PhD, University of Cincinnati, 2023, Arts and Sciences: Chemistry

Poly(ADP-ribosyl)ation (PARylation) is a reversible post translational modification that regulates an array of cellular processes such as cell signaling, DNA damage response (DDR), and apoptosis. ADP-ribosyltransferases (ARTs) catalyze this process by transferring ADP-ribose (ADPr) moiety from &beta-NAD+ to target proteins. Poly(ADP-ribose) polymerase 1 (PARP1) is the predominant ADP-ribosyltransferase upon DNA damage. Importantly, PARP1 is frequently upregulated in many human cancers that have compromised DNA repair functions, showcasing its main role in tumor development and progression. Consistently, pharmacological inhibition of PARP1 with PARP inhibitors has shown promise in tumor-selective therapy, for both BRCA (BReast CAncer gene)-deficient and BRCA-proficient tumors. However, in contrast to the structure and mechanism of PARP1, how PARP1 activity and functions are regulated remains poorly understood. Here, I have presented three projects to study the PARP1 functions and their regulation: (1) Regulation of DNA bridging activity of PARP1 by HPF1 (2) Selective monitoring of PARP1/2 mono-ADP-ribosylation (MARylation) by &beta-NAD+ analog (3) Identification of new regulator of PARP1 by human proteome microarray. The first project is focused on the bridging of DNA double-strand breaks by PARP1 and its regulation by PARP1-binding protein, HPF1 (Histone PARylation Factor 1). First, I investigated the damaged- DNA binding to PARP1 by Fluorescence Polarization (FP) and compared the DNA-binding activity of PARP1 to PARP1's ability to bridge two DNA ends together. This allowed us to further under- stand PARP1's role in damaged-DNA sensing and bridging, especially in the Non-Homologous End Joining (NHEJ) pathway. To quantitatively analyze DNA bridging activity of PARP1, I developed a time-resolved-FRET (Fluorescence Resonance Energy Transfer) assay using Tb3+ and fluoresceine as the FRET pair. Using this TR-FRET assay, (open full item for complete abstract)

Committee: In-Kwon Kim Ph.D. (Committee Chair); Pearl Tsang Ph.D. (Committee Member); George Stan Ph.D. (Committee Member) Subjects: Biochemistry

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

Recently, novel and unrecognized endogenous metabolites have been found to impact the risk of CVD not related to established risk factors (residual risk), including metabolites produced by gut microbes. Discovery platforms including metabolomics and metagenomics have identified new biomarkers associated with residual risk, but these platforms' usefulness is limited by the available methods of data analysis. This thesis aims to develop models of gut microbial metabolism using metagenomic data and new methods to identify unseparated structural isomers in metabolomics data. The gut microbial metabolism of trimethylamine-N-oxide, a metabolite associated with residual CVD risk, is used as a model system to develop predictive models of metabolism based on metagenomic information. An integrated analysis of metabolomics, metagenomics, and several other data types to predict circulating trimethylamine-N-oxide levels shows that while gut microbes play an essential role in trimethylamine-N-oxide synthesis, community composition does not quantitatively predict metabolism well enough to predict clinical risk. Analytical methods are developed to detect and identify structural isomers in metabolomics data. Two structural isomers, the terminal metabolites of niacin metabolism, are detected in human serum and characterized. Multiple clinical studies show these niacin metabolites are associated with residual CVD risk, and animal models show N1-methyl-4-pyridone-3-carboxamide (4PY) enhances vascular inflammation and thrombosis potential. Thus, new microbial and endogenous targets for therapy have been proposed, and new analytical methods have been introduced that may enable further study of residual CVD risk.

Committee: Mehmet Koyuturk (Committee Chair); Stanley Hazeen (Advisor); Daniel Rotroff (Committee Member); Andrew Pieper (Committee Member); Catherine Stein (Committee Member) Subjects: Analytical Chemistry; Animals; Biochemistry; Bioinformatics; Biology; Biostatistics; Food Science; Genetics; Health; Medicine; Microbiology; Molecular Biology; Molecular Chemistry; Molecules; Nutrition; Organic Chemistry; Public Policy

