PhD, University of Cincinnati, 2014, Medicine: Cancer and Cell Biology

Transcription factors and chromatin structure are master regulators of homeostasis during hematopoiesis. Regulatory genes for each stage of hematopoiesis are activated or silenced in a precise, finely tuned manner. Many leukemia fusion proteins are produced by chromosomal translocations that interrupt important transcription factors and disrupt these regulatory processes. Leukemia fusion proteins E2A-Pbx1 and AML1-ETO involve normal function transcription factor E2A, resulting in two distinct types of leukemia: E2A-Pbx1 t(1;19) acute lymphoblastic leukemia (ALL) and AML1-ETO t(8;21) acute myeloid leukemia (AML). E2A, a member of the E-protein family of transcription factors, is a key regulator in hematopoiesis that recruits coactivators or corepressors in a mutually exclusive fashion to regulate its direct target genes. In t(1;19) ALL, the E2A portion of E2A-Pbx1 mediates a robust transcriptional activation; however, the transcriptional activity of wild-type E2A is silenced by high levels of corepressors, such as the AML1-ETO fusion protein in t(8;21) AML and ETO-2 in hematopoietic cells. It is unclear how this context-dependent regulation of E2A-dependent transcription allows E2A protein to regulate transcription in response to variable intracellular levels of corepressor. In this study, we discovered that unlike HEB, another E-protein, the activation domain 1 (AD1) of E2A is inhibited for corepressor interaction by E2A-specific amino acid changes in the p300/CBP and ETO target motif, which interacts with only one of these targets at a time. At physiological levels of corepressor, E2A-Pbx1 escapes endogenous ETO-2 binding to confer its oncogenic ability via AD1 and AD2 cooperative activity to facilitate coactivator recruitment. In the presence of aberrantly high levels of AML1-ETO, E2A interacts with the corepressor to silence expression of E-protein target genes. This process requires the downstream ETO-interacting sequence (DES) domain of E2A to compensate for th (open full item for complete abstract)

Committee: Sohaib Khan Ph.D. (Committee Chair); Song-Tao Liu Ph.D. (Committee Member); Robert Brackenbury Ph.D. (Committee Member); Peter Stambrook Ph.D. (Committee Member); Jinsong Zhang Ph.D. (Committee Member) Subjects: Oncology

Doctor of Philosophy, Case Western Reserve University, 2013, Chemistry

Protein microarrays are becoming increasingly popular as platforms for applications ranging from bioanalyte detection (biosensors and diagnostics) to enzyme activity profiling and even the biosynthesis of natural products and their analogs in vitro. Selective and efficient protein immobilization methods are essential for such applications, and herein we describe the first example of such an immobilization that exploits the self-catalysis and binding properties of the commonly-used glutathione transferase (GST) purification tag. A library of 25 analogs of the known GST substrate 1-chloro-2,4-dinitrobenzene (CDNB) was synthesized, and 20 of these compounds bearing bioorthogonal linker substituents were screened for glutathione (GSH) conjugation activity with the GST from the helminth worm Schistosoma japonicum. The Michaelis-Menten kinetics for several enzymatic reactions were studied, and it was observed that improved aqueous solubility and strongly electron-withdrawing ring substituents accelerate the conjugation reaction, providing hints for the design of a second generation of GST substrates. The regiochemistry of the SNAr reaction was also studied; it was found that conjugation of GSH is favored at the 4-position of the aryl ring, a result that was rationalized by noting the increased electrophilicity of that position due to resonance effects. An alkyne-bearing CDNB analog was chosen to serve as a linker for the immobilization of SjGST on an azide-functionalized glass microscope slide. A “click” reaction was performed to modify the surface with the CDNB analog, and subsequent treatment with SjGST and GSH led to an array of immobilized protein that was characterized via contact-angle microscopy, fluorescence immunolabeling, and atomic force microscopy. Arrays of SjGST fabricated in this way showed an 18-fold higher fluorescence detection signal compared to non-oriented proteins, and a 6-fold increase in signal versus conventionally-immobilized GSH. As a firs (open full item for complete abstract)

