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  • 1. Alsulami, Seham Efficiency of Nitric Oxide and Peroxynitrite Release by Endothelial Nitric Oxide Synthase Variants - Implications for Cardiovascular Disease and Aging

    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)
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    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

  • 2. Rahman, Md Hasibur A Nanomedical Approach to Investigate and Mitigate Hypoxia-Induced Endothelial and Neural Damage: Clinical Implications for Heart Attack and Stroke

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

  • 3. Wagner, Michael The Nitroxidative Response to Traumatic Brain Injury

    Doctor of Philosophy (PhD), Ohio University, 2020, Chemistry and Biochemistry (Arts and Sciences)

    Traumatic brain injury (TBI) effects millions of Americans every year. Despite its relative commonality and frequency, there are no approved clinical treatments available for post-traumatic disease processes, which can lead to further pathologies such as post- traumatic epilepsy and chronic neurodegeneration. Further exacerbating the problem is the accidental nature of the insult. Thus, trying to understand the processes following mechanical insult and how they may develop into pathological processes is a promising general approach to developing new treatment strategies. Nitric oxide (NO), a small, gaseous messenger molecule, is released by both neural, glial, and vascular cells differentially following insult and is likely one of the most immediate responses the brain has to mechanical injury. Here, we use specialized nanonsensors implanted in mouse hippocampus to measure the real time in vivo production of NO and ONOO- following a modified controlled cortical impact injury. We then performed a proteomic analysis of S- nitrosylated proteins to examine the functional effects NO and ONOO- generated on the surrounding proteins. We found that post-traumatic NO and ONOO- generation has a significant impact on diverse cellular processes such as metabolism, autophagy and cell adhesion following TBI and may represent important proteins of interest for developing future treatments in the secondary phase following insult. At the same time, supplementation with NOS substrates like L-arginine may provide an effective prophylactic strategy by manipulating the post-traumatic redox environment, preventing secondary oxidative processes that can lead to neurodegeneration.
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    Committee: Tadeusz Malinski (Advisor); Michael Held (Committee Member); Lauren McMills (Committee Member); Sergio Ulloa (Committee Member) Subjects: Biochemistry; Chemistry; Neurosciences

  • 4. Zhao, Huizhi 3D Cell Culture Model Synthesized By Polycaprolactone Nanofiber Electrospinning

    Doctor of Philosophy (PhD), Ohio University, 2018, Chemistry and Biochemistry (Arts and Sciences)

    Previous studies indicated that nitric oxide (NO•) plays an important role in regulation of redox balance, cell damage and apoptosis of keratinocytes upon ultraviolet B light (UVB) exposure. Since endothelial cells also release NO• for a prolonged time post-UVB, which could diffuse to adjacent cells through membrane permeation or gap junctions, we determined whether endothelial cells (HUVEC) could influence UVB-induced DNA damage and transformation of their adjacent keratinocytes (HaCaT) using a 3D polycaprolactone-based nanofiber scaffold cell co-culturing system. We chose the 3D cell culture system because recent studies had proved more similarities between 3D cell culturing models and animal models compared with 2D cell culturing. Our purpose was to build an appropriate 3D cell culturing system in which the cells could grow in multiple layers and mimic the microenvironment in tissues and solid tumors with the supply of necessary nutrients and oxygen/carbon dioxide. The polycaprolactone (biofriendly and biodegradable polymer material) scaffold was prepared using high voltage electro-spinning technique to achieve nano to micro scale polymer fibers as skeletons for cell attachment and support. In the system, HaCaT cells were co-cultured with HUVEC (co-cultured HaCaT) or with HaCaT (mono-cultured HaCaT) as the control. Our data shows that the levels of DNA damages and cyclobutane pyrimidine dimer (CPD) level along with the phosphorylation of H2AX are higher in the co-cultured than in the mono-cultured HaCaT cells post-UVB irradiation. An analysis of NO• and peroxynitrite (ONOO-) reveals that the NO• level in the co-cultured cells is increased approximately three fold than in mono-cultured HaCaT cells within one-hour post-UVB but then is reduced much quicker and remains lower in co-cultured HaCaT cells comparing to mono-cultured cells from 6-24 hours post-UVB irradiation. However, the ONOO- level is higher in the co-cultured than in the mono-cultured HaCaT cells in th (open full item for complete abstract)
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    Committee: Shiyong Wu (Advisor); Xiaozhuo Chen (Committee Chair); Hao Chen (Committee Member); Justin Holub (Committee Member) Subjects: Biochemistry; Molecular Biology

  • 5. Dawoud, Hazem Nanomedical Studies of Angiographic Contrast-Induced Renal and Vascular Injury: Clinical Implications

    Doctor of Philosophy (PhD), Ohio University, 2018, Chemistry and Biochemistry (Arts and Sciences)

