Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2019, College of Sciences and Health Professions

Nitric oxide (NO) is an important physiological metabolite implicated in both health and disease states. Accurate determination of NO in tissues and cells is of paramount importance. The major challenges with NO measurement are its short half-life and often low nanomolar concentrations in tissues. In this work, we explore the performance of combined reference/working electrodes modified by a layer by layer method of electrodepositing transition metal ruthenium oxide on polymer supports for catalytic determination of NO at the level of single or collective cultured cells. We have characterized our sensors using electron microscopy, elemental analysis, and electrochemical characterization. We were able to confirm the successful modification of the carbon fiber surface with the transition metal and conductive polymers using surface characterization techniques. We were able to evaluate the sensor performance using potentiostatic techniques. Our sensor exhibited excellent sensitivity, linearity and response time as well as allowing for a very low limit of detection for NO making them ideal for detecting NO in biological samples. We tested the performance of our sensors in Madine Darby Canine Kidney (MDCK) epithelial cells and macrophage cell lines. Stimulation of the nitric oxide synthase enzyme was used to accurately detect NO released from MDCK cells as well as study the time course of NO release from the macrophage cells. Further confirmation of NO release was performed by fluorescence imaging using a probe that binds specifically to NO. Using this dual methodology for NO detection, we were able to achieve a temporal and spatial resolution of NO release from cell lines. The clinical significance of this work has been to detect and compare NO released directly from normal and Cystic Fibrosis (CF) cell line models. It has been found that exhaled NO levels remain unchanged or reduced in CF patients unlike other inflammatory lung diseases like asthma where it inc (open full item for complete abstract)

Committee: Mekki Bayachou Ph.D. (Advisor); David Anderson Ph.D. (Committee Member); John Turner Ph.D. (Committee Member); Yana Sandlers Ph.D. (Committee Member); Andrew Resnick Ph.D. (Committee Member) Subjects: Analytical Chemistry; Chemistry

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2009, College of Science

Nitric oxide (NO) is an important intercellular messenger that acts in many tissues to regulate a diverse range of physiological and pathological processes. The physiologically implications of NO function are far from being completely understood. The multifaceted reactivity of NO prompted the need for accurate determination of the concentration of this molecule. However, it is difficult to detect nitric oxide, particularly in biological media and near live cells due to its short half-life, a result of its reactivity and the low levels of NO produced in vivo. As a result, the accurate and reliable detection of NO under varying experimental conditions has always posed a challenging task. The main goal was to develop ultra-sensitive electrocatalytic sensors for accurate quantification of NO. We report the fabrication and characterization of improved NO sensors based on electrocatalytic platforms such as ruthenium (colloids, nanoparticles, and nanotubes) and carbon (pastes and nanotubes), acting as catalytic sites for NO oxidation. These sensors are characterized using various surface analytical tools. The electrocatalytic oxidation of NO is assessed by cyclic voltammetry and amperometry both in solution phase and gas phase. Excellent sensitivity and linearity are observed for our modified electrodes towards NO quantification. Our new NO detection sensors also show superior limit of detection and selectivity against common interference species. Our NO sensors are tested for various applications including in the measurement of NO released from human umbilical vein endothelial cells (HUVECs).

Committee: Dr. Mekki Bayachou PhD (Advisor); Dr. Lily Ng PhD (Committee Member); Dr. Robert Wei PhD (Committee Member); Dr. John Turner PhD (Committee Member); Dr. Petru Fodor PhD (Committee Member) Subjects: Chemistry

