PHD, Kent State University, 2024, College of Arts and Sciences / School of Biomedical Sciences

Wound healing is a very complex process requiring a well-orchestrated integration of multiple cellular and molecular events involving many players for antimicrobial activity and the promotion of new vascular formation. Acute wounds are healed by following the normal process of repair including inflammation, proliferation, and remodeling phases. If acute wounds fail to progress through the normal healing phases, it can develop into delayed healing or non-healing chronic wounds, particularly associated with the presence of bacterial biofilm (a community of bacteria encased in a protective matrix) and impaired angiogenesis. Conventional antibiotics frequently develop resistance and have limited efficacy against biofilm-associated wound infections. Approaches for promoting pro-angiogenic activity for wound healing have been relied on the use of bioactive molecules or growth factors, which have limitations in developing cost-effective treatment options. Additionally, thus far, each of the above aspects for antimicrobial and proangiogenic activities have been separately investigated to a great extent and an integrated approach to simultaneously addressing these three issues in a single drug delivery platform has yet to emerge. Wound scaffolds are biomaterial platforms designed to support tissue regeneration and enhance wound healing. In particular, nanoparticle-based scaffolds hold promise for treating chronic wounds due to their characteristics to exhibit higher reactivity due to high surface to volume ratio for improved cellular interactions, and easiness in surface functionalization, and controlled release of bioactive molecules. Additionally, their small size enables deep penetration into the wound bed. In this study, we propose to harness the unique characteristic of copper ion that can exhibit antibacterial and pro-angiogenic properties towards developing a cost-effective 33 scaffold for treating chronic wounds. Copper has emerged as an essenti (open full item for complete abstract)

Doctor of Philosophy, Miami University, 2024, Chemistry and Biochemistry

Pancreatic cancer remains a devastating disease with exceptionally poor prognoses due to inability to detect the disease when it is still surgically resectable and due to largely ineffective treatment modalities. Here, three engineered adenovirus vectors were developed to specifically target and suppress oncogenic activity of the HMGA1 architectural transcription factor that plays a crucial role in tumorigenesis, chemotherapy resistance and transformation of pancreatic cancer cells into pancreatic cancer stem cells. HMGA1 plays a similarly crucial role in many human cancers, including breast cancer. HMGA1 is an especially insidious oncoprotein because it is intrinsically disordered and therefore cannot be targeted by conventional small molecule drug therapy. Here, alternative adenoviral-mediated therapy approaches were designed and developed to suppress HMGA1 oncogenic activity in cancer cells and tested in MIA PaCa-2, PANC-1 and BxPC-3 human pancreatic cancer cell lines and in the ZR-75 breast cancer cell line. The engineered viruses characterized included: 1) a virus engineered to sequester overexpressed HMGA1 in cancer cells, 2) a virus engineered to express an artificial HMGA1 cis-antisense transcript and 3) a virus engineered to express an HMGA1-targeted shRNA transcript, the latter two viruses designed to suppress translation of HMGA1 mRNA into HMGA1 protein. Cancer cell characteristics after viral infection were evaluated using viability, toxicity, proliferation and wound healing assays and effects on HMGA1 mRNA transcript levels and HMGA1 protein levels were quantitatively evaluated. It was found that the virus designed to sequester HMGA1 proved more effective in suppressing HMGA1 oncogenic activity than the viruses designed to suppress HMGA1 translation.

Bachelor of Science, Marietta College, 2024, Chemistry

Parabens, a common antimicrobial agent used in a wide variety of industrial, cosmetic, and pharmaceutical products, have been used for many years due to their odorless and colorless properties. Structural modification of this traditional preservative may serve as a therapy for melanoma which has incidence and mortality rates that are predicted to increase by 78% and 73% respectively in the next two decades. In this study human melanoma cells and normal human epithelial cells were treated with 0.05-10mM paraben solution, consistent with LC50 values. Parabens were dissolved in ethanol or methanol solvent and added to complete DMEM for cell treatment. Methylparaben (methyl 4-hydroxybenzoate) and a paraben derivative, heptylparaben, were used. A colorimetric Caspase-3 microplate assay kit was used to assess the ability of these compounds to induce apoptosis. Previous research has shown parabens have induced apoptosis in human melanoma cells, while this study will assess parabens' ability to induce apoptosis in normal human epithelial HaCat cells versus human melanoma M624 cells and provide support for the continued study of paraben as a possible topical treatment for melanoma.

