Master of Science in Chemistry, Youngstown State University, 2020, Department of Chemistry

Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules and radicals mainly produced from the partial reduction of molecular oxygen. ROS have been associated with aging and several diseases such as atherosclerosis and cancer. Metal-catalyzed oxidation (MCO) systems are systems that produce free radicals using transition metal ions such as copper or iron and hydrogen peroxide. As such, MCO may cause oxidation of proteins. To study the correlation between protein structure and oxidative damage of proteins by MCOs, different mutants of hen egg white lysozyme (HEWL) have been developed. This research focuses on the optimization of the expression of the mutant HEWL H15S. The Pichia pastoris expression system was adapted for the expression of HEWL H15S. The P. pastoris X-33-pPICZαA-hewlH15S strain was subjected to different growth conditions in a glycerol and methanol buffered media under conditions for small scale expression. Both intracellular and extracellular protein expression were analyzed for enzyme activity. Increasing glycerol concentration from 0.5% to 1% did not show significant increase in yeast growth resulting in low protein concentration and enzyme activity at 28 °C. Also, protein expression at three different methanol concentrations at 28 °C: 0.5% (v/v), 1% (v/v), and 2% (v/v) showed an increase in enzyme activity but only small changes in total protein concentration. The addition of calcium chloride showed a significant effect on the expression of H15S to about 1mg/mL compared to the other conditions without CaCl2. Lysing of the cells grown at 28 °C for intracellular analysis by the Bradford assay showed a significant band of protein corresponding to the size of the H15S mutant. A lower temperature of 22 °C at different growth and expression conditions measured high protein concentration and an increase in enzyme activity for extracellular expression. Intracellular analysis on protein expression at 22 °C measured no lysozyme acti (open full item for complete abstract)

Committee: Michael Serra PhD (Advisor); Nina Stourman PhD (Committee Member); John Jackson PhD (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Chemistry; Microbiology

PhD, University of Cincinnati, 2024, Medicine: Cancer and Cell Biology

Epidermal vulnerabilities in Fanconi Anemia (FA) disease are an unexplored trait that may confer extreme susceptibility to squamous cell carcinomas (SCC) of the head and neck and esophageal region, anogenital tract and the skin, where keratinocytes are the cells of origin. Previously, we reported that FA epidermal rafts and skin biopsies from FA individuals harbor defects in desmosome cell junctions, which clinically contribute to accelerated skin blistering when induced by mechanical stress. However, relevant mechanisms that stimulate epidermal fragility and SCC predisposition in FA disease are poorly understood. Our findings demonstrate that functional loss of the FA pathway results in transcriptional repression and reduced protein expression of desmosomes in the stratified epidermis. Diminished desmosome expression occurs in part via transcriptional suppression of the master transcriptional regulator, TAp63, and post-translationally via accumulation of reactive oxygen species (ROS), membrane lipid peroxidation, and generation of the much stable reactive aldehyde, 4-hydroxy-no-nenal (4-HNE). Elevated ROS and lipid peroxidation were both sufficient and required to drive transcriptional repression and protein downregulation of TAp63 and subsequently desmosome proteins. TAp63 overexpression or treatment with a flavonoid antioxidant Quercetin restored TAp63 and desmosome transcript and protein levels as well as minimized ROS, lipid peroxidation, and 4-HNE in the FA epidermis. Quercetin supplementation over a period of 12 months in FA individuals improved epidermal integrity by increasing the time to form blisters upon induction with mechanical stress. Collectively, targeting the ROS-TAp63 signaling in FA individuals improves epidermal integrity and may help counteract SCC susceptibility both in the vulnerable FA population and in the general population where ROS plays a critical role in disease pathogenesis.

Committee: Susanne Wells Ph.D. (Committee Chair); Marie-Dominique Filippi Ph.D. (Committee Member); Joseph Palumbo (Committee Member); Dorothy Supp Ph.D. (Committee Member); Paul Andreassen Ph.D. (Committee Member) Subjects: Biology

Master of Science (M.S.), University of Dayton, 2024, Chemistry

Two BODIPY dyes with varying substituents have been synthesized and characterized. Coordination of [Ru(bpy)2Cl]+ complexes to different N-atoms, depending on the structure of the dye, resulting in a series of BODPIY-Ru(II) complexes, RuBDP-1 and Ru2BDP-2. The spectroscopic and electrochemical properties of the BODIPY dyes and their Ru(II) analogs have been characterized. The electronic spectra of these compounds reveal the intense absorption typical of the (π-π*) transition localized on the BODIPY core. Additional metal-centered charge transfer transitions associated with the [Ru(bpy)2Cl]+ moieties are observed at higher energy compared to the BODIPY transitions. RuBDP-1 exhibits significant singlet oxygen generation determined using a singlet oxygen trap in acetonitrile solutions. Ru2BDP-2 demonstrates the ability to generate singlet oxygen within the PDT window; however, it is limited in its quantum yield of singlet oxygen which translates to its limited ability to photocleave cpDNA.