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

The cardiovascular system is mainly regulated by nitric oxide (NO). A reduction in its synthesis or bioavailability might underlie the impaired endothelium-dependent vasodilatation, which is observed in the blood vessels of individuals with cardiovascular disease (CVD). The dysfunction of endothelium, which is a main characteristic of vascular aging, has been associated with low NO production and high production of cytotoxic peroxynitrite (ONOO-). Thus, the ratio of NO to ONOO- is an indicator of endothelial dysfunction. Moreover, vascular NO is produced by an enzyme called (endothelial nitric oxide synthase (eNOS), and its gene exhibits high polymorphism. However, it is unclear whether polymorphisms or haplotypes in the eNOS gene affect the NO production, ONOO- production, and eNOS coupling, as well as how aging impacts these haplotypes. The influence of the eNOS haplotype (consisting of single nucleotide polymorphisms (SNP) in the promoter region (T-786C) and (C-665T) and exon 7 (Glu298Asp) and a variable number of tandem repeats (VNTR) in intron 4 (4a/4b/4c)) on the production of NO and ONOO- and eNOS coupling was investigated. Sanger sequencing and DNA electrophoresis were used to detect SNPs and VNTRs in the samples, respectively. To evaluate the production of NO and ONOO- , nanosensors were used to determine the maximal concentrations of NO and ONOO- and traditional and low-temperature SDS-PAGE to evaluate the expression of eNOS and the eNOS dimer/monomer ratio, respectively. Interestingly, these results indicated that the eNOS haplotype (H5) combining the “T T/C C 4 4b” of the G894T, T-786C, C-665T, and 27 bp VNTR a/b/c is more susceptible to endothelial dysfunction. Compared with other haplotype samples, it had lower [NO]/[ONOO-] and higher eNOS expression with reduced eNOS dimer/monomer (P < 0.005). These findings have important implications for understanding the genetic basis of cardiovascular disease and aging and may lead to new (open full item for complete abstract)

Committee: Tadeusz Malinski (Advisor); Howard Dewald (Advisor); Michael Held (Committee Member); Katherine Cimatu (Committee Member); Faiz Rahman (Committee Member) Subjects: Analytical Chemistry; Biochemistry; Biomedical Research; Genetics; Molecular Biology; Nanotechnology

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

Doctor of Philosophy (PhD), Ohio University, 2015, Chemistry and Biochemistry (Arts and Sciences)

Hypertension can be triggered by abnormal concentration of angiotensin-II; and diabetes mellitus is induced by hyperglycemia. Both hypertension and diabetes mellitus show adverse effects on endothelial function. Dysfunctional endothelium may produce less bioavailable NO and high level of oxidative stress. We hypothesized that endothelial dysfunction may be reversed by 1α,25-Dihydroxyvitamin D3. Using nanomedical methods and techniques we elucidated a multifunctional effect of 1α,25-Dihydroxyvitamin D3 on dysfunctional endothelium in cellular model of hypertension and diabetes mellitus. 1α,25-Dihydroxyvitamin D3 increased NO bioavailability and reduced nitroxidative stress by coupling eNOS dimer and downregulating expression of NADPH oxidase 4, eNOS, and iNOS in cultured HUVEC's. NO and ONOO- concentrations were measured in near real-time with nanosensors in HUVEC's of Caucasian American (CA) and African American (AA). ELISA was performed to measure the expression of eNOS, iNOS and NADPH oxidase 4 in HUVEC's of CA and AA. Exposure of HUVEC's to elevated concentration of angiotensin-II [1 µmol/L] and D-glucose [250 mg/dL] for 1-4 hours caused endothelial cells dysfunction manifested by eNOS uncoupling, reduced NO bioavailability and increased ONOO- concentration. Both angiotensin-II and D-glucose upregulated eNOS, iNOS and NADPH oxidase 4 expression level in HUVEC's of both ethnic groups. 1α,25-Dihydroxyvitamin D3-treatments of dysfunctional HUVEC's reduced eNOS, iNOS and NADPH oxidase 4 expression levels followed by significant reduction in ONOO- release and induction in NO bioavailability indicating that NADPH oxidase 4 plays a major role in endothelial dysfunction. The NADPH oxidase 4 role in endothelium dysfunction was confirmed by the use of NADPH oxidase inhibitor, VAS2870 [10 µmol/L]. VAS2870 increased NO bioavailability and reduced ONOO- release. The ratio of NO to ONOO- concentrations, [NO]/[ONOO-], was used as an indicator of eNOS coupling/uncoupling and (open full item for complete abstract)