Committee: Rajesh Viswanathan (Advisor); Michael Zagorski (Committee Chair); Mary Barkley (Committee Member); Gregory Tochtrop (Committee Member); John Mieyal (Committee Member) Subjects: Biochemistry; Chemistry; Organic Chemistry

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2022, Biomedical Sciences (Medical Microbiology and Immunology)

Burkholderia pseudomallei is a Gram-negative bacterium that causes the severe febrile disease melioidosis. The wide variety of signs and symptoms associated with melioidosis hinders timely diagnosis of this illness, which often allows the infection to progress to fatal septicemia before this bacterium is accurately identified. Successful treatment of melioidosis patients is further complicated by the extensive repertoire of antibiotic resistance mechanisms employed by B. pseudomallei. Due to the low dose required to cause fatal infection and the difficult nature of properly diagnosing and treating melioidosis, B. pseudomallei has great potential for deliberate misuse in acts of bioterrorism and is therefore classified a Tier 1 select agent by the Centers for Disease Control and Prevention. Thus, there is great interest in finding therapeutic targets to prevent and/or treat melioidosis, as no FDA-approved vaccine currently exists. Previous studies from our lab and others demonstrated that B. pseudomallei must evade the complement system in order to successfully persist within the host. More recently, unpublished work from our lab demonstrated that B. pseudomallei binds Factor H, the host's complement regulatory protein, to improve B. pseudomallei survival within the host. Importantly, the proteins involved in this immune evasion strategy are considered to function as effective therapeutic targets, as loss of binding Factor H may incur a fitness cost to the pathogen. Preventing the interaction between B. pseudomallei and Factor H may enhance serum susceptibility and/or bacterial clearance by host phagocytic cells. A novel and promising therapeutic approach for serum-resistant pathogens is the recombinant fusion protein Factor H-Fc. These chimeric constructs are composed of either Factor H domains 6-7 or 18-20 linked to constant regions (Fc) of immunoglobulin G isotypes 1 or 3 (Fc1 and Fc3, respectively). Potential mechanisms for how these might act as a therapeutic (open full item for complete abstract)

Committee: Mark Wooten (Advisor); Viviana Ferreira (Committee Member); Robert Blumenthal (Committee Chair) Subjects: Biomedical Research; Health Sciences; Immunology; Microbiology

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

Ewing sarcoma is an aggressive bone and soft tissue-associated cancer affecting pediatric, adolescent, and young adult patients. Despite general improvement in pediatric cancer outcomes due to novel therapeutic options, Ewing sarcoma treatment, which consists of high-dose chemotherapy, radiation, and/or local surgical control, has remained largely unchanged for several decades and patients with metastatic disease continue to see poor outcomes. Although pediatric cancers often have far fewer mutational events than adult cancers, Ewing sarcoma is particularly interesting as the disease is often characterized by a sole chromosomal translocation event: These chromosomal translocations fuse one of the FET protein family members, a group of putative RNA-binding proteins, to a member of the ETS transcription factor family. As these FET/ETS fusion proteins have been determined to function as oncogenic transcription factors responsible for driving Ewing sarcomagenesis, it is critical that the biological mechanisms these fusions utilize to facilitate this process are elucidated. Despite discovery of several FET/ETS translocations, the majority of studies in the field focus on EWS/FLI, as it is the most common fusion observed in patients. Although these studies have provided a breadth of knowledge surrounding oncogenic function of the protein, there is a great deal of uncertainty how alternative FET/ETS fusions should be diagnosed and treated in the clinic. Herein, we characterize a novel FET/ETS fusion and perform the first comparative analysis on EWS/FLI and alternative, rarer FET/ETS fusion proteins. Our results reveal general similarities in DNA-binding and transcriptional regulation properties between the broad FET/ETS fusion group and provide the first tangible body of evidence to support that these fusions should indeed be classified as bona fide Ewing sarcoma tumors. Furthermore, we sought to characterize contributions of the FLI protein to overall EWS/FLI funct (open full item for complete abstract)

Committee: Stephen Lessnick MD/PhD (Advisor); Timothy Cripe MD/PhD (Committee Member); Lawrence Kirschner MD/PhD (Committee Member); Mark Parthun PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology; Molecular Biology; Oncology

Master of Science (MS), Wright State University, 2015, Biochemistry and Molecular Biology