Committee: Shawn Swavey (Advisor); Justin Biffinger (Committee Member); Vladimir Benin (Committee Member) Subjects: Biochemistry; Chemistry

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

Mitochondria are the major sites of cellular energy production. Electrons from reduced metabolites flow through the ETC consisting of complexes I through IV that creates the mitochondrial membrane potential (MMP) which is harnessed by complex V, culminating in ATP synthesis. Any disruption in the activity of the ETC complexes hampers formation of MMP and consequently ATP synthesis. ETC in the inner mitochondrial membrane during oxidative phosphorylation also serves as the primary source of reactive oxygen species (ROS), which if not fully neutralized causes oxidation of major biomolecules. While the consequences of DNA, protein and lipid oxidation have been explored, the effect of RNA oxidation on cellular processes in general is rather poorly understood. It's all the more important because the mechanisms for repair of oxidized RNA are still under debate. Various diseases including major neurological ailments and certain cancers are associated with RNA oxidation. Also, mitochondrial dysfunction is a hallmark of major neurodegenerative diseases including, Parkinson's disease (PD), Alzheimer's disease (AD), and Multiple Sclerosis (MS). However, the connection between the mitochondrial dysfunction and RNA oxidation is yet to be investigated. We hypothesize that “mitochondrial dysfunction can be a result of oxidation of the mRNAs encoding the subunits of the ETC complexes, which would impede subunit production leading to compromised ETC and decreased ATP production.” To systematically and comprehensively address the hypotheses, we focused on addressing three specific aims in this study: i) identify the oxidized mitochondrial mRNAs in neuronal cells, and determine if they lead to lowering of the cognate protein subunit levels and measure function of the ETC complexes and ATP level, ii) investigate if nuclear mRNAs encoding ETC complex subunits are oxidized and consequence of that on function of ETC complexes and ATP synthesis, and iii) detect oxidized mRNAs in EAE mice, (open full item for complete abstract)

Committee: Soumitra Basu (Committee Chair); Sanjaya Abeysisrigunawardena (Committee Member); Yaorong Zheng (Committee Member); John Johnson (Committee Member); Jennifer McDonough (Committee Member) Subjects: Biochemistry; Molecular Biology

Master of Sciences, Case Western Reserve University, 2023, Pathology

Noise-induced hearing loss (NIHL) is a major health issue with no treatment to prevent it. Loss of cochlear sensory ‘hair' cell function is one common cause of NIHL. Studies show that exposure to noise is a major cause of oxidative stress in hair cells, and the mitochondria generates reactive oxygen species (ROS) under stress. Since Szeto-Schiller (SS-31) peptide is a proven inhibitor of ROS production from the mitochondria, it was hypothesized that SS-31 treatment would prevent noise-induced loss of hair cell function. The thesis project was designed to test the hypothesis in a mouse model of NIHL. CBA/J mice exposed to 8-16 kHz noise at 98 dB SPL for 2 hours displayed hearing loss from synaptopathy (reduced hair cell-afferent synapse activity); however, SS-31 treatment initiated immediately after noise exposure prevented the noise-induced phenotype. The results support the hypothesis and highlight the potential of SS-31 to prevent NIHL in humans.

Committee: Ruben Stepanyan (Advisor); Pamela Wearsch (Committee Member); Brian McDermott (Committee Member); Neena Singh (Committee Chair) Subjects: Biomedical Research; Pathology; Pharmacology; Physiology

PhD, University of Cincinnati, 2022, Medicine: Systems Biology and Physiology

Ventricular tachyarrhythmias resulting in sudden cardiac death (SCD) continue to claim over 350,000 lives per year in the United States. Though several modes of treatment exist, these are palliative and do not address the root pathology of the disease. Treatment is further complicated by adverse side effects which increase mortality and decrease patient quality of life. Recent clinical trials have explored vagus nerve stimulation (VNS) as a potential therapy for patients with heart failure, though findings are mixed. VNS has yet to be explored in the context of SCD in the clinical setting. VNS has been shown to augment antioxidant capacity and may confer protection from ROS-induced cardiac remodeling, preventing SCD. However, VNS is known to decrease cardiac contractility and heart rate while prolonging ventricular repolarization, all of which could exacerbate symptoms of heart failure, increasing patient susceptibility to SCD. To better discern whether VNS is a potential candidate for patients with heart failure or SCD, insight into the molecular mechanisms of both VNS and SCD is essential. The results of this dissertation suggest that vagal signaling is cardioprotective. Using a unique guinea pig model of SCD, it was observed that animals dying from SCD experienced tachyarrhythmias resulting from prolonged and disperse ventricular repolarization, both of which are hallmarks of SCD. Further, animals that did not reach premature endpoints in the SCD group were observed to have undergone cardiac remodeling, resulting in prolonged and heterogeneous cardiac conduction. Reactive oxygen species (ROS) have been shown to play a critical role in the mechanism of SCD, leading to widespread damage, electrical instability, and cardiac electromechanical dysfunction. Using a ROS scavenger, we were able to abolish arrhythmia in ex vivo failing hearts. Importantly, studies have found that vagal stimulation confers anti-oxidant activity, suggesting a potential means of alle (open full item for complete abstract)