Committee: Tadeusz Malinski (Advisor) Subjects: Biochemistry

Master of Science, University of Toledo, 2014, Exercise Science

This study examined pulmonary function, ventilation, exercise tolerance and the fractional concentration of exhaled nitric oxide (FeNO) in healthy controls (CON; n=7) and asthmatics (ASTH; n=7) following two-weeks of high intensity interval training (HIIT). An increase in FeNO above basal levels is associated with lung inflammation and is gaining popularity as a reliable diagnostic tool for the treatment of asthma. PURPOSE: The extent that HIIT decreases FeNO and thus, exercise tolerance in asthmatic subjects has not been previously investigated. METHODS: Pulmonary function was assessed using peak expiratory flow [PEF], forced vital capacity [FVC], and the ratio of forced expiratory volume in one second to FVC [FEV1/FVC]. FeNO was measured noninvasively using a NIOX portable device. Both groups performed a progressive exercise test (20 W/min) on a cycle ergometer to determine peak values of O2 uptake (VO2peak), CO2 output (VCO2peak), ventilation (VE) and time to exhaustion (TTE). Each subject completed six sessions of HIIT, which included 8 minutes of loadless cycling warm-up, 10 x 60 s bouts of exercise interspersed with 60 s periods of recovery at a low intensity of cycling. The intensity of the HIIT protocol was set at a target equivalent to 75% peak WR for ASTH and 80% peak WR for CON. Each training session was followed by 5 minutes of loadless cycling cool down. Following completion of all HIIT sessions, each subject repeated the pulmonary and exercise tests that were performed prior to training. A two-way analysis of variance with repeated measures (ANOVA-RM) was used to examine main effects, group (CON vs. ASTH) and time (Pre- vs. Post-HIIT) and significant interactions (Group x Time). A Student Newman Kuels post hoc test was used to determine specific differences as appropriate. Statistical significance was set at p < 0.05 RESULTS: Significant differences were found between the groups in FeNO, VO2peak, TTE, and peak WR. No differences were found between or w (open full item for complete abstract)

Committee: Barry Scheuermann (Committee Chair); Craig Black (Committee Member); Suzanne Wambold (Committee Member) Subjects: Anatomy and Physiology; Experiments; Health; Health Sciences; Kinesiology

PhD, University of Cincinnati, 2008, Medicine : Epidemiology (Environmental Health)

Asthma is the most common chronic and disabling disease of childhood. Unfortunately, there are limited tools for primary care asthma management, and these tools are poor predictors of a patient's disease status, airway inflammation, and future disease. The first part of the dissertation reviews the inflammatory biomarkers that are currently being investigated as potential tools to assess airway inflammation in children with asthma. Of these exhaled markers, the most data is available for exhaled nitric oxide (FeNO). The second part of the dissertation details an evaluation of the association of environmental exposures and FeNO levels among children with asthma. Higher baseline FeNO levels, atopy, and fall season were associated with increased FeNO levels, measured 6 and 12 months after study initiation, whereas inhaled steroid use, summer season, and increasing nicotine exposure were associated with lower FeNO levels. This suggests that FeNO is responsive to some of the same moderators and risk factors associated with asthma control. Genetic differences influence the susceptibility and response to environmental exposures. The third part of the dissertation provides information on basic genetic epidemiology. This information will help interpret the fourth part of the dissertation, an exploration of the association of nitric oxide synthase genes (NOS) and environmental exposures with FeNO levels among children with asthma. There was no association of genetic polymorphisms in NOS1 or NOS3 with FeNO levels in this cohort. Individuals with the GT or TT genotypes of NOS3 had decreased FeNO when exposed to nicotine. The differing genetic susceptibilities may explain some of the conflicting results in studies that evaluated the effects of tobacco exposure on FeNO levels without considering genetic differences. Together these findings suggest that FeNO holds promise for use in the management of asthma in children.

Committee: Bruce Lanphear MD, MPH (Committee Chair); Ralph Buncher ScD (Committee Member); Richard Hornung DrPH (Committee Member); Michelle Lierl MD (Committee Member); Robert Rinsky PhD (Committee Member) Subjects: Environmental Science; Epidemiology; Health Care