Doctor of Education , University of Dayton, 2022, Educational Leadership

The following is a mixed-methods action research study entitled, “Increasing Retention and Graduation Rates of BIPOC and/or Male Students in ASL Interpreting at Sinclair Community College.” This action research broadly focuses on increasing student retention in the American Sign Language and Interpreting for the Deaf (IEP) program for students of color and/or males by increasing equity, cultural competency, a sense of belonging, and diversity of curriculum in the Sinclair College ASL IEP program. The study has a foundational framework in the Critical Theory of Love, Sociolinguistic Theory, Catholic Marianist values and by (Re)Framing the Deaf Heart Theory. This research addresses the fact that BIPOC and/or male interpreters are not well represented in the field or classroom. For this study, both qualitative and quantitative data was gathered through surveys, interviews, and focus groups. The participants were alumni, and current students in the SCC IEP program. The participants' responses identified four themes which guided the SCC IEP program towards creating a more equitable, and inclusive IEP. Listening to the alumni and students' voices throughout this research was powerful and demonstrated where the program is doing transformation work, as well as identified gaps. This action research can be easily replicated in one's own program, by following the action plan. Having hard conversations in the classroom, with the Deaf community and out in the field to address racism, and microaggressions against BIPOC and/or male ASL interpreters make way for transformational change to the field of ASL interpreting.

Committee: Matthew Witenstein, Ph.D. (Committee Chair) Subjects: Educational Leadership; Language

Doctor of Philosophy, The Ohio State University, 2019, Biomedical Sciences

Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults affecting almost 12,000 people each year in the US. This disease is collectively characterized by an accumulation of rapidly proliferating neoplastic cells of the myeloid lineage with differentiation defects. In spite of the vast amount of information known about AML and the identification of favorable prognosis factors, a large percentage of patients relapse and succumb to this disease. In addition, the inter- and intra-tumor heterogeneity of AML makes the identification of therapeutic targets for this disease particularly challenging. Future studies are warranted to identify multi-targeted agents that could influence AML as a composite disease. A target that shows promise in targeting the bulk AML leukemic cell population is nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is a protein involved in the generation of NAD+ in tumor cells, an important mediator of enzymatic reactions involved in various functions of leukemic disease progression. Leukemic blasts show a higher NAD+ turnover rate than normal cells, suggesting that NAD+ biosynthesis could be critically required in hematologic malignancies and therefore targeting the regeneration of NAD+ offers an attractive alternative strategy in AML. Inhibitors of NAMPT that have been described by others have shown potent anti-tumor activity and selectivity of several tumor models, including AML, while preserving the viability and functionality of normal tissues. While two agents targeting NAMPT have been tested in Phase I clinical trials, dose-limiting toxicities including thrombocytopenia and gastrointestinal toxicities led to their clinical discontinuation. Novel compounds with improved tolerability are needed. We sought to determine the mechanism of anti-tumor activity on AML leukemic cell population using a novel compound, KPT-9274, targeting NAMPT. We will also highlight several mechanisms used to antagonize AML disease progression v (open full item for complete abstract)

Committee: John Byrd (Advisor); Rosa Lapalombella (Advisor); Sameek Roychowdhury (Committee Chair); Vinay Puduvalli (Committee Member) Subjects: Biomedical Research; Oncology; Pharmacology

Doctor of Philosophy (PhD), University of Toledo, 2018, Medicinal Chemistry

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate-ribose (cADPR) are the two fraternal twins that act as Ca2+ mobilizing second messengers apart from inositol triphosphate (IP3). These Ca2+ mobilizing second messengers regulate various cellular functions like fertilization, insulin secretion, muscle contraction, etc. but the binding proteins for NAADP and cADPR are unknown. There is a high requisite to know about the binding proteins of NAADP and cADPR. To isolate and purify soluble mammalian NAADP binding proteins we propose to use ligand-affinity chromatography. Affinity chromatography is an isolation technique for proteins using `specific-ligand' approach, by immobilizing an analog of NAADP to a hydrophilic water insoluble resin using a hydrophilic tetraethyleneglycol spacer arm. This affinity resin absorbs the specific proteins on to the resin which can be then eluted by higher ionic strength solvent or using natural ligand NAADP. Affinity chromatography will enable us to obtain the binding proteins of NAADP to identify the proteins by sequencing. To isolate and purify the cADPR binding proteins we propose to synthesize the bifunctional photoaffinity probes consisting of a photoaffinity label and purification site (clickable site). These photoaffinity probes should contain most of structural properties of naturally occurring substrate plus an added photoreactive group (aromatic azide) which has the potential to form an irreversible covalent bond with an amino acid residue located within the active site of the binding protein and an acetylene moiety to conjugate with an azide linked fluorescent or affinity tags for further purification of the protein. This affinity tag will then permit us to rapidly and efficiently enrich for the derivatized target protein. As a bifunctional cADPR analog we synthesized 8-N3-2'-O-propargyl-cADPR by alkylation of 8-bromoadenosine by propargyl bromide, followed by substitution of bromide with (open full item for complete abstract)