Committee: Roger Worrell Ph.D. (Committee Member); Karthickeyan Chella Krishnan Ph.D. (Committee Member); Margaret Powers-Fletcher Ph.D. (Committee Member); Sarah Pixley Ph.D. (Committee Member); Onur Kanisicak PhD (Committee Member) Subjects: Physiological Psychology

PhD, University of Cincinnati, 2022, Arts and Sciences: Chemistry

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive ions and molecules derived from molecular oxygen (O2), which can cause DNA damage and lead to skin cancer. High levels of ROS can promote cancer development, cancer cell survival, and resistance to chemotherapeutics. NADPH oxidase (NOX) is a significant producer of ROS in the cell. NOX1 generates two superoxide molecules by reducing NADPH. This only occurs when the membrane-bound NOX cytochrome p450 alpha chain (CYBA) binds to the organizer subunit NOXO1 from the cytosolic portions of the holoenzyme on the cell surface. We propose that stopping NOX1 complex subunits from coming together at this CYBA-NOXO1 junction is a potential way to prevent ROS production in human skin cells when exposed to ultraviolet rays. This dissertation investigates potential small-molecule inhibitors of the crucial NOX1 holoenzyme to solve these issues. We designed and synthesized NOX1 specific Inhibitor 1 using a diapocyin backbone structure. Computational docking studies were used to optimize inhibitor design and evaluate the NOXO1 protein subunit specificity. Due to increased binding interaction with NOXO1 protein and to improve solubility of solution preparation for further physical binding studies, we modified Inhibitor 1 and synthesized Inhibitor 2 by adding the NHS-ester Biotin polyethylene glycol chain to the piperidine ring. Both inhibitors were found to be non-toxic in human keratinocyte cells. The Inhibitor 2 reduced the cyclobutene pyrimidine dimer (CPD) DNA mutation in a human skin explant model. Finally, the isothermal calorimetric (ITC) binding assay and MALDI-TOF mass spectrometry were used for physical binding studies to evaluate the critical molecular interaction, leading to the decreased binding affinity of Inhibitor 1, Inhibitor 2, resulting in additional modifications seen in Inhibitor 3 and Inhibitor 4. The results demonstrate that Inhibitor 2 and Inhibitor 3 reduced the binding affinity bet (open full item for complete abstract)

Committee: In-Kwon Kim Ph.D. (Committee Member); Peng Zhang Ph.D. (Committee Member); Pietro Strobbia Ph.D. (Committee Member); Edward Merino Ph.D. (Committee Member) Subjects: Pharmaceuticals

Master of Science (MS), Wright State University, 2021, Pharmacology and Toxicology

In skin connective tissue, dermal fibroblasts appear to be the most prevalent cell type. They are in charge of making the extracellular matrix that makes up the skin's connective tissue, and also involved in wound healing. Moreover, they produce Insulin-like growth factor-1 (IGF-1) which helps in activation of Insulin growth factor-1 receptor (IGF-1R). This receptor helps to control cell proliferation and responses to DNA-damaging substances such as UVB radiation, reactive oxygen species (ROS), and therapeutic drugs. According to our findings, lack of IGF-1 expression in the dermis of elderly patients due to fibroblast senescence (senescence is characterized by which cells enter a condition of irreversible growth arrest after irreversibly avoiding dividing without enduring cell death) has been linked to an increased incidence of skin cancer in the epidermal keratinocyte. Our group resolved that pretreatment with creatine monohydrate and nicotinamide shows a protective effect on oxidative-stress senescence. Based on this study, the present project was designed to study the effect of creatine and nicotinamide on stress-induced reactive oxygen species (ROS) generation as a possible mechanism for their protective effects. Similarly, the present study also examined how the pro-energetics acts on senescence as a post-treatment. Using primary human dermal fibroblasts exposed to H2O2 in vitro, via ROS staining, beta-galactosidase staining, and RT-qPCR, we discovered that pre-treatment with creatine and nicotinamide reduces oxidative stress-induced ROS levels, while post-treatment with creatine or nicotinamide after H2O2 had no effect on stress-induced senescence.