PhD, University of Cincinnati, 2005, Pharmacy : Pharmaceutical Sciences

Intestinal P-glycoprotein (P-gp) expression and luminal nitric oxide (NO) levels are significantly greater in patients with refractory inflammatory bowel disease (IBD). We investigated whether the proinfammatory cytokine IFN-γ, the transcription factor NF-κB, and the signaling intermediate NO regulate the P-gp encoding ABCB1 gene using the Caco-2 cells as an in vitro model of human intestinal epithelial cells. To identify cytokine-mediated activation of signal transduction pathways, phosphorylation of JAK-2, cytosolic IκBα degradation and nuclear binding of NF-κB were determined in Caco-2 cells stimulated with 10 ng/ml of IFN-γ using immunoblot analysis and electrophoretic mobility shift assays, respectively. Changes in ABCB1 mRNA were measured by realtime PCR, total cellular P-gp protein was quantitatively assessed using immunoblot analysis, and digoxin uptake was used to determine P-gp efflux activity at the apical membrane. To further evaluate underlying molecular pathways, similar experiments were performed using Caco-2 cells expressing phosphorylation-deficient IκBα (Caco-2/ NF-κB-/-) in the presence or absence of the NF-κB inhibitor parthenolide (25 iM), the iNOS inhibitor L-NIL (1 mM), and the NO donor SNAP (0.1-5 mM). Transactivation of the ABCB1 promoter was determined using a 1 kb ABCB1-luciferase construct that contained a functional or mutated NF-κB response element. Intracellular NO levels were determined by the Griess reaction. IFN-γ stimulation of Caco-2 cells enhanced phosphorylation of JAK-2, cytosolic degradation of IκBα, increased nuclear binding of NF-κB, and augmented NO production by 9-fold. In parallel, ABCB1 promoter activity was increased by 4-fold, ABCB1 mRNA by at least 2-fold, total cellular P-gp protein expression by 2-fold, and digoxin uptake reduced by ~40%. Significant NF-κB activation, NO generation, and changes in ABCB1 transcription and P-gp function were absent in Caco-2/NF-κB-/- or parental Caco-2 cells coincubated with parthenol (open full item for complete abstract)

Committee: Giovanni Pauletti (Advisor) Subjects: Health Sciences, Pharmacy

Doctor of Philosophy, The Ohio State University, 2007, Biophysics

EPR oximetry is a technique based on the paramagnetic distinctiveness of molecular oxygen. It is a non-consuming technique that allows repeated measurements of low levels of oxygen. Using ~10 µl of sample, molecular oxygen from the oxygen-induced EPR line broadening of a suitable paramagnetic probe such as lithium phthalocyanine (LiPc) may be determined. In the first portion, a new quantitative methodology for cellular respiration using EPR oximetry is established; where three phases of respiration can be observed: pO2-independent respiration, pO2-dependent respiration and a static equilibrium. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. Following, we introduce the application of the developed EPR procedure to study NO-regulated respiration. We conclude that at a low pO2, the reduction of respiration by endogenous NO does not occur through direct reversible inhibition of cytochrome c oxidase (CcO) at complex IV, by competing with O2. Lastly, heat shock protein 90 (Hsp90) and endothelial nitric oxide synthase (eNOS) are evaluated, based on their influential role on cellular respiration in various pathophysiological conditions such as heat shock, hypoxia and hyperglycemia. A mild heat shock increases the Hsp90-eNOS complex. The association of Hsp90 and eNOS enhances the production of NO, leading to a reduction in respiration. Exposure to extreme hypoxia can also minimize cellular respiration. It was hypothesized that the p50 increases in the presence of NO; however, we have found that the p50 remains unchanged. Similarly, the Hsp90-eNOS interaction enhances during hyperglycemia (up to 4 h). At a longer exposure, the Hsp90-eNOS complex diminishes. During a moderate hyperglycemic treatment (~8 h), the maximum O2 consumption rate is attenuated; where the p50 was unaffected. At longer exposures, this behavior is not present. To enhance the presence of the Hsp90-eNOS complex, heat shock was applied pr (open full item for complete abstract)

Committee: Govindasamy Ilangovan (Advisor) Subjects: Biophysics, Medical

Doctor of Philosophy (PhD), Ohio University, 2007, Molecular and Cellular Biology (Arts and Sciences)

The aim of this work was to study an imbalance between nitric oxide (NO) bioavailability and nitroxidative stress during intracerebral hemorrhage stroke (ICH). Microliters of blood, platelets and leucocytes were injected directly into the caudate putamen of Sprague-Dawley Kyoto rats. Nitric oxide, superoxide (O 2 . -),and peroxynitrite (ONOO -) were measured with nanosensors implanted into the caudate putamen. After injection of 5-50 microliters of blood, significant decrease in NO concentration was recorded at 6 hour post stroke and resulted in excessive clot formation. The concentrations of both O 2 . -and ONOO -reached their maxima 12 hours after ICH. A main sources of O 2 . -and ONOO -were uncoupled endothelial (eNOS) and neuronal (nNOS) nitric oxide synthases. As a consequence, endothelial dysfunction as well as defective circulation of platelet and leucocytes were observed. ICH modeling performed with the injection of platelet, resulted in a larger infarct size than that observed after the injection of the whole blood or leucocytes. ICH induced nitroxidative stress caused differences in enzymatic expression. Maximal expression of eNOS and nNOS were shifted in time (1 hour and 3 hour post-stroke, respectively). Expression of MnSOD was the highest at 12 hour post-stroke, and correlated positively with a highest concentration of ONOO -and protein nitration. L-arginine treatment before ICH resulted in decrease of ONOO -concentration, increase in bioavailable NO and improved the locomotor function in post-stroke animals. The increase in NO bioavailability by exogenous L-arginine and decrease in ONOO -associated with improved coupling of constitutive (eNOS and nNOS) nitric oxide synthases. Therefore, a supplementation of L-arginine may be of significance in treatment of ICH.

