member); Rolando Ramirez (Committee Member); Brian Bagatto (Committee Member); Zhong-Hui Duan (Committee Member) Subjects: Anatomy and Physiology; Bioinformatics; Biology; Developmental Biology; Molecular Biology

MS, University of Cincinnati, 2022, Medicine: Molecular and Developmental Biology

Transcription factors (TFs) interpret instructions encoded by the genome. However, related transcription factors often have similar binding site preferences but not necessarily similar functions. Two such TFs are Gsx2 and Dlx2; both are members of the homeodomain family and have highly similar DNA binding motifs. However, Gsx2 can bind as both a monomer (M) and a homodimer (D), whereas Dlx2 has only been shown to bind as a monomer. Once bound to DNA, Gsx2 can stimulate or repress transcription according to its binding mode, but the Dlx factors are thought to predominantly function as transcriptional activators. This leads to divergent transcriptional outcomes based on which TF is bound. In the present work, we show that both Dlx2 and Gsx2 can bind to the same M-sites from known regulatory elements, but they mediate opposing outcomes with Dlx2 activating transcription and Gsx2 repressing transcription. In contrast, both Gsx2 and Dlx2 could stimulate gene expression via D-sites, but they bind these sites via different mechanisms: Gsx2 cooperatively and Dlx2 non-cooperatively. These findings highlight the need to identify high confidence enhancer elements that share binding events among multiple TFs. To test potential regulatory elements, we employed an immortalized rat Multipotent Neural Stem cell line (MNS70). By characterizing its chromatin accessibility and transcriptome and intersecting with additional existing mouse forebrain data, we predict potential enhancers for future study. Further, we found that the expression of the dorsal-telencephalic factor, Pax6, in MNS70 cells is not uniform, but varies from cell-to-cell. Since prior studies showed that Gsx2 represses Pax6 during mouse forebrain development, the MNS70 cells provide a useful tool for evaluating the ability of Gsx2 to regulate Pax6 expression as a model of the pallial-subpallial boundary.

Committee: Brian Gebelein Ph.D. (Committee Member); Hee-Woong Lim Ph.D. (Committee Member); Kenneth Campbell Ph.D. (Committee Member) Subjects: Developmental Biology

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

Neurons develop remarkably long, complex processes that integrate into circuitry critical for neuronal communication. Once mature, postmitotic neurons must survive for decades of life and continuously function to ensure normal, lifelong cognition, perception, behavior and movement. Due to their long and complex fibers, neurons face a unique challenge to supply distal synapses and axons with materials and energy needed to compensate for turnover, conferring a vulnerability to aging and neurodegenerative diseases should the mechanisms controlling these processes break down. Remarkably little is known regarding the transcriptional regulation of postmitotic neuronal survival and maintenance of long range axons. Here we found an adult stage specific transcriptional program controlled by terminal selectors Lmx1b and Pet1 for the survival of serotonergic synapses and axons in vivo. Adult conditional targeting of Lmx1b/Pet1 in mouse serotonergic neurons resulted in dysregulation of hundreds of genes enriched for functions related to synapses, axonal transport and structure, and mitochondrial functions. Mutant neurons presented with fragmented mitochondria, and progressive degeneration of serotonergic synaptic baskets and fiber pathology characterized by dramatically thinned axons with swollen varicosities and spheroids containing unusual aggregates of amyloid precursor protein, alpha-synuclein, and phosphorylated neurofilament. Lmx1b/Pet1 targeting induced fiber degeneration without cell body loss, is independent of 5-HT levels, and is accompanied by neuroinflammation and altered physiological and behavioral responses. This work provides substantial evidence for transcriptional programs required in the adult mammalian brain for survival of long range axonal architectures.