The best characterized eukaryote replication model is of the budding yeast Saccharomyces cerevisiae. Replication origins of S.cerevisiae are 100 to 200 bp in size and contain an essential 11-bp autonomous replicating sequence (ARS) consensus sequence (ACS). The origin recognition complex (ORC) binds to the ACS in order to recruit additional replication factors (Cdt1, Cdc6, MCM, Cdc45) and together they form the pre-replication complex (pre-RC). Unlike budding yeast, the mammalian cells contain dispersed replication origins in which multiple elements distributed over large distances act as replication start sites. Mammalian DNA replication origins, such as the c-myc origin, contain a DNA unwinding element (DUE), which is an AT-rich region that contains10-of-11 matches to ARS consensus. Our lab primarily studies DNA replication of the human c-myc locus. We have successfully integrated the wild-type 2.4 kb c-myc replication origin and its various inactive mutants in a known ectopic chromosomal site in HeLa/406 cells in order to study the multiple functional elements of this origin. My thesis focused on investigating the minimal sequence required in mammalian replication origin for replication activity. To address this question, I assessed the effects of replication protein tethering on chromatin accessibility of inactivated c-myc origins which are 930 bp or less and contain an intact DUE. In part I of my thesis, I describe how I made two new HeLa/406 cell lines containing two different deletion mutants of the cmyc replicator core (which are 607 bp and 228 bp long) at the known ectopic chromosomal site. In part II of my thesis, I describe how I created plasmids expressing GAL4 DNA-binding domain (GAL4DBD)-BRG1 fusion proteins. BRG1 is a catalytic core subunit of SWI/SNF mammalian chromatin remodeling complex. Wild-type BRG1 and catalytic inactive BRG1K798R mutant were used to make GAL4DBD fusion proteins. In part III of my thesis, I evaluated the effects of (open full item for complete abstract)

Committee: Michael Leffak Ph.D (Advisor); Weiwen Long Ph.D (Committee Member); John Paietta Ph.D (Committee Member) Subjects: Molecular Biology

Master of Science, The Ohio State University, 2015, Anatomy

Cancer immunotherapy is a treatment approach aimed towards harnessing and enhancing the immune system's intrinsic ability to eliminate malignant cells. One feature which lends to the attractiveness of cancer immunotherapy is the extensive capacity of the immune system to recognize specific molecular targets expressed on pathogenic or malignant cells. Natural-killer group 2, member D (NKG2D) ligands are “stress-induced” proteins which are not typically expressed in most normal, healthy tissues, but are upregulated in response to conditions of cellular stress, such as those arising from malignant transformation. These ligands have been shown to be widely expressed on a variety of cancer types, and thus, represent auspicious targets for the development of novel immunotherapies. To capitalize upon the tumor-specific expression of NKG2D ligands, a novel NKG2D ligand-targeting fusion protein was created, in which the extracellular domain of NKG2D, the receptor for NKG2D ligands, is fused to the Fc domain of human immunoglobulin G1 (IgG1). For this NKG2D-Fc fusion protein (NKG2D-Fc), the NKG2D region serves as the binding domain, intended to target NKG2D ligands expressed on tumor cells, while the Fc region serves as the effector domain, providing a site for interaction with various immune components which can mediate destruction of tumors. This thesis aims to describe this novel immunotherapeutic fusion protein and to examine its binding ability. NKG2D-Fc was hypothesized to be able to recognize and bind to NKG2D ligands expressed on human cancer cells. Results of flow cytometry analysis and ELISA demonstrated that NKG2D-Fc was able to bind to NKG2D ligands expressed on a human breast cancer cell line and a human melanoma cell line. These results are promising as they demonstrate that this novel construct can effectively bind to NKG2D ligands on cancer cells and warrant further investigation of this fusion protein as a potential cancer immunotherapeutic agent.