Committee: Tadeusz Malinski (Advisor) Subjects:

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2009, College of Sciences and Health Professions

Nitric oxide (NO) is a freely diffusible, gaseous free radical, associated with many physiological and pathological processes that include neuronal signaling, immune response, and inflammatory response. NO is produced from L-arginine in an NADPH-dependent reaction catalyzed by a family of nitric oxide synthase (NOS) enzymes. A deficiency in NO plays a role in hypertension, hyperglycaemia, and arteriosclerosis, among other pathological states. Conversely, increased NO levels contribute to arthritis, septic shock, and hypotention. Therefore, measuring and quantifying NO production in biological systems and matrices may be vital in elucidating physiological and pathological processes. The goal of this work is to develop an ultra-sensitive and selective electrochemical sensor taking advantage of NO-sulfur chemistry. In particular, electropolymerizing 3,4-ethylenedioxythiophene (EDOT) monomers on the surface of our electrodes yield a suitable sulfur-based polymer PEDOT to be used as an affinity matrix for NO sensing. In other work, we have shown that the ruthenium (Ru) mediates the catalytic oxidation of NO. In this work, we tried to achieve improved sensitivity by combining both Ru nanoparticles and PEDOT using the layer-by-layer (LBL) modification method. Further, to eliminate interferences the Ru-PEDOT-Ru modified carbon fiber was coated with a nafion layer, which acts as an anionic filter. We used our NO-sensor to accurately monitor NO release from mouse embryonic fibroblast cells as well as isolated single human umbilical vein endothelial cells. A second part of this work focused on testing the performance of our sensors in characterizing NO release from liposomes with confined NOS enzyme. Liposomes are spherical, closed, self-assembled phospholipids, which enclose part of the surrounding solvent in their interior. Liposomes can enclose an aqueous medium separate from the external aqueous medium. Therefore, liposomes can be used as carriers of enzymes (NOS in this (open full item for complete abstract)

Committee: Mekki Bayachou (Advisor); Robert Wei (Committee Member); Alan Riga (Committee Member); Xue-Long Sun (Committee Member); Orhan Talu (Committee Member) Subjects: Chemistry

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2009, College of Science

Nitric Oxide releasing biopolymers have the potential to prolong vascular graft and stent potency without adverse systemic vasodilation. It was reported in literature that eNOS-overexpressing endothelial cell seeding of synthetic small diameter vascular grafts decreased human platelet aggregation by 46% and bovine aortic smooth muscle cell proliferation by 67.2% in vitro. We hypothesized that incorporating the enzyme nitric oxide synthase (NOS) in biocompatible polymeric matrix will provide a source of NO that utilizes endogenous compounds to maintain an unlimited supply of NO. To test this hypothesis, we have incorporated the enzyme nitric oxide synthase into a polyethyleneimine film using a layer-by-layer electrostatic deposition. This approach will provide a source of NO that utilizes endogenous compounds available in the blood matrix to maintain a constant supply of NO at the blood/device interface. When coated onto the surface of various blood-contacting implantable medical devices, it will provide NO fluxes at levels equal or greater than the normal endothelial cells, and for extended time periods. This configuration will help solve the issues of both thrombosis and stenosis that occur as side effects for several types of biomedical implants. Our results indicate a proof of principle of a new approach for making antithrombotic coatings for medical devices and implants based on NO release. We have demonstrated that NOS-based polymetric films successfully generate NO under physiologic conditions at small levels equal to and higher than those observed for endothelial cells. The level of NO release can be fine-tuned through varying the number of NOS layers in the film buildup. We have shown that NO fluxes from our NOS-based PEI films are sustained for prolonged periods of time, which has the potential of producing efficient, short and long-term, antithrombotic coatings for medical devices and blood-contacting tools such as stents and catheters. We also show that N (open full item for complete abstract)