Committee: Evan Deneris (Advisor) Subjects: Neurobiology; Neurosciences

Doctor of Philosophy (Ph.D.), University of Dayton, 2021, Biology

There are 36 recognized animal phyla that are characterized by a set of morphological features considered to be a body plan. Upon these 36 body plan themes, there has evolved an interminable amount of diversity ranging from the underwhelming and simplistic to the most extraordinarily ornate. These body plans and the diverse traits that decorate them are the products of orchestrated gene expression during the developmental process. Underlying the development of morphological traits is a hierarchy of regulatory gene expression, chiefly comprised of transcription factor proteins, organized into a gene regulatory network (GRN) that ultimately impart instructions on so-called differentiation genes responsible for building the physical trait. Expression for genes within the GRN is controlled by cis-regulatory elements (CREs), which instructs a gene as to when and where it is to be active during development and how much of the protein product it encodes for is needed. The goal of my thesis research was to understand how changes to a GRN drive the development and evolution of animal morphology. Chapter 2 presents research on the repeated evolution of male-specific abdominal pigmentation in fruit fly species of the Sophophora subgenus. This research focused on analyzing the phylogenetic distribution of male-specific pigmentation and characterizing the expression for genes involved in the pigment metabolic pathway. We found dimorphic pigmentation to be widespread among distantly related fruit fly species. Using in situ hybridization and immunohistochemistry, we show that there is conservation in the expression of the pigmentation enzymes responsible for constructing the trait and flexibility at the level of the regulatory genes that control their expression. In Chapter 3, we investigate the extent to which mutations within the CREs (cis-evolution) or the CRE-interacting transcription factors (trans-evolution) for pigment metabolic enzymes contribute to the origin, diversifica (open full item for complete abstract)

Committee: Thomas Williams PhD (Advisor); Mark Nielsen PhD (Committee Member); Mark Rebeiz PhD (Committee Member); Madhuri Kango-Singh PhD (Committee Member); Ryan McEwan PhD (Committee Member) Subjects: Biology; Evolution and Development; Genetics

Doctor of Philosophy, Case Western Reserve University, 2021, Genetics

Gene regulation of CFTR is complex. Cell type-specific enhancers and transcription factors orchestrate its precise expression, at the appropriate time and in the appropriate tissue. Decades of research at this locus have generated a rich dataset to interrogate the mechanisms of such regulation. In this study, I performed enhancer deletions to study the combinatorial effect of two intestine-selective enhancers on CFTR expression in their native context. I reveal the cooperative interactions involving enhancers and other elements in gene regulation and maintenance of 3D structure at the CFTR locus, using gene expression profiling and 4C-seq post dual enhancer deletion. In addition to studying cis elements, I also interrogate the contribution of trans factors by knockdown of TFs and investigating their effect on chromatin structure. My study demonstrates that TF binding is necessary for looping at the CFTR locus. Besides homing in on the CFTR locus, I also investigate the global differences in functional genomics between intestinal cell models. Organoids are a valuable 3D model to study the differentiated functions of the human intestinal epithelium. Here I combine genome-wide analysis of open chromatin by ATAC-seq with transcriptome mapping by RNA-seq, to define the genomic signature of human intestinal organoids (HIOs). I next compared the transcriptome and open chromatin profiles of HIOs with equivalent datasets from the Caco2 colorectal carcinoma line. My results define common gene regulation features of the intestinal epithelium in HIO and Caco2. Generation of Caco2 cysts enabled interrogation of the genomic basis for2D and 3D cultures. Over-represented motif analysis of open chromatin peaks identified CDX2 as a key activating transcription factor in HIO, but not in monolayer cultures of Caco2. However, the CDX2 motif becomes overrepresented in open chromatin from Caco2 cysts, reinforcing the importance of this factor in intestinal epithelial differentiation and (open full item for complete abstract)

Committee: Fulai Jin (Committee Chair); Ann Harris (Advisor); Jonathan Haines (Committee Member); Angela Ting (Committee Member) Subjects: Genetics

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2020, College of Sciences and Health Professions

Prostate cancer (CaP) accounts for more than 30,000 deaths annually in the United States. Sustained reliance on androgen receptor (AR) after failure of AR-targeting androgen deprivation therapy (ADT) prevents effective treatment of castration-resistant CaP (CRPC). Interfering with the molecular machinery on which aberrantly activated AR relies to drive CaP progression may be an alternative therapeutic strategy. The feasibility of such approaches remains to be tested. Here, I explore targeting the mechanism by which AR, via RhoA, conveys androgen-responsiveness to serum response factor (SRF), a secondary transcription factor. The AR-SRF signaling axis controls aggressive CaP behavior and is maintained in CRPC. Following an siRNA screen and candidate gene approach, RNA-Seq studies confirmed that the RhoA effector Protein Kinase N1 (PKN1) transduces androgen-responsiveness to SRF. PKN1 overexpression occurred during clinical CRPC progression, and hastened CaP growth and shortened CRPC survival in orthotopic CaP xenografts. Mechanistically, PKN1 was found to form a complex with SRF upon AR activation. Via biotin-based proximity ligation assay coupled with mass spectrometry, novel members of the PKN1-SRF transcriptional complex were isolated. PKN1's effects on AR-SRF signaling relied on its kinase domain. The multikinase inhibitor lestaurtinib inhibited PKN1 action and preferentially affected androgen regulation of SRF over direct AR target genes. PKN1 inhibition decreased viability of CaP cells before and after ADT, and in patient-derived xenograft models inhibited CaP growth. These results provide a proof-of-principle for selective forms of ADT that preferentially target different fractions of AR's transcriptional output to inhibit CaP growth.