Committee: Jeffrey B. Travers M.D., Ph.D. (Advisor); Michael G. Kemp Ph.D. (Committee Member); Ravi P. Sahu Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

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

Background: Mitochondria play a key role in immune defense pathways, particularly for macrophages. We and others have previously demonstrated that cystic fibrosis (CF) macrophages exhibit weak autophagy activity and exacerbated inflammatory responses. Previous studies have revealed that mitochondria are defective in CF epithelial cells, but to date, the connection between defective mitochondrial function and CF macrophage immune dysregulation has not been fully elucidated. Here, we present a characterization of mitochondrial dysfunction in CF macrophages. Methods: Mitochondrial function in wild-type (WT) and CF F508del/F508del murine macrophages was measured using the Seahorse Extracellular Flux analyzer. Mitochondrial morphology was investigated using transmission electron microscopy and confocal microscopy. Mitochondrial membrane potential (MMP) as well as mitochondrial reactive oxygen species (mROS) were measured using TMRM and MitoSOX Red fluorescent dyes, respectively. All assays were performed at baseline and following infection with Burkholderia cenocepacia (B. cenocepacia), a multi-drug resistant bacterium that causes detrimental infections in CF patients. Results: We have identified impaired oxygen consumption in CF macrophages without and with B. cenocepacia infection. We also observed increased mitochondrial fragmentation in CF macrophages following infection. Lastly, we observed increased MMP and impaired mROS production in CF macrophages following infection with B. cenocepacia. Conclusions: The mitochondrial defects identified are key components of the macrophage response to infection. Their presence suggests that mitochondrial dysfunction contributes to impaired bacterial killing in CF macrophages. Our current study will enhance our understanding of the pathobiology of CF and lead to the identification of novel mitochondrial therapeutic targets for CF.

Committee: Amal Amer MD, PhD (Advisor); Narasimham Parinandi PhD (Committee Member); Mark Wewers MD (Committee Member); Anne Strohecker PhD (Committee Member) Subjects: Biology; Cellular Biology; Immunology

PhD, University of Cincinnati, 2020, Arts and Sciences: Chemistry

Cancer remains one of the leading causes of mortality in the world. Even with the improvement of treatments in multiple type of cancer, current chemotherapies are highly toxic and associated with severe acute side effects due to the low selectivity of the existing drugs. Additionally, multiple cancers develop chemotherapeutic resistance and new targets need to be investigated. Amongst them, Ras is one of the most common aberration found in cancer with approximately 30% of cancer containing the mutated oncogenes. Ras-targeted therapy with small molecule inhibitor could be the key to better and safer treatments. Moreover, the use of targeted therapy with drug conjugate and delivery vehicles such as aptamers, antibodies or targeting peptides could help bring selectivity to treatments and reduce off-target toxicity. This dissertation focuses on the development of both targeted therapy strategies for cancer treatment. In the first half, we report the identification of IODVA1 as the active compound of NSC124205 for the treatment of Ras-driven cancer models in cellulo and in vivo. Identification of the IDOVA1 side products led me to synthesize GUPR-195 followed by the synthesis of several related molecules. In the other half, we focus on decreasing toxic off-target inhibitors effect using targeting therapy. Here, we developed two new reactive oxygen species (ROS)-sensitive linkers for selective drug release to leukemic cells. Indeed, cancer cells are known to have higher levels of ROS than their healthy counterparts. By using a ROS-sensitive linker for the conjugation of a cytotoxic payload to a delivery vehicle, it is possible to double the selectivity of drug release. We report in the last chapter of the dissertation the development of a self-cyclizing ROS-sensitive linker and an oxalamide ROS-sensitive linker.

Committee: Edward Merino Ph.D. (Committee Chair); In-Kwon Kim Ph.D. (Committee Member); David Smithrud Ph.D. (Committee Member) Subjects: Pharmaceuticals

PhD, University of Cincinnati, 2020, Pharmacy: Pharmaceutical Sciences

Melanoma is the deadliest form of skin cancer. Although melanoma cases represent only 4% of all skin cancers, it accounts for 80% of all skin cancer-related deaths [1]. The identification of BRAF mutants in cancer piloted a new era in the treatment of advanced melanomas. Approximately one-half of melanomas contain a BRAFT1799A transversion, encoding the constitutively active BRAF V600E oncoprotein [3]. This discovery led to the clinical development of selective ATP-competitive RAF kinase inhibitors, including vemurafenib and dabrafenib, targeting the mutant BRAF protein. Treatment of melanoma patients with BrafV600E with either BRAF inhibitor resulted in a response rate of about 50% and median progression-free survival of about 5 months [4, 5]. With the success of clinical trials [6, 7] BRAF-inhibitor and MEK-inhibitor combination therapy has become the standard of care for BRAF-mutant melanoma with response rates of ~70% and disease control in almost all patients. Despite these successes, most treated patients will eventually exhibit disease progression. The acquired resistance to these inhibitors has limited their long-term efficacy, and has stirred interest in understanding the mechanisms underlying resistance. Recent observations demonstrate that BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells. A100, identified from a library screen, is a ROS-activated prodrug that self-cyclizes into a stable bicyclic ring and causes DNA double strand breaks. We proposed to examine if ROS activated therapy will inhibit tumor growth and evade resistance to BRAF inhibitors. In this study, the BRAF inhibitor dabrafenib was used to generate resistant cell lines (A375DR, SK-MEL-24DR and WM-115DR). Furthermore, we observed that resistant cells had increased levels of the mitochondrial antioxidant enzymes superoxide dismutase 2 (SOD2) and peroxiredoxin 1 (PRDX1). We found that A100 sensitized the resistant melanoma cells to dabrafenib and induced DNA damage. C (open full item for complete abstract)