Committee: Amanda Agnew (Advisor); Eileen Kalmar (Committee Member) Subjects: Anatomy and Physiology; Medicine; Oncology

MS, University of Cincinnati, 2014, Medicine: Immunology

Autoimmune disorders are the second leading type of chronic illness in the US, affecting about 50 million individuals. In 2001, the National Institute of Allergy and Infectious Diseases (NIAID) estimated that the annual autoimmune healthcare cost is greater than $100 billion, although this value may be significantly underestimated. Autoimmune diseases such as hypersensitivity reactions are a result of impairment of this FcR regulatory system. Here, we focus on the interaction between Immunoglobulin A (IgA), the most predominant antibody in mucosal sites, and its principal myeloid receptor, FcaRI (CD89). Cross-linking of multiple FcaRI molecules at the cell surface by immune complexed IgA, initiates pro-inflammatory immune responses such as phagocytosis, antigen presentation and antibody-dependent cellular cytotoxicity. These findings led to perceiving FcaRI as a solely activating receptor. In 2005, Pasquier et al. identified that FcaRI, when interacting with monomeric serum IgA, can also drive powerful anti-inflammatory responses through the ITAMs in the FcR γ-chain and thus behave as an inhibitory receptor that controls inflammation. This revealed the dual nature of the ITAM motif, which otherwise is typically considered to be involved in immune cell activation. This inhibitory signaling pathway mediated by FcaRI in association with FcRγ ITAMs was termed the Inhibitory ITAM or ITAMi signaling pathway. Several research groups have shown that some but not all anti-FcaRI monoclonal antibody Fab fragments are capable of triggering the ITAMi pathway via FcaRI. The anti-FcaRI monoclonal antibodies most widely used to study the ITAMi pathway include MIP8a, A3, A59 etc. and these antibodies recognize different extracellular domains in the FcaRI ectodomain. Hence FcaRI can be considered as a 3 state system: a resting state in which it does not mediate signaling, an activating state in which it triggers pro-inflammatory responses via recruitment of a Syk kinase, and an inhi (open full item for complete abstract)

Committee: Andrew Herr Ph.D. (Committee Chair); Jonathan Katz Ph.D. (Committee Member); William Miller Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy, The Ohio State University, 2013, Integrated Biomedical Science Graduate Program

Respiratory syncytial virus (RSV), a paramyxovirus, is the most significant respiratory pathogen in infants and causes 90,000 emergency hospitalizations in the United States and 160,000 deaths worldwide every year. It is also a leading respiratory pathogen in the elderly. RSV infection provides weak immunity, and the virus infects individuals repeatedly throughout life. The only effective antiviral compound commercially available is a monoclonal antibody, palivizumab (Synagis®), which is given prophylactically to at-risk infants. Despite intense efforts, no other therapeutic and no vaccines have been approved for use, although several are in development. Most drugs developed against RSV target the fusion (F) glycoprotein. The F protein is responsible for fusing the host cell and viral membranes together to initiate infection. The RSV F protein is unique among other paramyxoviruses in that it does not require its partner attachment (G) glycoprotein to function as do most other paramyxovirus F proteins. It is the subject of extensive research in hopes of determining its mechanism of action and developing anti-RSV compounds. The mature, cleaved RSV F protein contains N-glycans at asparagine residues 27, 70, and 500. It has previously been reported that none of these N-glycans are required for protein processing or cell surface expression, but that the N500 glycan is required for cell-to-cell fusion in an assay serving as a proxy for virion-cell fusion. In this study, we replicated these results in the full-length protein. We also built N27Q, N70Q, N500Q, N27/70Q, and N/27/70/500 mutations into a soluble version of the RSV F protein to determine the role, if any, that these N-glycans have in triggering. Following purification, each of the N-glycan mutants migrated near the top of a sucrose velocity sedimentation gradient, similar to the wild type (WT) sF protein, indicating that these proteins were produced and secreted from the cell in the prefusion form. We determined (open full item for complete abstract)