Master of Science in Engineering, Youngstown State University, 2024, Department of Civil/Environmental and Chemical Engineering

Wearable biosensors have become a valuable tool for their promising applications in personalized medicine. Cortisol is a biomarker for various diseases and plays a key role in metabolism, blood pressure regulation, and glucose levels. In this study, we fabricated an interdigitated laser-induced graphene (LIG) biosensor for the non-faradaic impedimetric detection of cortisol in sweat. A direct laser writing technique was used to produce the LIG. Gold nanoparticles (AuNPs) were electrochemically deposited onto the surface to enhance impedance response. A Self-Assembled Monolayer (SAM) was formed with on the AuNPs via 3-Mercaptopropionic acid (MPA) thiol chemistry. The carboxylic acid (-COOH) groups of the MPA were activated using EDC/NHS chemistry. Following activation, anti-cortisol antibodies were immobilized on the surface. Lastly, the LIG was incubated in the blocking agent bovine serum albumin (BSA) to avoid unwanted detection. Surface characterization of the LIG was performed at each step of modification by Electrochemical impedance spectroscopy (EIS) in a phosphate buffered saline (PBS) solution containing a 5 mM Fe(CN)3-/4- (1:1) redox couple. Further characterization of the modified LIG electrode was achieved through Fourier transform infrared (FT-IR), surfaced-enhanced Raman spectroscopy (SERS), and X-ray diffraction (XRD). The detection experiment using EIS was conducted in increasing concentrations of cortisol (0.1 pM-100 nM) in PBS. The ZMod decreased logarithmically (R2=0.97) with a 0.0085 nM limit of detection. Reproducibility was examined by percent change of ZMod at 100 nM and a 5.93%RSD (n=5) was observed. Additional analysis of sensor specificity and interference studies showed no substantial effect on detection. This research establishes the feasibility of using the gold nanoparticle decorated LIG electrode for flexible, wearable cortisol sensing devices, which would pave the way towards an end-user easy-to-handle biosensors as point-of-care diagno (open full item for complete abstract)

Doctor of Philosophy (PhD), Ohio University, 2024, Chemistry and Biochemistry (Arts and Sciences)

The human growth hormone (hGH) is a 191 amino acid polypeptide hormone synthesized and secreted by anterior pituitary somatotrophs, that plays a pivotal role in regulating postnatal growth, development, bone density, and metabolism. Upon secretion into bloodstream, hGH binds to the extracellular domain of the human growth hormone receptor (hGHR), a transmembrane protein belonging to the cytokine receptor superfamily. Binding of hGH to the hGHR induces a conformational change in the receptor, leading to the activation of hGHR-associated Janus kinase 2 (JAK2) in the cytoplasm and subsequent phosphorylation of signal transducer and activator of transcription 5 (STAT5). This phosphorylation event in turn initiates downstream signaling pathways that regulate the expression of hGH-associated genes, such as IGF1, FOS, SOCS2, involved in stimulating growth and regulating metabolism. Unfortunately, dysregulated hGH signaling has been linked to various disease phenotypes, such as acromegaly, gigantism, diabetes, cardiovascular diseases and cancer, highlighting the therapeutic relevance of modulating hGH-hGHR interactions. To date, the only growth hormone receptor antagonist approved by the FDA for clinical use is pegvisomant (Somavert® for injection), which is used for the treatment of acromegaly. Although pegvisomant has shown success in improving symptoms of acromegaly, it comes with several disadvantages. For example, high production costs, end-product heterogeneity, and the requirement for daily injections, collectively impact efficacy of the treatment regimen. Therefore, the development of next-generation antagonist of the hGHR has become a necessity. The work outlined in this dissertation addresses this gap in knowledge by developing peptide-based antagonists that target the hGHR and aims to provide new insight into expanding novel therapeutic strategies for growth hormone-associated diseases. The use of peptide-based antagonists for modulating t (open full item for complete abstract)

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2024, College of Arts and Sciences