Committee: Joan Garrett Ph.D. (Committee Chair); Zalfa Abdel-Malek Ph.D. (Committee Member); Pankaj Desai Ph.D. (Committee Member); Nalinikanth Kotagiri Ph.D. (Committee Member); Timothy Phoenix Ph.D. (Committee Member) Subjects: Pharmaceuticals

PhD, University of Cincinnati, 2020, Arts and Sciences: Chemistry

Reactive oxygen species(ROS) are a family of radical and non-radical byproducts of aerobic metabolism. It plays essential roles as secondary signaling molecules in cell proliferation, differentiation, sentence, and apoptosis. Ultraviolet radiation (UVR) overexposure can upregulate ROS in skin cells and results in further damage to deoxyribonucleic acid (DNA), protein, and lipid. As UVR is an essential risk factor for the development of premalignant skin lesions as well as of melanoma and nonmelanoma skin cancer, sunscreen agent was developed to prevent it. The chemoprevention strategy is found and developed since 1976, which is being developed to present. It includes two different types: one is applying the chemical which can absorb or reflect UVR to prevent UVR radiation on the skin surface, the other type affects the metabolism of skin cells to stop the cell damage and malignant initiation. The second category is developing fast in the most recent 20 years to meet the need of human beings. A series of antioxidants and natural products prove to effectively prevent UVR by reducing the ROS level by ROS scavenging or as an inhibitor of the ROS generator. And a ROS-activated prodrug strategy is developed to enhance the selectivity of chemoprevention. The first project was finished by designing a novel ROS-activated moiety attached with apocynin, which is not only an antioxidant but a nicotinamide adenine dinucleotide phosphate oxidase(NOX) inhibitor. Releasing manner about it was studied by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). The skin protection function of it was further proved by gel electrophoresis, dichlorofluorescein diacetate(DCFDA) assay, MTT assay, western blot, and cyclobutane pyrimidine dimers (CPD) quantification. To sum, this prodrug was proved to release the NOX1 inhibitor and protect the DNA from UVR radiation by lowing the ROS level. Considering the drug attached has a controversial mechanism to inhibit (open full item for complete abstract)

Committee: Edward Merino Ph.D. (Committee Chair); Ana Luisa Kadekaro Ph.D. (Committee Member); David Smithrud Ph.D. (Committee Member); Peng Zhang Ph.D. (Committee Member) Subjects: Pharmaceuticals

PHD, Kent State University, 2020, College of Arts and Sciences / Department of Chemistry and Biochemistry

The efficacy of treating bacteria infection by conventional, reliable, powerful, and abundant antibiotics has been increasingly weakened due to the appearance of ever-growing resistance to these drugs in the past several decades. The multidrug-resistant (MDR) pathogens are especially indulged in the hospitals globally, which leads to a rapid emergence and dissemination of such microorganisms and threatens the inappropriate usage of antibiotics in infected patients. As one of the most lethal gram-positive pathogens in the world, methicillin-resistant staphylococcus aureus (MRSA) was first discovered five decades ago. Since then, they have overwhelmingly spread worldwide, causing more than tens of thousand deaths per year in the US due to its ability to resist most clinical antibiotics. On the other hand, the gram-negative MDR Pseudomonas Aeruginosa causes hundreds of deaths per year in the US, and even nearly all antibiotic resistance has been found in some strains. Therefore, traditional antibiotics that possess one target can easily generate bacterial resistance through chromosomal mutation to form different mutants or via obtaining resistance genes from other organisms. In this study, we have designed and synthesized a naphthoquinone derivative as a potent antimicrobial and validated its therapeutic efficacy against MRSA as well as the ability to disabling the development of drug resistance. We have synthesized the derivatives based on the classical anticancer scaffold--lawsone (2-hydroxy-1,4-naphthoquinone) and evaluated their antimicrobial efficacy using the in vitro culture of MRSA and the in vivo model of both wound infection and systemic infection by MRSA. We effectively tuned the structure of lawsone by changing functional groups on it to improve the cell membrane permeability of lawsone, while retaining the functions of redox activity and chelation of metal ions in order for the molecule to exhibit antibacterial activity by either generating ROS (r (open full item for complete abstract)