Committee: James Slama (Committee Chair); Zahoor Shah (Committee Member); viranga Tillekeratne (Committee Member); David Giovannucci (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2009, Physiology and Biophysics

The ATP-gated P2X7 purinergic receptor (P2X7R) is predominantly expressed in cells of hematopoietic origin including macrophages and T lymphocytes. Roles for the P2X7R have been identified in the regulation of various pro-inflammatory and immune responses. An unusual feature of the P2X7R is its high threshold for activation by extracellular ATP (EC50 ~ 500 μM); this contrasts with much lower activation thresholds for the other six members of the P2X family (EC50 ~ 10 μM). Recent studies have demonstrated that extracellular NAD induces the ATP-independent activation of the P2X7R in murine T lymphocytes via ADP-ribosylation of arginine residues on the P2X7R ecto-domain. This modification is catalyzed by ART2.2, a GPI-anchored ADP¬ribosyltransferase (ART) that is constitutively expressed in murine T cells. Although the NAD-induced, ART2-dependent mechanism is clearly a major pathway for P2X7R activation in mouse T lymphocytes, it is unclear whether this mechanism is operative in macrophages, another class of leukocytes which natively express P2X7R at high levels. The studies described in this dissertation addressed this question in following two folds. The first series of studies (Chapter 3) demonstrated that bone-marrow derived macrophages (BMDM) from BALB/c mice selectively up-regulate thiol-dependent ART2.1 in response to multiple proinflammatory mediators including agonists for toll-like receptors (TLR) and type-1/2 interferons. LPS-induced ART2.1 expression involved regulation by multiple kinase pathways or possibly IFN-β autocrine signaling. A related group of studies (Chapter 5) showed expression of ART2.1 in a wide range of freshly isolated or tissue-cultured murine myeloid and lymphoid antigen-prensenting leukocytes. In contrast with T cells, stimulation of naive or inflammatory macrophages with NAD alone did not activate the P2X7R. Rather, NAD potentiated ATP-dependent P2X7R activation in inflammatory macrophages as indicated by a left-shift in the ATP dose- (open full item for complete abstract)

Committee: George R. Dubyak PhD (Advisor); Ulrich Hopfer PhD (Committee Chair); Stephen Jones PhD (Committee Member); Michael Simonson PhD (Committee Member); Margaret Chandler PhD (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy, Case Western Reserve University, 2009, Chemical Engineering

The goal of this research was to apply thick-film screen printing technology to produce a single-use, disposable, cost-effective electrochemical biosensor prototype in large scale. Iridium nanoparticles supported by carbon were selected to modify the electrode of the biosensor for its excellent catalytic effect towards the commonly detected electrochemical active species, i.e., hydrogen peroxide, nicotinamide adenine dinucleotide (reduced form, NADH), and nicotinamide adenine dinucleotide (oxidized form, NAD+). The development of this electrochemical biosensor prototype can establish a platform technology for various analytes of clinical importance. This study focused on the biosensing of the following analytes related to diabetes, i.e., 3-hydroxybutyrate (3HB), fructosyl valine, and the HbA1c, providing an analytical tool for diabetic patient management. The first part of this study discusses the importance of developing a point-of-care amperometric biosensor to detect 3-hydroxybutyrate (3HB) for diabetic patient management. Current electrochemical detection methods for 3HB require at least two stepwise reactions or a mediator. The detection method in this study only requires a single reaction step without any mediator, which can potentially also be more accurate, sensitive, cost-effective and stable over the long term. In this detection method, the enzyme D-3-hydroxybutyrate dehydrogenase (3HBDH, EC 1.1.1.30) was immobilized on the iridium–modified sensor prototypes which detect the NADH produced by the reaction of 3HB with NAD+ in the presence of 3HBDH. This microelectrode quantified the NADH electrochemically, which produced an electrical current that would then be used to quantify the concentration of 3HB. The interferences from uric acid, NAD+, and serum were measured. It was concluded that the level of 3HB could still be quantified well in the presence of these interfering species. Spectrometric measurements of NADH and 3HB were performed in both PBS and bov (open full item for complete abstract)