Hypoxia Inducible Factor 1 (HIF-1) is a heterodimeric transcriptional factor that plays a physiological role in low oxygen concentration or hypoxia. HIF-1 consists of two dimers: HIF-1alpha (HIF-1α) and HIF-1beta (HIF-1β). HIF-1α is the active oxygen-sensing domain in the cytoplasm that leads to stabilizing and overexpression of HIF-1 in the cells during hypoxia. On the other hand, HIF-1β is a stable domain in the nucleus that is required to form a dimer with HIF-1α in the DNA to express the HIF-1 gene. Upregulation of HIF-1α by either hypoxia or drug molecules has been elucidated to overexpress more than 100 tumor genes. These genes are involved in developing angiogenesis (vascularization), metastasizing, cellular proliferation, switching to anaerobic metabolism, and cellular survival. Hepatocellular carcinoma (HCC) is one of the solid tumors that have a hypoxic intratumor environment and relies on overexpression of HIF-1α to overcome hypoxia and allow cancer cells to survive, proliferate, and metastasize in these harsh conditions. Targeting or downregulating the HIF-1α gene in HCC with chemical compounds may provide a treatment for this cancer. However, inducing and overexpression of HIF-1α has many of benefits, such as accelerating wound healing in diabetic patients. Diabetic patients suffer from hyperglycemia and thick blood that delay wound healing and may cause infections. Upregulation of HIF-1α expression in diabetic wounds will increase the speed of the repair process of wound healing. HIF-1α plays a vi crucial role in all phases of wound healing by facilitating cell division, growth factor secretion, cell migration, survival in hypoxic environments, and matrix synthesis. We screened the LOPAC drug library to discover several chemical compounds that either inhibit or stimulate HIF-1α expression. These drug candidates have been further investigated to confirm their activity against HIF-1α expression. These findings suggest that up or downregulation of HIF-1 (open full item for complete abstract)

Master of Science in Chemistry, Youngstown State University, 2024, Department of Biological Sciences and Chemistry

Beta-galactosidases are enzymes that hydrolyze β-glycosidic bonds with galactose at the nonreducing end. These enzymes have been used in the food and pharmaceutical industries for processing waste into environmentally friendly compounds and the synthesis of prebiotics. GalB2 is a novel β-galactosidase from Enterobacter sp. YSU that was expressed in Escherichia coli. The enzyme was isolated with ammonium sulfate precipitation and purified by an anionexchange chromatography. The optimal pH for GalB2 was found to be 7.4. There was no loss in enzymatic activity when GalB2 was incubated at temperatures 37 ºC and 40 ºC, but activity significantly diminished after incubation at temperatures greater than 45 ºC. The window of substrates is narrow with only o-nitrophenyl-β-D-galactopyranoside (o-NPGal) and lactose being evident. The catalytic parameters were determined for both substrates. For the substrate o-NPGal, the Michaelis constant, KM was determined to be 0.18 mM and a catalytic constant, kcat was 44 s-1. With respect to lactose, the KM was found to be 2.5 mM with a kcat of 51 min-1. Glucose inhibited the enzyme in an uncompetitive manner, but galactose demonstrated competitive inhibition of GalB2. The enzyme shows dependence from magnesium ions. Native gel electrophoresis with exposure to fluorogenic substrate 4-methylumbelliferyl-β-D-galactopyranoside indicated that enzyme is active as a dimer. No transglycosylation activity for GalB2 was observed. Transglycosylated products from immobilized commercially available lactase were confirmed using LC/ESI-MS. Attempts to isolate oligosaccharides using a silica-gel column with an acetonitrile:water eluting solvent were unsuccessful.

Doctor of Philosophy, Miami University, 2024, Cell, Molecular and Structural Biology (CMSB)