Committee: Songping Huang (Advisor); Min-Ho Kim (Advisor) Subjects: Biochemistry; Chemistry; Materials Science

PhD, University of Cincinnati, 2019, Arts and Sciences: Chemistry

Chemotherapy for cancer always has many off-target effects that damage healthy cells. Many chemotherapeutic drugs can affect the function of normal cells in the heart, kidneys or nervous system cause another health problem. Reactive Oxygen Species (ROS) are molecules produced in cell metabolism. ROS are important for proliferation, differentiation, senescence, apoptosis, and chemoresistance of cancer cells. Compared to normal cells, it has been found that ROS concentration is elevated in lots of cancers. These differences between cancer and normal cells are the basis for the development of new chemotherapy agents that selectively target the cancer cell but not impair the function of normal cells. Based on these observations, we made several ROS activated compounds and found a lead compound, MA14, which has an IC50 of 2 µm against AML cancer cells. I hypothesized that the high selectivity of MA14 is due to its unique oxidation mechanism in the presence of H2O2. Herein, the oxidation mechanism of MA14 was investigated using MS and NMR spectroscopy. In the presence of ROS, MA14 goes through oxidative-cyclizing to form two isomers. These isomers could be further oxidized to non-toxic products. I also synthesized several molecules to study the relationship between structure and activity. It is shown that the a-carbon of the side chain is essential for cytotoxicity. The size of the cyclic amine has a moderate influence on activity. I took advantage of the finding that the phenol group to the MA14 could be modified. In particular, I designed and synthesized several compounds based on the MA14 structure that released substituted phenols in the presence of ROS. It is shown that the oxidation mechanism is influenced by the electron density of the phenol. Further, we found that these compounds could selectively target AML cells while the normal cell can be surviving. To further develop the concept, we designed and synthesized prodrugs, termed self-cyclizing reagents, w (open full item for complete abstract)

Committee: Edward Merino Ph.D. (Committee Chair); Patrick Limbach Ph.D. (Committee Member); David Smithrud Ph.D. (Committee Member) Subjects: Biochemistry

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2019, Photochemical Sciences

Ever since the discovery of the DNA molecular structure, this molecule became a desirable target for the researchers aiming to develop anticancer drugs and therapies. The main emphasis of this dissertation was placed on the design, development and studies of photochemical properties of a novel photo activated DNA damaging agent. Upon light irradiation of compound the release of highly reactive 9,10-phenanthrenequinone was observed. Studies of phenanthrene dihydrodioxin interaction with DNA demonstrated the intercalative mode of binding and slightly preferential binding affinity to the AT-rich DNA sequences. The synthesized agent induced a partial transition from B to Z form of DNA. The phenanthrene dihydrodioxin compound proved to be an efficient single-strand DNA photocleaver upon visible light irradiation. This further demonstrates the potential of photomasked ortho-quinones as efficient DNA damaging agents. This work also describes an unusual transformation from pyridinium substituted methane to the corresponding gem-diol in the mild conditions in the presence of air. Proposed mechanism of the reaction involves the formation of reactive oxygen species (ROS). ROS are known to be highly toxic to biomolecules, including DNA. It is demonstrated in this work that the production of ROS intermediates in the reaction of the gem-diol formation can lead to DNA damage in the dark. In the continuous interest of our research group, a series of bis(pyridinium) and bis(3-carboxamidepyridinium) alkane salts were synthesized in order to study through-space electron delocalization and formation of dimer radical cation species upon electrochemical and photoreduction. Investigation of photoinduced charge separation is important for the development of artificial photosynthetic systems and molecular electronics. Studies of unsubstituted and meta-substituted pyridines connected by the alkyl linker of different length provided additional information on the efficiency of the pyridi (open full item for complete abstract)

Committee: R. Marshall Wilson Prof. (Advisor); Farida Selim Prof. (Other); Pavel Anzenbacher Prof. (Committee Member); Alexander Tarnovsky Prof. (Committee Member) Subjects: Biochemistry; Chemistry; Organic Chemistry; Physical Chemistry

Doctor of Philosophy, University of Toledo, 2018, Biology (Cell-Molecular Biology)