Committee: Hannelore Heemers (Advisor); Robert Silverman (Committee Chair); Angela Ting (Committee Member); Girish Shukla (Committee Member); Nima Sharifi (Committee Member); Justin Lathia (Committee Member); Ian Mills (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology; Oncology; Therapy

Doctor of Philosophy, Case Western Reserve University, 2021, Neurosciences

Alzheimer's disease (AD) is a highly prevalent neurodegenerative disorder characterized by extracellular deposition of β-amyloid (Aβ) containing plaques, intracellular neurofibrillary tangles, neuronal loss, and progressive cognitive decline. Robust Aβ-induced neuroinflammation is a hallmark of AD. Through execution of immune functions, myeloid cells, such as brain-resident microglia and peripheral monocytes, impact disease progression in various mouse models of AD. Microglial phagocytic clearance of Aβ fibrils and plaques becomes diminished with time, but microglia still interact with plaque perimeters throughout disease and exhibit a characteristic transcriptional signature. It is unclear if microglial engagement of plaques plays a protective or detrimental role during peak plaque formation. To test whether microglia exert disease-modifying effects during peak pathology, we selectively depleted microglia cells with a CSF1R antagonist in an AD mouse model. CSF1R inhibition depleted microglia, reduced plaque burden, and curtailed inflammation. Microglia depletion altered plaque compaction, increasing diffuse-like plaque morphologies. Plaque-associated neuritic dystrophy was also enhanced, most likely owing to fewer microglia-engagement of plaques. Importantly, this effect was dependent on CSF1R signaling and reversible, as withdrawal of antagonist facilitated plaque remodeling in regions where microglia repopulated. Contrary to other works, we show microglia exert neuroprotective roles by limiting diffuse plaque expansion and neuritic dystrophy during peak pathology in an AD mouse model. Aβ clearance and resolution of inflammation is mediated by activation of nuclear receptors in myeloid cells. Although nuclear receptor agonists produce salutary outcomes in mouse models of AD, it is unknown whether deletion of nuclear receptors in myeloid cells alters AD pathology. It is also unclear if nuclear receptor deletion in peripheral monocytes or microglia (open full item for complete abstract)

Committee: Gary Landreth (Advisor); Richard Zigmond (Committee Chair); Heather Broihier (Committee Member); George Dubyak (Committee Member) Subjects: Neurosciences

PhD, University of Cincinnati, 2020, Medicine: Molecular and Developmental Biology

Notch signaling is a form of short-range communication between neighboring cells that regulates many aspects of animal development and alterations in this signaling pathway have been associated with numerous disease states. At its simplest, ligand-receptor interactions at the cell membrane trigger sequential proteolytic cleavages of the Notch receptor to release the Notch intracellular domain (NICD) into the cytoplasm. Once released, NICD translocates into the nucleus, binds to the transcription factor CSL, recruits the co-factor Mastermind (Mam), and activates the transcription of target genes. In the absence of NICD, the same transcription factor CSL can bind to co-repressors to turn off transcription of the same target genes. Many Notch pathway genes show haploinsufficiency phenotypes, meaning that halving the dosage of Notch pathway genes fails to accomplish normal developmental processes. Haploinsufficiency implies that Notch-dependent processes are highly sensitive to alterations in signal strength. While all cells utilize the same Notch transcriptional cascade, haploinsufficiency phenotypes for the different Notch pathway genes manifest in distinct tissues, suggesting that Notch-dependent cell types have differential sensitivity to alterations in signal strength. However, the underlying mechanisms of such differential sensitivity are not well understood. Ultimately, Notch signaling has been shown to result in changes in gene expression via two types of CSL binding sites. The CSL monomer sites bind independent Notch transcription complexes, whereas CSL dimer sites, which are composed of two CSL binding sites orientated head-to-head and spaced 15 to 17 bp apart (known as Su(H) paired sites or SPSs), bind two cooperative Notch transcription complexes via NICD-mediated dimerization. Although it has been shown that NICD dimerization is required for optimal transcription of some Notch target genes, the impact of the NICD dimerization on SPSs versus NICD recruitment (open full item for complete abstract)