Cells require a robust quality control system to maintain proper function. Molecular chaperones function in protein quality control by promoting proper protein folding, preventing protein aggregation, and targeting terminally misfolded proteins for degradation. In response to stress or cytotoxic conditions, misfolded or aggregated proteins escape the protein quality control system and can lead to neurodegenerative diseases like Alzheimer's disease and Parkinson's disease. The heat shock protein (Hsp) molecular chaperone family is highly conserved from bacteria to man and upregulated in response to cellular stressors. In the endoplasmic reticulum (ER), Grp94 (Hsp90), BiP (Hsp70), and ERdj co-chaperones (Hsp40s) function to maintain proteostasis. Grp94 promotes the proper folding of complex secretory and membrane proteins, along with playing a role in stress signaling. BiP and ERdj proteins work together for protein import, folding, and degradation. Grp94 and the BiP chaperone system have been shown to collaborate in vitro to refold denatured client proteins, however open questions still remain surrounding mechanistic details of Grp94 and ERdj molecular chaperones in the ER. Chapter 2 of my dissertation characterizes Grp94 chaperone activity using in vitro aggregation prevention studies. Aggregation prevention by Grp94 is independent of ATP binding and hydrolysis by Grp94 and does not require a specific Grp94 conformation. Additionally, this work highlights the importance of all domains in chaperone activity. Chapter 3 describes the purification and in vitro characterization of several ERdj co-chaperones. Together, this work provides insight to the cellular mechanisms of ER chaperone networks and their roles in protein homeostasis. These findings expand our current understanding of ER chaperone mechanisms and provide insight for targeting chaperone-specific mechanisms for disease management.

Doctor of Philosophy, University of Akron, 2024, Chemistry

Cell communication is crucial for regulating cell growth, proliferation, survival, and function. The process of cell communication and cell signaling involves the recognition of exogenous or endogenous signaling molecules by receptors expressed on the surface of the plasma membrane. The activation of surface receptors such as enzyme-linked receptors results in the activation of the receptor's intracellular domain, which might have an intrinsic kinase activity like receptor tyrosine kinase (RTK). The activation of RTK triggers multiple downstream signaling pathways that regulate cell proliferation, angiogenesis, apoptosis, motility, adhesion, and metastasis. Therefore, a thorough understanding of RTK activation mechanisms and subsequent signaling events is crucial to provide insights into the control of RTK activity. The work presented in this dissertation is studying two different receptors from two subfamilies of RTKs, c-KIT, and EGFR/HER receptors family. The first project in this dissertation is focused on studying the SCF-mediated c-KIT signaling in mast cells (MCs) and the role of the underlying mechanisms in modulating mast cell viability and proliferation. The number of MCs in tissues usually remains constant, inflammation and asthma disturb this homeostasis, leading to proliferation of MCs. Understanding the signaling events behind this proliferative response could lead to the development of novel strategies for better management of allergic diseases. MC survival, proliferation, differentiation, and migration are all maintained by a MC growth factor, stem cell factor (SCF) via its receptor, KIT. Here, we explored how protein kinase C (PKC) redundancy influences MC proliferation in bone marrow-derived MC (BMMC). We found that SCF activates PKCα and PKCβ isoforms, which in turn modulates KIT phosphorylation and internalization. Further, PKCα and PKCβ activate p38 mitogen-activated protein kinase (MAPK), and this axis subsequently regulates SCF-induced MC cel (open full item for complete abstract)

Master of Science, Miami University, 2024, Biology

Floral traits, including color, morphology, and scent, play a crucial role in attracting specific pollinators, with floral scent being a significant component for both short- and long-range pollinator attraction. In dioecious systems, where male and female flowers are present on separate plants, sexual dimorphism of floral displays is driven by differing selective pressures on male and female reproductive success. In some dioecious species, females engage in Bakerian mimicry—offering no nectar rewards yet mimicking male pollination syndromes to deceive pollinators. Research has observed Bakerian mimicry in several species, but its evolutionary conservation within the largely dioecious Caricaceae family remains underexplored. We used Vasconcellea parviflora as a model to examine the characteristics of Bakerian mimicry within the Caricaceae. We characterized sexual dimorphism in floral display, collected and analyzed floral volatile quantity and composition, and assessed nectar production in males and females. We found that V. parviflora females have smaller floral displays and produce no nectar rewards unlike males. There is, however, increased emission rates of floral scent compounds in females relative to males, potentially representing an evolutionary trade-off in females between producing no nectar rewards at the cost of increased volatile production to ensure pollinator attraction.