Committee: Mekki Bayachou PhD (Advisor); David Anderson PhD (Committee Member); Xue-Long Sun PhD (Committee Member); Yan Xu PhD (Committee Member); Gary Wnek PhD (Committee Member) Subjects: Analytical Chemistry; Biomedical Research; Chemistry; Polymers

Doctor of Philosophy, University of Akron, 2008, Chemistry

In general, a dressing is used to protect and help heal wounds. There are several types of dressings on the market such as hydrocolloid, hydrogel, and medicated dressings. One technique for making a dressing is electrospinning, which is a very simple procedure used to produce fibers. Due to much smaller fiber-diameters than produced with the conventional technique, the fibers from electrospinning have unique properties: high porosity and high surface areas, which are advantageous for wound healing. In this research, the fibers were electrospun using polyurethane, Tecophilic® or Tecoflex®, with various additives. First, multilayer transdermal electrospun dressings, four and five-layers, were developed that allowed for the controlled release of nitric oxide (NO) from a NO2-/ascorbic acid system encapsulated in polymer nanofibers. The amount of NO released from each wound dressing was investigated. Both the four and five-layer dressings were tested for morphology of fibers, water absorption, nitrite distribution, NO release profile after sterilization by gamma radiation, and stability. In the case of the four- layer transdermal dressing, the dressing was tested in diabetic, periodontal, and leishmaniasis patients. Furthermore, the color change of dressing was investigated. Tecophilic® was also spun with an antimicrobial agent, which was added to the Tecophilic® solution to electrospin an antimicrobial dressing. The morphology of fibers was tested using an optical microscope and the water absorption, uniformity, and percent extraction of dressing were also determined. In addition, the efficiency of the antimicrobial agent in the dressing was determined according to SN 195920-1994 and ASTM 2149-01. Another NO-releasing dressing was developed employing the NO donor molecules, sodium salt of linear polyethlenime NONOates (LPEINO-Na) and calcium salt of linear polyethlenime NONOates (LPEINO-Ca), which were electrospun with Tecophilic®. The NO release profiles for the LPE (open full item for complete abstract)

Committee: Daniel J. Smith Ph.D. (Advisor) Subjects: Biochemistry; Biomedical Research; Chemistry; Materials Science; Polymers

Doctor of Philosophy, The Ohio State University, 2012, Chemistry

This dissertation involves the study of the interaction of carbon monoxide (CO) and nitric oxide (NO) on derivatives of low temperature conducting metal oxides, ruthenium and vanadium oxides. The interactions of these gases with the metal oxides lead to changes in conductivity which show promise for possible applications as a new class of resistive based ambient gas sensors that alleviate the current limitations of CO and NO sensors that operate at elevated temperatures. These sensors are based on hydrated ruthenium oxide (RuOx(OH)y) and vanadium pentoxide (V2O5) . RuOx(OH)y was prepared a wet precipitation reaction involving ruthenium chloride with a base. This material was amorphous, made up of 20–50nm particles and contains Ru(III) and Ru(IV), as determined by XPS. Thick films were made of air and supercritical dried particles of RuOx(OH)y. The conductivity of these films decreased in the presence of CO in air and this change was reversible. Infrared spectroscopy showed the formation of carbonates and water in the presence of CO, which disappeared upon replacement of CO with air. Upon thermal treatment of RuOx(OH)y above 200°C, a decrease in the conductivity change in the presence of CO at room temperature is observed. These changes were accompanied by a conversion of the amorphous RuOx(OH)y to a crystalline RuO2 and consequently a conversion of Ru(III) to Ru(IV). This dissertation proposes the oxidation of CO on RuOx(OH)y leads to reduction of the ruthenium and subsequently a decrease in conductivity of the thick films. With the conversion to crystalline RuO2, the material becomes metallic and conductivity changes are diminished. Changes in RuOx(OH)y conductivity with CO provides an opportune platform for an ambient CO sensor. The interferences from ambient concentrations of hydrocarbons, ammonia, CO2, NO and NO2, were shown to have no effect on the conductivity . This dissertation also discusses the study of the interaction of NO with vanadium oxides. The V (open full item for complete abstract)

Committee: Prabir Dutta (Advisor); Sheikh Akbar (Committee Member); Susan Olesik (Committee Member) Subjects: Chemistry