Committee: Chung-Chiun Liu (Committee Chair); Vernon Anderson (Committee Member); Heidi Martin (Committee Member); Harihara Baskaran (Committee Member) Subjects: Biochemistry; Biomedical Research; Chemical Engineering; Engineering

Doctor of Philosophy, Case Western Reserve University, 2007, Pharmacology

Beta-Lapachone (Beta-Lap; a.k.a. ARQ 501) is a novel antitumor quinone currently in Phase II clinical trials for the treatment of pancreatic and head/neck cancers. Beta-Lap has been shown to be an effective cancer chemotherapeutic agent both in vitro and in vivo against a number of human cancers that express NAD(P)H:quinone oxidoreductase-1 (NQO1). Bioactivation of beta-lap by NQO1 caused a futile oxidoreduction, leading to reactive oxygen species generation (ROS). Unique to this compound was its ability to kill cancer cells regardless of abnormalities in commonly altered apoptosis-related proteins, such as p53, Bcl-2, and Bax/Bak. This thesis describes in more mechanistic detail the cell death pathway activated after beta-lap treatment, with particular focus on its involvement with, and resistance by, DNA repair. We demonstrated that the NQO1-dependent reduction of beta-lap caused ROS generation, DNA breaks, and triggered calcium (Ca 2+)-dependent gamma-H2AX formation and PARP-1 hyperactivation. PARP-1 hyperactivation was an integral part of cell death triggered by this compound, causing NAD +and ATP losses that suppressed DNA repair and caused cell death. PARP-1 inhibition or intracellular Ca 2+chelation protected cells from beta-lap-induced cell death. Similarly, hydrogen peroxide (H 2O 2), but not N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), caused Ca 2+-mediated PARP-1 hyperactivation and death. Thus, Ca 2+appears to be an important co-factor in PARP-1 hyperactivation after ROS-induced DNA damage. To explore DNA repair as a resistance factor(s) that might impede cell death, we explored the contribution of DNA double-strand break (DSB) repair following beta-lap exposure. Beta-Lap treatment resulted in the NQO1-dependent activation of the MRE11-Rad50-Nbs-1 (MRN) complex, as well as ATM Serine 1981, DNA-PKcs Threonine 2609, and Chk1 Serine 345 phosphorylation, indicative of ATR activation. These data suggested the simultaneous activation of both homologous recombi (open full item for complete abstract)

Committee: David Boothman (Advisor) Subjects:

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2010, Biological Sciences

Midgut mitochondria from fifth larval instar Manduca sexta exhibited a transhydrogenase that catalyzes the following reversible reaction: NADPH + NAD+ ↔ NADP+ + NADH. The NADPH-forming transhydrogenation occurred as a non energy- and energy-linked activity. Biochemical characterization for reversibility, energy-linkages, pH optima, stability to dialysis/heat denaturation, transmembrane proton translocation and localization were accomplished. During the ten day developmental period preceding the larval-pupal molt (fifth larval instar), significant peaks in the mitochondrial transhydrogenase activities of midgut and fatbody tissues were noted and these peaks were coincident with the onset of wandering behavior and with the 50-fold increase in ecdysone 20-monooxygenase (E20-M) activity previously reported for M. sexta midgut. Since E20-M preferentially uses NADPH in catalyzing ecdysone conversion to the physiologically active molting hormone, 20-hydroxyecdysone, the physiological and developmental significance of the mitochondrial, NADPH-forming energy-linked transhydrogenations are apparent. Using isolated mitochondrial membranes, the M. sexta transhydrogenase was subjected to kinetic analysis pertaining to the NADPH → NAD+ as well as non energy-linked and the ATP-dependent, energy linked NADH → NADP+ reactions. Kinetic analysis demonstrated that the reversible insect transhydrogenase is subject to site-specific inhibition, contains two substrate binding sites (viz., NADP(H) and NAD(H)), and is susceptible to end-product inhibition. The effect of various allelochemicals on the M. sexta transhydrogenations was also evaluated. Taken together, the findings of this dissertation support a distinct physiological role of mitochondrial transhydrogenase in M. sexta post-embryonic development.