PhD, University of Cincinnati, 2024, Arts and Sciences: Chemistry

SLX1-SLX4 is a structure-specific endonuclease that plays a pivotal role in resolving Holliday-Junctions, which manifest during homologous recombination-mediated DNA repair (or homology-directed repair). The SLX1-SLX4 complex emerges as a key player in regulating cellular response to PARP inhibitors. In cells afflicted with homologous recombination deficiencies, such as those carrying BRCA1 or BRCA2 mutations, PARP1 inhibitors instigate synthetic lethality by impeding the mending of DNA single-strand breaks, culminating in unrepaired double-strand breaks. However, heightened expression or activity of the SLX1-SLX4 complex can facilitate the resolution of these DNA structures, enabling cell survival notwithstanding PARP1 inhibition. In the first project, I characterized and compared the DNA nuclease activities of human SLX1-SLX4 and yeast Slx1-Slx4 complex. The DNA substrate specificity, HJ cleavage site mapping, models of DNA cleavage, and the role of C-terminus in human SLX1-SLX4 DNA were extensively investigated in my study. Interestingly, I found that the hSLX1-SLX4 complex, containing the full-length SLX1 and a truncated SLX4 (residues 1632-1834: SLX1-binding domain CCD), efficiently cleaved the branched DNA structures including HJ, 5`-Flap, 3`-Flap, splayed arm, and replication fork DNA in the presence of Mg2+. In addition, endonuclease activity of hSLX1-SLX4 is initiated through DNA-binding to three different sites (site I, II, and III) of this complex. Binding of at least one continuous DNA strand between sites I and II appears to be essential for the DNA nuclease activity of hSLX1-SLX4. In the second project, to quantitatively analyze DNA and RNA nuclease activities, I developed a time-resolved-FRET (Fluorescence Resonance Energy Transfer) assays. I successfully and quantitatively analyzed the kinetics of the nuclease reactions. Using this assay, I found that human hSLX1-SLX4 prefers 5`-Flap DNA rather than 3`-Flap DNA for nuclease activity. I (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Chemistry

Organophosphorus (OP) compounds are highly toxic, causing over 200,000 deaths annually. These compounds inhibit acetylcholinesterase (AChE) by covalently modifying its catalytic serine residue, preventing the breakdown of the neurotransmitter acetylcholine (ACh). The resulting ACh buildup can cause severe symptoms and, if untreated, death. Traditional treatments using oximes aim to reactivate OP-inhibited AChE but are limited by their inability to cross the blood-brain barrier and their lack of broad-spectrum efficacy. Moreover, OP-inhibited AChE can undergo a spontaneous O- dealkylation event termed aging, and the OP-aged form is recalcitrant to oximes. Quinone methide precursors (QMP) provide a plausible strategy to realkylate the OP-aged cholinesterase adducts, and then, by subsequent water activation or nucleophilic addition to the phosphorus center, restore native enzyme function. Studies were performed to both determine efficacious compounds and their mechanism of action. Additionally, research was done to explore different enzymatic model systems and develop methodologies to investigate high binding QMPs. Phenolic QMPs can both reactivate OP-inhibited AChE and resurrect OP-aged AChE with both electron-donating and electron-withdrawing groups, primarily off of the 4-position. Electron-donating groups should theoretically promote quinone methide formation, whereas electron-withdrawing groups should hinder it. The fact that OP-aged AChE was resurrected by QMPs with both types of groups indicates that the realkylation or reactivation steps in the resurrection mechanism of the phosphylated oxyanion moiety are likely mediated by the enzyme or water. ii 6-Alkoxypyridin-3-ol QMPs are able to restore activity to both OP-inhibited AChE as well as OP-aged AChE. Resurrection kinetic studies show that efficacy of QMPs with (R)-2-methylpyrrolidine as the leaving group is due to their lower Km values, while QMPs with diethylamine are effective because of their high phos (open full item for complete abstract)