Doctor of Philosophy, Miami University, 2024, Biology

This dissertation investigates the sexually dimorphic expression of the nos gene, which encodes for the enzyme Nitric oxide synthase (NOS). NOS activation produces the gaseous signaling molecule nitric oxide (NO), which can impact immune function and behavior. Despite the crucial role of NO in insect physiology, the mechanisms underlying nos expression in both sexes and in different social environments remain poorly understood. Our primary aim was to elucidate the impact that a lack of social interaction can have on nos expression and immune function, providing new insights into insect physiology and behavior. We cloned and characterized the Ad-nos gene, revealing a close phylogenetic relationship with other orthopteran insects. Quantitative PCR analysis showed that early adult females exhibited significantly higher Ad-nos expression in nervous and thoracic fat body tissues than males, while males had higher expression in hemolymph. Following lipopolysaccharide (LPS) injection, males showed increased Ad-nos expression in brain and hemolymph, whereas females showed elevated expression in fat bodies and hemolymph. These findings suggest sex-specific immune strategies, with males prioritizing cellular immunity and females employing both humoral and cellular responses. We also examined the effects of social interaction during different developmental stages on NO levels and immune function. Comparing adult-isolated and nymph-isolated crickets, we found significant sex-dependent differences in Ad-nos expression. Nymph-isolated females had higher Ad-nos expression in brain and hemolymph, while nymph-isolated males showed higher expression in brain fat body and thoracic fat body tissues. NO activity assays indicated lower activity in nymph-isolated crickets compared to adult-isolated crickets. In addition, nymph-isolated females had fewer circulating hemocyte than males, while males had higher hemolymph protein content. Survival analysis post-LPS injection showed that adult (open full item for complete abstract)

Committee: Kathleen Killian (Advisor); Dawn Blitz (Committee Member); Jennifer Quinn (Committee Chair); Yoshinori Tomoyasu (Committee Member); Paul James (Committee Member) Subjects: Biology; Immunology; Molecular Biology; Neurosciences

Master of Science, The Ohio State University, 1974, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, University of Toledo, 2023, Biology (Cell-Molecular Biology)

Millions of women across the world are affected each year with a breast cancer diagnosis. Depending at what stage the cancer is detected, the chance of survival is greatly influenced. While great improvements in technology have led to earlier detection of the cancer, hundreds of thousands of women die from this disease each year. Interestingly, many risk factors of this disease are linked to a decrease in production of the gaseous signaling molecule nitric oxide (NO). NO signaling could play a key role in maintaining a normal breast phenotype and when reduced could lead to an increase in breast cancer and progression. One way the presence of NO could protect against breast cancer formation is by controlling the activity of protein signaling, specifically the activity of transforming growth factor beta (TGFβ). TGFβ is an important signaling protein that has many vital roles in the body. Like many others, however, if the protein becomes overly active, the signaling cascade that follows could lead to serious consequences. TGFβ, along with all of its other roles, is responsible for the production of fibrous proteins. An overactivation of TGFβ could therefore lead to an increase in fibrous proteins and result in complications. The direct association between NO and TGFβ activity has not been clearly defined in the context of breast cancer and is the goal of this study. Previously, our lab has shown that decreasing the amount of NO in a mouse model led to an increase in phospho-Smad3 signaling, the signal transducer of TGFβ. To further elucidate these observations, the MCF10A progression series cell lines were utilized as a model throughout the investigation. In order to model an increase in NO in our experimentation, we used the NO donor S-nitroso-N-acetylpenicillamine (SNAP). To model an environment that has low NO, we used the nitric oxide synthase (NOS) inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME). Using these compounds allowed us to visualize how (open full item for complete abstract)

Committee: Saori Furuta (Committee Chair); Ivana de la Serna (Committee Chair); Dragan Isailovic (Committee Member); Yue Zou (Committee Member); Kam Yeung (Committee Member) Subjects: Biomedical Research; Cellular Biology

Master of Science (MS), Wright State University, 2023, Biochemistry and Molecular Biology

Duchenne muscular dystrophy (DMD) is a severe and progressive muscular dystrophy that develops in the skeletal muscles because of mutations in the dystrophin gene. Dystrophin stabilizes sarcolemma and assembles neuronal nitric oxide synthase (nNOS) into the dystrophin-associated protein complex on the sarcolemma. The absence of dystrophin triggers the delocalization of nNOS and contributes to the misregulation of muscle development, blood flow, muscle fatigue, and inflammation. Lipin1 was reduced in the skeletal muscles of patients with DMD and the mdx mouse model of DMD. In this study, we explored the role of lipin1 in the regulation of nNOS expression and function. We found that Lipin1 deficiency leads to the downregulation of nNOS protein and gene expression levels, while overexpression of lipin1 elevated nNOS protein and gene expression levels. We also found that Lipin1 upregulated nNOS gene expression by coactivating PPARα and binding to the promoter region of nNOS. Lipin1 deficiency leads to muscle fatigue in lipin1Myf5cKO mice, possibly due to the downregulation of nNOS. Most importantly, overexpressing lipin1 in dystrophic muscle improved muscle fatigue in our mdx:lipin1 transgenic mice which may be through the restoration of nNOS expression.