Committee: Carmen Fioravanti PhD (Advisor); Raymond Larsen PhD (Committee Member); Martin Mitchell PhD (Committee Member); Mark Munson PhD (Committee Member); Jill Zeilstra-Ryalls PhD (Committee Chair) Subjects: Biochemistry; Biology; Cellular Biology; Entomology

Master of Science, University of Akron, 2006, Chemical Engineering

In cystic fibrosis airway infection, Pseudomonas aeruginosa forms microaerobic biofilm and undergoes significant physiological changes. It is important to understand the bacterium's metabolism at microaerobic conditions. Continuous cultures of P. aeruginosa (ATCC 9027) maintained at different dissolved oxygen concentrations (DO) and three different dilution rates (D) were studied for the effects of DO and D on various culture properties, especially on aerobic respiration and denitrification. The DO was varied from 0 mg/L (completely anoxic condition) to 2.2 mg/L, and measured with optical sensors that could accurately determine very low DO based on oxygen-quenched luminescence. The studied dilution rates were 0.026 h-1, 0.06 h-1 and 0.13 h-1. The strain was found to perform aerobic denitrification; while the specific nitrate and nitrite reduction rates decreased with increasing DO, denitrification persisted even at relatively high DO levels (1-2.2 mg/L) at different D. In the presence of nitrate, the Monod constant for DO (i.e., the critical DO at which the specific oxygen uptake rate (OUR) is half of the maximum rate) was practically zero (< 0.001 mg/L) for this P. aeruginosa strain. Aerobic denitrification appeared to function as an electron-accepting mechanism supplementary or competitive to aerobic respiration. The shift of culture's respiratory mechanism was also clearly detected with a fluorometer targeting at intracellular NAD(P)H, i.e., the reduced coenzymes nicotinamide adenine dinucleotides (phosphate). Comparatively, the NAD(P)H fluorescence was highest at the anoxic, denitrifying condition (NFUDN), lowest at fully aerobic conditions (NFUOX), and intermediate fluorescence (NFU) at conditions where both denitrification and aerobic respiration occurred. Representing a quantitative measure of the culture's “fractional approach” to the fully denitrifying state, the normalized fractions (NFU - NFUOX)/(NFUDN - NFUOX) were correlated with the calculated fraction (open full item for complete abstract)

Committee: Lu-Kwang Ju (Advisor) Subjects:

Master of Science (MS), Bowling Green State University, 2013, Biological Sciences

The adult intestinal cestode, Hymenolepis diminuta, is energetically anaerobic and displays mitochondria that physiologically function anaerobically. These organelles require an inner membrane-associated NADPH→NAD+ transhydrogenase as well as other membrane-associated, NADH-utilizing activities that reflect anaerobic electron transport. Plant flavonoids are known to affect both mammalian and invertebrate systems and, more specifically, the ecdysone 20-monooxygenase, mitochondrial transhydrogenase and electron transport-linked systems of the tobacco hornworm, Manduca sexta. However, with the exception of the isofavonoid, rotenone, no data existed as to the potential effects of plant flavonoids on parasitic helminth anaerobic, mitochondrial enzymes. Thus, the effects of chrysin, quercetin, morin, juglone, and plumbagin on the H .diminuta transhydrogenase as well as NADH dehydrogenase, NADH-cytochrome c reductase, and an NADH→NAD+ transhydrogenation were evaluated. Although lacking an NADPH→NAD+ system, comparisons with the other corresponding mitochondrial activities of another anaerobic, intestinal, adult helminth, viz., the nematode, Ascaris suum, were made. Activities were assessed spectrophotometrically employing isolated cestode or nematode mitochondrial membranes as the source of enzyme activities. While not all flavonoid treatments proved to significantly affect the activities tested, a suggested inhibition by chrysin was noted and stimulations by juglone and plumbagin were noted for the helminth transhydrogenase. The H. diminuta NADH dehydrogenase was inhibited by plumbagin, but stimulation was apparent at higher concentrations whereas morin stimulated (lower concentrations) and inhibited (higher concentration) and plumbagin stimulated the A. suum enzyme. Both the cestode and nematode NADH-cytochrome c reductase activities appeared to respond positively to the presence of juglone. A tendency towards stimulation of the NADH→NAD+ transhydrogenation of H .diminut (open full item for complete abstract)

Committee: Carmen Fioravanti Dr. (Advisor); Lee Meserve Dr. (Committee Member); Vipaporn Phuntumart Dr. (Committee Member) Subjects: Biochemistry; Biology; Parasitology