Master of Science, The Ohio State University, 2024, Biochemistry

Aminoacyl-tRNA synthetases (aaRSs) ensure translational fidelity by charging tRNAs with their cognate amino acids. Mistakes made by aaRSs lead to mischarged tRNAs, thereby increasing the error rate of protein synthesis. Mutations in aaRSs can also increase the protein translation error rate and lead to severe neurological disorders. Here, we investigated the effect of human glutamyl-prolyl-tRNA synthetase (EPRS1) biallelic disease-associated point mutations and the role of EF-Tu in Pro-tRNAPro protection from ProXp-ala. EPRS1 is a dual-functional aaRS encoding ERS and PRS separated by a linker encoding three helical WHEP domains, named after the four aaRSs first discovered to encode this motif. We characterized one compound heterozygous (CHet) EPRS1 mutation in an individual with diseases affecting multiple organ systems and one homozygous recessive (HMZ) EPRS1 mutation in an individual with severe hypomyelination leukodystrophy (HLD). We purified recombinant EPRS1 mutants in the context of the ERS-RC (where RC refers to random coil, as this construct encodes a short unstructured region of the linker) and ProRS-0.5W (where 0.5W refers to half of a WHEP domain). The HMZ variant, Y1130S, caused the most significant decrease in catalytic activity and altered the protein structure, the latter determined by a limited proteolysis assay. The CHet mutations, N443S in the ERS catalytic domain and M1037V in the PRS catalytic domain, also decreased the enzyme's catalytic function, but caused a less severe reduction in enzyme activity, compared to the HMZ mutation. The Y1130S mutation resulted in a loss in catalytic activity while the M1037V mutation caused a 2.2-fold decrease in catalytic efficiency relative to the wild-type (WT) protein. In comparison to WT ERS-RC, the N443S mutation caused a 1.1-fold decrease in catalytic efficiency. AaRSs sometimes mischarge cognate tRNAs with the wrong amino acid. PRS enzymes from all domains of life have been shown to mischarge (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

The composition of diet has a significant impact on both stem cells and cancer cells by regulating nutrient-sensing pathways that affect their metabolic state and survival. High- proliferating cells tend to increase lipid usage and activate several lipid synthesis enzymes. Previous research both from our lab and others has shown that lipids play a role in regulating stem cell function through fatty acid oxidation (FAO), which controls access to lipid intermediates and, in turn, mediates stem cell proliferation and energy production. However, it is not well understood whether intestinal stem cells require de novo lipogenesis (DNL) under homeostatic conditions or in response to dietary changes. The balance between saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) is governed by the rate-limiting Stearoyl-CoA Desaturase (SCD) enzymes in the DNL pathway. SCD1 expression is elevated in various types of cancer, including colorectal cancer, and is associated with poor patient survival. While previous studies have linked increased DNL with tumorigenesis, the necessity of DNL in adult stem cells remains unclear. Recently, we discovered that Stearoyl Desaturases (Scds) are regulated in intestinal stem cells in a nutrients and growth factor-dependent manner, and they influence stem cell function, which prompted us to explore the regulation and role of murine Scd1 and Scd2 isoforms in the intestinal stem and progenitor cells. Briefly, Chapter 2 examines the regulatory effects of varying nutrient availability states (fasting and refeeding) and ietary fatty acid content on the Scd1 and Scd2 enzymes. Scd1 and Scd2, protein and RNA levels, become upregulated under nutrient abundance while downregulated in nutrient- deplete conditions. We also found that both the Scd1 and Scd2 isoforms exhibit high sensitivity to dietary fatty acid content. While a high saturated fatty acid diet upregulates their expression throughout the entire gut epithelium, an unsaturated fatt (open full item for complete abstract)

PhD, University of Cincinnati, 2024, Arts and Sciences: Chemistry

Deltex (DTX) family of E3 ubiquitin ligases are RING-containing E3 enzymes that catalyze the transfer of ubiquitin (Ub) from an E2-conjugating enzyme to a target substrate. Interestingly, DTX enzymes were discovered to mediate crosstalk between ubiquitination and ADP-ribosylation, which are two independent and reversible post-translational modifications (PTMs) that regulate a wide-array of cellular signaling pathways, including DNA repair. Ubiquitination is catalyzed by an enzymatic cascade that results in the covalent modification of a target substrate with a single- or multiple Ub units. And ADP-ribosylation involves the addition of an ADP-ribose (ADPR) moiety from nicotinamide adenine dinucleotide (NAD+) to a target substrate by ADP-ribosyl transferases (ARTs), such as poly-ADP-ribosyl polymerases (PARPs). PARP1, the primary ART enzyme in humans, is crucial for the initiation of DNA damage response (DDR) pathways. Previous research identified Ub as an ADP-ribosylation target by DTX enzymes, but the mechanism through which this reaction occurred was poorly understood. Through my research, I have instead proposed that DTX enzymes do not catalyze the ADP-ribosylation of Ub but the ubiquitination of ADP-ribose. This distinction is important as the chemistry involved in both pathways is distinct and leads to different products. DTX2 and DTX3L are primarily nuclear and have interactomes including DDR enzymes like PARP1, PARP9, and XRCC5, so I have hypothesized that DTX activity potentially has a functional linkage to DDR through the direct modification of ADP-ribosylation. Due to the recency of these discoveries, there is limited information on the mechanism controlling this activity, and thus my research has three main objectives: (I) Determine the substrate specificity of DTX-mediated Ub-ADPR conjugate formation, (II) Define the role of Ub-ADPR conjugate formation on the ADP-ribosylation reversal (ADP-ribose capping), and (III) Develop quantitative and real-time TR-F (open full item for complete abstract)