Silicon (Si) supplementation can provide plants with several benefits: increased photosynthetic activity, crop yield, and resistance to abiotic and/or biotic stress. While most research examining these benefits mainly focused on high Si accumulators such as certain monocotyledonous plants, low Si accumulators are thought not to benefit from Si supplementation. In the present study, I show that the low Si accumulator, Nicotiana tabacum, benefits from Si supplementation, although these effects were time dependent. Si supplementation reduces metal stress in N. tabacum propagated hydroponically under copper (Cu) toxicity after 3 weeks of treatment. Plants displayed increased shoot and root biomass almost to that of control. Furthermore, Si supplementation reversed the reduction in maximum quantum yield of photosystem II caused by Cu toxicity. However, Si supplementation showed no beneficial effect on growth after 1 week in Cu-treated plants but did affect other plant processes. The foliar tissue of Si-supplemented plants under Cu toxicity accumulated more Si than plants provided with Si alone after 1 week. In this work, I have termed this phenomenon stress-induced Si accumulation (SISA), and similar responses in N. tabacum infected with Tobacco ringspot virus have been reported. These data suggest that foliar SISA may be part of the N. tabacum stress response. I observed SISA in roots but only after three weeks of treatment, which corresponded with recovery from Cu toxicity. Silicon might influence Cu toxicity by affecting metal uptake, chelator expression, redox homeostasis, and phytohormone signaling. Therefore, I investigated these processes via a transcriptomic analysis. I found that Si-mediated alleviation of Cu toxicity corresponded to reduced mRNA expression of COPT1, the major Cu transporter in roots. This could, in part, play a role reducing root Cu concentrations as observed in Si-supplemented plants under Cu toxicity conditions after 3 weeks of (open full item for complete abstract)

Committee: Scott Leisner Ph.D. (Committee Chair); Lirim Shemshedini (Committee Member); John Gray (Committee Member); Donald Ronning (Committee Member) Subjects: Molecular Biology; Plant Biology

PhD, University of Cincinnati, 2018, Arts and Sciences: Chemistry

Anticancer agents that modify DNA are a mainstay of chemotherapy regimens, but development of new classes of these agents has slowed because of the modifications of DNA in non-cancerous cells. This is what gives rise to serious side effects via poor selectivity. The Merino Lab has developed a pro-drug strategy to achieve specificity by translating the finding that levels of reactive oxygen species (ROS) are elevated in cancers, such as Acute Myeloid Leukemia (AML). This pro-drug approach allows cellular ROS to oxidize the pro-drug into its active form to achieve selective cytotoxicity. Our current lead agent (A100) is shown to have 10-fold selectivity between AML cells over normal CD34+ blood cells in vitro and showed some efficacy in the in vivo AML mouse model. This work started with computational analysis to determine what parts of the molecule were a target for metabolic enzymes. The first step taken to improve the molecule was to add polyethylene glycol (PEG) to the free phenol, increasing both its solubility and metabolic stability. To prove metabolic stability, binding assays against CYP1A2 (Cytochrome P450, Isoform 1A2) were done, as CYP1A2 is known to attack alcohols. The synthetic addition of the PEG increased stability against CYP1A2 by almost 50%. To prove stability in a more complex matrix, total stability was measured via half-life in pooled human liver microsomes. The PEGylated compound (A100-PEG) showed a 7-fold increase in the half-life of A100, as compared to A100 alone. A100 alone was not detectable in mouse blood samples after 15 minutes; however, in all three mouse models, A100-PEG was detectable even after 100 minutes and was calculated to have a 6-fold increase in half-life, as compared to A100. Though stable, A100-PEG only showed similar efficacy in the in vivo AML mouse model; however, an IV injection was used instead of the previous IP injection. Having improved its solubility and metabolic stability, the next step was to improve agen (open full item for complete abstract)

Committee: Edward Merino Ph.D. (Committee Chair); Patrick Limbach Ph.D. (Committee Member); Laura Sagle Ph.D. (Committee Member) Subjects: Pharmaceuticals

MS, Kent State University, 2018, College of Arts and Sciences / School of Biomedical Sciences

Epidemiological studies have given rise to possible etiologies of Parkinsons disease (PD), especially farmers exposed to toxicants. The herbicide paraquat is similar in structure to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and redox cycles with microglial NADPH-oxidase to form reactive oxygen species (ROS). Excessive ROS and microglial activation can damage neurons and is thought to contribute to the initiation and progression of neurodegenerative diseases, including PD. Hv1 is a voltage-gated proton channel selectively found in microglia and other immune cells where it regulates NADPH-oxidase-dependent production of ROS. Deletion of Hv1 in mice has been found to protect neurons from oxidative damage during ischemic stroke, but there have been no studies of Hv1 in PD. The purpose of this study was to determine the role of Hv1 in paraquat-induced neuroinflammation. In vitro studies with the BV2 mouse microglial cell line and primary microglia demonstrated that while paraquat does not directly activate pro-inflammatory microglia, it induces Hv1 gene and protein expression by 2-fold and 60% respectively. Direct paraquat treatment also resulted in a 25% increase in ROS production both in the BV2 microglial cell line and C57 primary microglia. However, Hv1KO (Hv1 knockout) primary microglia show complete abolishment of paraquat-induced ROS production. Direct paraquat treatment was further shown to prime the microglial NLRP3 inflammasome via up-regulation of NLRP3 and IL-1beta gene expression allowing microglia to become sensitized to further insults. This paraquat-induced priming effect was shown to be abolished in Hv1KO microglia. To assess the effect of Hv1 in the microglial response to paraquat-induced neuronal damage, we exposed the BV2 mouse microglia cells and primary microglial cells from C57 and Hv1KO mice to conditioned media from N27 dopaminergic neuronal cells treated with paraquat. The paraquat-treated N27 conditioned me (open full item for complete abstract)