Committee: Hongmei Ren Ph.D. (Advisor); Michael Markey Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member) Subjects: Biochemistry; Biology

Doctor of Philosophy (PhD), Ohio University, 2023, Chemistry and Biochemistry (Arts and Sciences)

The regulation of various genes associated with cell cycle, proliferation, and apoptosis relies significantly on NF-κB (Nuclear Factor Kappa-light-chain-enhancer of activated B cells) transcription factors. These transcription factors are critical for the UV-induced production of ROS, which is linked to skin disease pathophysiology. Our previous study showed that UV radiation can activate NF-κB through the activation of constitutive nitric oxide synthase(s) (cNOS) in human keratinocyte HaCaT cells in the early phase (within 6 h) post-UVB. One of the upstream NF-κB proteins, I kappa B kinase alpha (IKKα), is also involved in the pathway post-UVB. However, the regulation of IKKα after UVB irradiation is not clear. In this study, I used HEK293 cells to study the role of cNOS as it is known for the cNOS-null cell line in comparison with the overexpression of cNOS in HEK293cNOS cells. My evidence showed that the expression of cNOS increases the IKKα promoter activities in the presence and absence of UVB. In addition, second p53 and ETS-1 binding sites that are elements in IKKα promoter are critical for maintaining the IKKα activity and UVB inducibility. In addition to the promoter, our data also demonstrated that UVB inhibits the elongation of IKKα mRNA transcription but not mRNA stability. These results indicated that while IKKα promoter is activated by UVB, the elongation inhibition of the mRNA synthesis limited the expression of IKKα. Later, we checked their protein levels in the presence and absences of cNOS both in vitro and in vivo. The protein level also confirmed the presence of cNOS can increase the basal level of IKKα in order to prevent skin tumor formation. In addition to IKKα, the effect of cNOS was also studied in skin aging and wrinkle using L-NAME, a selective inhibitor of NOS, with UV treatment. Our data indicated cNOS could play a protective role in skin aging post-UV irradiation through activation of PERK/GCNE and p-eIF2α. Meanwhile, cNOS KO (nNOS+/- (open full item for complete abstract)

Committee: Shiyong Wu (Advisor); Michael Held (Committee Member); Jixin Chen (Committee Member); Xiaozhuo Chen (Committee Chair) Subjects: Biochemistry; Molecular Biology

Doctor of Philosophy (PhD), Ohio University, 2023, Chemistry and Biochemistry (Arts and Sciences)

Stroke and heart attack are among the leading causes of death in the United States and worldwide. The high mortality rate is alarming given the wide availability of therapies for these illnesses. The mechanisms of action of these two diseases are remarkably similar, despite the fact that they are linked to two quite distinct organs in our body; in both, there is significant oxygen deprivation at the cellular level. Nitorooxidative stress occurs in physiological system due to the imbalance between Nitric Oxide (NO) and peroxynitrite (ONOO-). Using electrochemical nanosensors1,2 and a hypoxic chamber (which can control O2 from 21% to 0%), nitrooxidative stress in the endothelial and neural systems was studied, as well as two potential treatments (L-arginine and vitamin D3). Additionally, a connection was made between the protein HIF-1α and nitrooxidative stress. According to experimental data, L-arginine and vitamin D3 treatments can restore the [NO]/[ONOO-] balance in the endothelial system by up to 73 % and 69%, respectively. L-arginine and vitamin D can help the neural system's balance by up to 61% and 8%, respectively. Both L-arginine and vitamin D3 were also found to be effective in downregulation of HIF-1α in severe hypoxic conditions.

Committee: Tadeusz Malinski (Advisor); Michael Held (Committee Member); Katherine Cimatu (Committee Chair); Krisanna Machtmes (Committee Member); Howard Dewald (Committee Member) Subjects: Biochemistry; Chemistry