Master of Science, The Ohio State University, 2024, Chemistry

The plasma membrane barrier poses a substantial obstacle to drug development, as it makes it difficult to deliver therapeutics to 75% of prospective targets various diseases with conventional methods. Although small molecules can target intracellular components and pass through the plasma membrane via passive diffusion, their efficacy is restricted to macromolecules with a well-defined hydrophobic binding site. Proteins, peptides, and oligonucleotides are examples of biologics that can provide a more varied and highly selective method of targeting a broad range of molecules, preventing protein-protein interactions, and possibly substituting for missing or defective proteins. However, because most biologics are unable to permeate the plasma membrane, their efficacy as therapeutic agents are hampered. In this study, by leveraging a bismuth-cyclized proteinogenic bicyclic cell-penetrating peptide (pBCP), we genetically fused pBCP15 at the N-terminus of a super-ecliptic phluorin (SEP) protein to render it cell-permeable. In comparison to the wild-type SEP, the recombinant protein showed an increase in cellular entry and in cytosolic entry. Moreover, a class of disulfide cyclized cell-penetrating peptide (DCPs) were developed, and their cytosolic delivery was tested. The DCPs, which cyclize on-resin through a disulfide bond between two cysteines, have shown a cytosolic entry up to 12-times that of Tat, a highly basic peptide from the transactivator of transcription protein of human immunodeficiency virus. The DCPs should enable the delivery of cell-permeable proteins and be leveraged as a new class of therapeutic agents.

Doctor of Philosophy, The Ohio State University, 2024, Chemistry

Organophosphorus (OP) compounds are highly toxic to the human body via the inhibition of the essential enzyme acetylcholinesterase (AChE). After phosphylation of the active site serine residue, AChE can no longer hydrolyze the neurotransmitter acetylcholine, therefore overstimulating the nervous system. OP compounds also covalently modify the nonessential enzyme butyrylcholinesterase (BChE). If these covalently inhibited enzymes are left untreated, the phosphylated serine residue can spontaneously dealkylate to the OP-aged state, a non-functional (dead) enzymatic state for which there are no approved medical therapeutics for recovering (resurrecting) the native activity. My dissertation research focuses on the synthesis and evaluation of >200 novel quinone methide precursors (QMPs) that can resurrect native enzyme activity from OP-aged AChE and BChE. The resurrection of OP-aged AChE may improve patient care and outcomes after exposure to various OP compounds. Furthermore, BChE is a known stoichiometric bioscavenger of OP compounds found in high concentrations in human blood. Resurrection of this nonessential enzyme would transform endogenous BChE into a pseudo-catalytic bioscavenger, potentially allowing it to protect AChE from OP exposure. 6-Alkoxypyridin-3-ol QMPs are an excellent scaffold for resurrecting the aged form of both cholinesterase enzymes, with interesting structural differences that are necessary in order to achieve this feat. 6-Methylpyridin-3-ol QMPs are also capable of resurrecting OP-aged AChE with small acyclic amine leaving groups, yet when the leaving group is based on a benzylamine, these QMPs are outstanding compounds for the reactivation of OP-inhibited BChE. Finally, there has been initial investigation on 5,6-bicyclic heterocycles, fluorinated QMPs, di-substituted QMPs, and 2-alkoxypyridin-3-ol QMPs to better understand the impact of QMP structure on biochemical activity. Several of the QMPs reported herein have been selected for in vi (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