Committee: Brian Gebelein Ph.D. (Committee Chair); Kenneth Campbell Ph.D. (Committee Member); Stacey Huppert Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Masato Nakafuku M.D. (Committee Member) Subjects: Developmental Biology

PhD, University of Cincinnati, 2020, Medicine: Molecular and Developmental Biology

Rapid growth of the embryonic brain requires accurate cell specification and sufficient proliferation to generate a diversity of neuronal subtypes. Neurogenesis in the developing mammalian forebrain is controlled via precise spatial and temporal expression of key transcription factors. The homeodomain transcription factor, Gsx2, is important for three key functions during the development of the mouse forebrain: 1) Dorso-Ventral patterning of neural progenitors by establishing the pallio-subpallial boundary; 2) Balancing the maintenance of proliferative progenitors with neurogenesis in the Lateral Ganglionic Eminence (LGE); and 3) Defining progenitor identity to specify distinct neuronal subtypes. Despite its clear functional importance, little is known about the mechanisms by which Gsx2 performs these functions during forebrain development. Here, I will describe two new molecular functions of the Gsx2 protein. First, we found a novel direct interaction between Gsx2 and the proneural basic helix-loop-helix (bHLH) transcription factor Ascl1, which promotes neurogenesis during forebrain development. We show that physical interactions between Gsx2 and Ascl1 within dividing neuronal progenitors prevents Ascl1 from forming homodimers or heterodimers with other bHLHs, and thereby inhibits Ascl1 binding to DNA. Based on our data, we propose a model in which Gsx2 induces Ascl1 gene expression, but limits Ascl1's ability to promote neuronal differentiation through direct Gsx2-Ascl1 protein-protein interactions that inhibit the activation of neurogenic target genes. Therefore, Gsx2-Ascl1 co-expressing cells are primed for neurogenesis, but will not differentiate until Gsx2 is down-regulated. Second, we have identified two types of Gsx2 DNA binding sites within LGE enhancers: monomer sites (M-sites) that independently bind Gsx2 and dimer sites (D-sites) that cooperatively bind Gsx2. We define the DNA binding site features required for cooperative DNA binding and demonstrate it (open full item for complete abstract)

Committee: Brian Gebelein Ph.D. (Committee Chair); Kenneth Campbell Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Masato Nakafuku M.D. (Committee Member); James Wells Ph.D. (Committee Member) Subjects: Developmental Biology

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

Since being discovered in the early 1990s, the primary functions of classical serine-arginine rich (SR) proteins and non-classical SR-related proteins have been demonstrated in gene regulatory processes such as pre-mRNA processing, mRNA metabolism, and nuclear export of mRNAs. Bcl-2-associated transcription factor 1 (BCLAF1, also called Btf) and Thyroid hormone receptor associated protein 3 (THRAP3, also called TRAP150) are homologous non-classical SR-like splicing factors. In this thesis, I have identified endogenous binding partners of Btf and TRAP150. I used statistical analysis to select common versus distinct protein partners and compared previously documented subcellular localization of these identified proteins with studies for Btf/TRAP150. Previous studies indicate that Btf and TRAP150 have overlapping roles in maintaining cell cycle progression and DNA damage repair, but different roles in regulating subcellular mRNA distribution. Therefore, I hypothesized that Btf and TRAP150 interact with a common set of protein partners for pathways in which they share functions yet have distinct protein partners that impart unique functions. MS-based proteomic analysis of immunoprecipitated Btf and TRAP150 complexes revealed novel interactions with FXR1P and FXR2P, autosomal paralogs of Fragile X mental retardation protein (FMRP). FXR1P is a common partner for both Btf and TRAP150 whereas FXR2P is distinct to TRAP150. Future studies will confirm interaction between these binding partners as well as others from the analysis. Also, siRNA depletion studies will investigate the mechanisms for how Btf/TRAP150 impact subcellular distribution and function of Fragile X proteins.

Committee: Paula A. Bubulya Ph.D. (Advisor); Labib Rouhana Ph.D. (Committee Member); Quan Zhong Ph.D. (Committee Member); David L. Goldstein Ph.D. (Other) Subjects: Biochemistry; Bioinformatics; Biology; Biomedical Research