Committee: Mark Peeples Ph.D. (Advisor) Subjects: Biomedical Research; Virology

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2013, College of Medicine

Restriction-modification (R-M) systems play key roles in controlling gene flow among bacteria and archaea, and their own genetic mobility depends critically on their regulation, but the regulation of these systems is poorly understood. The PvuII R-M system is a Type IIP R-M system in that the protective DNA methyltransferase (MTase) is a separate and independently-active protein from the potentially lethal restriction endonuclease (REase). PvuII is one of the best studied of the R-M systems that use a positive feedback regulatory loop, involving a transcriptional regulator called C protein, to delay expression of the REase relative to that of the MTase. This allows protective methylation of a new host cell's DNA before the REase is produced. In searching for R-M systems related to PvuII, in order to study evolution and variation of its regulatory system, a putative system was found in the genome sequence of the bacterium Niabella soli strain DSM 19437, in which the regulatory C protein and the REase are translationally fused. The hypothesis is that N. soli truly produces a fused C-R protein, and that it is active as both a REase and as an autogenous regulator. The genes for the N. soli R-M system were synthesized, produced and purified with affinity tags, and the production of full-length C-REase fusion protein was confirmed. The dual activity of the fusion protein was determined by in vitro restriction of known DNAs, and in vivo transcriptional activation of a lacZ fusion to the promoter on which the C protein acts.

Committee: Robert Blumenthal Ph.D. (Committee Chair); Jason Huntley Ph.D. (Committee Member); Steve Patrick Ph.D. (Committee Member) Subjects: Biomedical Research

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

Aberrant DNA replication, including over-replication or under-replication may lead to life-threatening mutation or even cause human diseases. This thesis focused on three issues related to abnormal DNA replication in human chromosomes including: I) to define the function of DNA unwinding element (DUE) and DNA unwinding element-binding protein (DUE-B) to maintain an active c-myc replicator; II) to determine the role of trans-acting factors in defining a replication origin on human chromosomes; III) to investigate the mechanism by which hairpins affect DNA replication and instability of (CTG)n•(CAG)n trinucleotide repeat tracts in human cells. Our laboratory previously demonstrated that both DUE and DUE-B, are essential in c-myc DNA replication initiation. In part I, I have shown that the increased binding of DUE-B and Cdc45 correlated with the decrease of the DUE helical stability and increased origin activity for the chimeric c-myc/SCA10 replicators. However, tethered binding of DUE-B on a mutant c-myc replicator with DUE deletion could not confer the DNA replication activity. In part II, I explored the induction of DNA replication origin via trans-acting factors. My data suggested that tethered binding of transcription factor, E2F1, could induce replication activity likely by changing the chromatin structure. Tethered binding of pre-RC component, Orc2 and Cdt1 also can induce replication origin activity while Mcm7 and Cdc45 could not. Unlike episomal systems, our system revealed that the induction of replication origin activity on human chromosome also required the essential cis-acting elements including the DUE. Although it is widely accepted that unstable triplet nucleotide repeat (TNR) caused instability, it still remains elusive how and when the hairpins form during DNA replication. In part III, engineered zinc finger nucleases (ZFNs) and small pool PCR (spPCR) were applied to probe the hairpin formation in vivo in human cells. In our system, it was demonstra (open full item for complete abstract)

Committee: Michael Leffak PhD (Advisor); Steven Berberich PhD (Committee Member); Paula Bubulya PhD (Committee Member); John Paietta PhD (Committee Member); Courtney Sulentic PhD (Committee Member); Michael Leffak PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2011, Comparative and Veterinary Medicine

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, Pneumovirinae subfamily is the most significant respiratory pathogen in infants and second only to influenza virus in the elderly. Despite extensive efforts, no vaccines or small molecule antiviral drugs are available. The RSV fusion (F) glycoprotein has been a major target for vaccine and antiviral drug development because of its importance in the viral replication cycle, its conserved sequence and structure, its exposed position in the virion, and its strong immunogenicity. Like other paramyxoviruses, the RSV F protein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. However, the Pneumovirinae F protein is unique in that it, alone, is sufficient to mediate membrane fusion and virus infection. It is, therefore, the simplest F protein to study. It likely has the ability to attach to target cells from which position it is triggered. Neither the trigger site on the F protein nor the triggering molecule/event has been identified. To begin to study the triggering mechanism of the RSV F protein biochemically, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. This sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopy, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni2+ column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming “hatpin” shaped molecules that aggregated as rosettes, characteristic of the po (open full item for complete abstract)

Committee: Mark Peeples PhD (Advisor); Michael Oglesbee PhD (Committee Member); Stefan Niewiesk PhD (Committee Member); Jianrong Li PhD (Committee Member) Subjects: Virology