Heart failure (HF) is one of the leading causes of morbidity and mortality globally. A growing body of evidence has indicated the 5-year mortality rates are about ~50% after initial HF diagnosis. Electrolyte imbalances have been implicated in predicting the outcome of HF patients, where lower serum sodium levels (hyponatremia; serum sodium <135mEq) have been strongly associated with increased mortality in HF. Hence, serum sodium levels have been a well-established adverse prognostic marker for patients with chronic HF. Recent evidence has suggested that lower serum chloride (hypochloremia, <97mEq) is associated with increased mortality risk in patients with chronic HF independent of serum sodium levels. Even though hypochloremia is associated with increased mortality in HF patients, the exact role of chloride (Cl ) homeostasis in cardiac function and how hypochloremia contributes to cardiac injury is unknown. Hence, our study focuses on understanding the mechanism of hyperchloremia-mediated ischemia-reperfusion (IR) injury and the role of Cl- channel in mediating hypochloremia effects. In the first part of this dissertation, we showed that hypochloremia increases mortality in left ventricular assist device (LVAD) placement and acute decompensated HF (ADHF) patients. This increase in mortality was associated with aggravated myocardial infarction post IR injury from our ex vivo studies with isolated rat hearts. In cardiac physiology, hypochloremia increases the beating of hiPSC-CM, which is attributed to increased intracellular calcium (Ca2+) cycling. Since hypochloremia affects the Cl- homeostasis, the mitochondrial functions: membrane potential, and reactive oxygen species production are affected post IR injury. In the second part of this thesis, we have explored the involvement of Cl- channel downstream of hypochloremia on intracellular Ca2+ cycling. Two Cl- channel inhibitors, 4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt (DIDS), non-specific pl (open full item for complete abstract)

PHD, Kent State University, 2024, College of Arts and Sciences / Department of Chemistry and Biochemistry

DNA origami structures have provided versatile tools in a wide variety of applications. Due to higher stability, tunability and mechanical strength compared to single stranded or double stranded DNA, origami-based DNA nanostructures have garnered special interest in the scientific community. Many of these biomedical applications exploit mechanical properties and therefore, need a discrete tool/method to analyze the dynamic structural changes in these nanoassemblies. Herein, we synthesized different origami based nanosprings and mechanically characterized their properties and eventually applied them to halt the cancer cell motions. To that end, the nanosprings were synthesized from 6 helix bundles of DNA with specific bridge elements made up of either i-motifs, or G-quadruplexes or duplex DNAs. The bridge elements served as junctions which pulled/pushed nearby elements to bend the structures; multiple of which exhibited a coiled spring. To characterize the mechanical properties, we employed optical tweezers as a single molecule force manipulation tool that record the extension behaviors under tensile force in real time, assisting to compute spring constant, kinetics, recoil and uncoil distances, and pitch of nanosprings. With the strength 17 times more than previous nanosprings, these nanostructures were exploited to modulate cell migrations. To that end, we modified nanosprings with arginyl-glycyl-aspartate (RGD) domains with a spacing such that when the nanospring is coiled, the RGD ligands trigger the clustering of integrin molecules, which changes cell motions. The coiling or uncoiling of the nanospring is controlled, respectively, by the formation or dissolution of pH responsive i-motifs. Results showed significant inhibition to the migration of HeLa cells in acidic extracellular environment under the influence of nanosprings. Likewise, to divulge the structural-property relationship and the modulation factors behind the long-range, higher order arrangement of s (open full item for complete abstract)

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

Activation of Toll-like Receptors (TLRs) induces inflammation regulated by members of the nuclear factor kappaB (NF-κB) transcription factor family. NF-κB c-Rel, a subunit of NF-κB, is a key regulator of inflammatory signaling, and is critical in specific dendritic cell functions. Dendritic cells bridge the innate and adaptive immune response through the secretion of cytokines, sampling and presentation of antigens, and activation of naive T cells during immune response. In this work, we describe the role of c-Rel following TLR activation as an important regulator of dendritic cell function. We describe the TLR7/c-Rel signaling axis and its critical role in the expression of inflammatory cytokines needed for psoriasis pathogenesis. We found that TLR7 agonism, and not application of its vehicle compound isostearic acid, is the primary driver of the Aldara-induced psoriasis mouse model, thus elucidating important mechanisms behind disease models. Finally, we describe how the increase in O-GlcNAcylation, and the enhancement of c-Rel specific O-GlcNAcylation, during TLR4 signaling regulates dendritic cell activation. Together, this work expands on the fundamental understanding behind how c-Rel acts as a transcriptional regulator during TLR signaling, and the downstream consequences in disease propagation.