Committee: Jason Richardson (Advisor); Samuel Crish (Committee Member); Sheila Fleming (Committee Member) Subjects: Biomedical Research; Neurosciences

Doctor of Philosophy, The Ohio State University, 2018, Biomedical Sciences

Pseudomonas aeruginosa is an opportunistic pathogen that causes devastating, chronic pulmonary infections in patients with cystic fibrosis (CF). During persistent infection of the CF lung, P. aeruginosa acquires adaptive mutations that confer resistance to antimicrobials and host responses. Most strikingly, mutation of mucA results in the conversion of initially colonizing nonmucoid strains to the mucoid phenotype, which is defined by overproduction of the exopolysaccharide, alginate. Though mucoidy provides advantages to P. aeruginosa in withstanding environmental pressures within the airway, mucoid strains often revert back to a nonmucoid phenotype in vitro and in vivo . Importantly, mixed populations of both mucoid and nonmucoid variants are often isolated from chronically-infected CF patients, suggesting a selective advantage for the coexistence of these variants within the host. In Chapter 2, we report that within mixed-variant communities, P. aeruginosa exhibits enhanced resistance to innate immune effectors, LL-37 and hydrogen peroxide (H2O2). Immune evasion is mediated by the production and sharing of “public goods” by both P. aeruginosa variants: While mucoid constituents provide protection from LL-37 via alginate production, nonmucoid revertants shield the population from (H2O2) via catalase (KatA). We further demonstrate that katA expression is negatively regulated by AlgT and AlgR, two transcription factors that are essential for alginate biosynthesis. Additionally, we provide evidence that an endolysin encoded by lys, which is implicated in P. aeruginosa autolysis and extracellular DNA release, is also responsible for catalase release from nonmucoid revertants. Given these findings, we wanted to better understand how mixed-variant P. aeruginosa communities interact with the host in vivo. Tissue damage to the CF lung is heterogeneously manifested across the organ, wherein the upper lobes of the lung are typically more damaged than the lower lobes. (open full item for complete abstract)

Committee: Daniel J. Wozniak PhD (Advisor); Kevin M. Mason PhD (Committee Chair); Amal O. Amer MD/PhD (Committee Member); Santiago Partida-Sanchez PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy (PhD), University of Toledo, 2017, Experimental Therapeutics

Ischemic stroke is caused by a blockage of the blood flow to the brain resulting in neuronal and glial hypoxia leading to inflammatory and free radical-mediated cell death. Reactive oxygen species (ROS) formed in excess under hypoxic conditions cause protein, DNA and lipid oxidation. Nitric oxide (NO) formed by NO synthase (NOS) is known to be protective in ischemic stroke, however NOS has been shown to `uncouple' under oxidative conditions to instead produce superoxide. Nitrones are antioxidant molecules that are shown to trap ROS to then decompose and release NO. In this study, the PBN-type nitrone 5 was designed such that its decomposition product is a NOS inhibitor, effectively leading to NOS inhibition specifically at the site of ROS production. The ability of 5 to spin-trap radicals and decompose into the putative NOS inhibitor was observed using EPR and LC-MS/MS. The pro-drug concept was tested in vitro by measuring cell viability and inhibitor formation in SH-SY5Y cells subjected to oxygen glucose deprivation (OGD). 5 was found to be more efficacious and more potent than PBN, and was able to increase pAkt while reducing nitrotyrosine and cleaved caspase-3 levels. Doppler flowmetry on anesthetized mice showed an increased cerebral blood flow upon intravenous administration of 1 mg/kg 5, but a return to baseline upon administration of 10 mg/kg, likely due to its dual nature of antioxidant/NO-donor and NOS-inhibition properties. Mice treated with 5 after permanent middle cerebral artery occlusion (pMCAO) performed better in neurobehavioral assessments and exhibited a > 30% reduction in infarct volume. This efficacy is proposed to be due to higher formation of the NOS inhibitor decomposition product in ischemic tissue observed by LC-MS/MS, resulting in region specific effects limited to the infarct area.

Committee: Zahoor Shah Ph.D. (Committee Chair); Isaac Schiefer Ph.D. (Committee Member); F. Scott Hall Ph.D. (Committee Member); Wissam AbouAlaiwi Ph.D. (Committee Member) Subjects: Biochemistry; Neurobiology; Neurosciences; Organic Chemistry; Pharmacology; Physical Chemistry