    Endothelial dysfunction is a common denominator of several cardiovascular diseases (CVDs). It is fundamentally necessary to apply imaging system-angiography for diagnosis and invasive cardiology. Iodinated contrast media (CM) are widely used in angiography to visualize the cardiovascular system. Adsorption of CM molecules is necessary to attain the medical images; on the other hand, the adsorption process may contribute to toxic effects, cellular dysfunction, and serious vascular and renal injury. The incidence of CM-induced renal and vascular toxicity is about 1-2%; however, this toxic effect can increase more than 50% in patients with CVDs. The molecular mechanism of CM induced-cytotoxicity is not well understood. In this study, we hypothesized that CM induced-cytotoxicity could be due to dysfunction/uncoupling of endothelial nitric oxide synthase (eNOS) followed by a decrease of bioavailable vasodilator nitric oxide (NO) and an enhancement in the production of vasoconstrictor peroxynitrite (ONOO-). A well-established nanomedical approach, utilizing systems of nanosensors (diameter ~ 300nm) with high sensitivity, selectivity, accuracy, and a detection limit of about 1.0 nmol/L was applied to measure simultaneously CM induced NO and ONOO- concentrations in normal and dysfunctional endothelial cells. To elucidate the role of cytoprotective NO and cytotoxic nitroxidative stress induced by ONOO-, NO and ONOO- measurements were made at different concentrations of CM and at different incubation times with normal and cellular model of different diseases including diabetes, hypertension, and hypercholesterolemia. Also, animal models were used to elucidate the effect of CM on normal, diabetic and aged Wistar rats. The ratio of concentrations [NO]/[ONOO-] was used as an indicator to evaluate the level of CM induced-nitroxidative stress, coupling/uncoupling of eNOS, and function/dysfunction of endothelial cells. Exposure of endothelial cells to CM induced endothelial (open full item for complete abstract)
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    Committee: Tadeusz Malinski Distinguished Professor (Advisor); Marcia Kieliszewski Professor (Committee Member); Michael Held Professor (Committee Member); Savas Kaya Professor (Committee Member) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology; Pharmacology

  • 6. Kalil, Haitham NANOMATERIALS-BASED SENSORS FOR PEROXYNITRITE DETECTION AND QUANTIFICATION

    Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2017, College of Sciences and Health Professions

    Peroxynitrite (ONOO-, PON) plays a crucial role in several cardiovascular dysfunctions and other diseases triggered by oxidative stress. PON is a strong oxidizing agent produced from the diffusion-controlled reaction between nitric oxide radical and superoxide anion-radical. It is also a member of the reactive oxygen-nitrogen species family, which attacks vital components inside the body and initiates deleterious effects via direct and indirect interactions. PON reacts directly with lipids, DNA, and proteins, whereas indirectly, it acts as an initiator of radical-chain reactions. In this work, we have explored various interfaces of manganese-oxide-decorated graphene/hemin and selenium-containing compound for PON detection and quantification. The combination of manganese oxide nanoparticles with the graphene/hemin matrix has allowed for more hemin molecules to be adsorbed on the final composite matrix. As a result, the adsorbed hemin has enhanced the catalytic activity of the final composite and improved the sensitivity towards PON detection. In the same context, a selenium-containing compound (aniline-selenide) has been synthesized and grafted on the electrode surface using the chemistry of diazonium salt. The aniline-selenide-modified electrode showed an increase of approximately 40 times the catalytic current as the aniline-modified electrode. Throughout this project, the preparation methods of the electroactive nanomaterials were described in detail. Moreover, the characterizations of the prepared-materials have been investigated by various physicochemical methods using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), ultraviolet/visible measurements (UV/Vis), and X-ray photoelectron spectroscopy (XPS). This study showed the importance of using selenium and manganese interfaces as sensitive platforms for PON detection. It also provides the initial stage to extend the use of these interfaces to ultramicroelectrodes sensors for use (open full item for complete abstract)
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    Committee: Mekki Bayachou Ph.D. (Committee Chair); Aimin Zhou Ph.D. (Committee Member); John Turner Ph.D. (Committee Member); W. Christopher Boyd Ph.D. (Committee Member); Petru S. Fodor Ph.D. (Committee Member) Subjects: Chemistry

  • 7. Mahmud, Farina Nitric Oxide and Peroxynitrite Imbalance Triggers Cortical Hyper-Excitability and Migraine Headaches

    Doctor of Philosophy (PhD), Ohio University, 2017, Chemistry and Biochemistry (Arts and Sciences)

    Migraine is a neurovascular disorder which exhibits as debilitating pain and other auxiliary symptoms. The headache is incapacitating enough to render the patient inactive and unable to carry on with their daily activities. Excitable neurons that are susceptible to noxious stimulus and ongoing activity in their circuit promotes the manifestation of pain. Current treatments only treat acute nociception and are non-functional when the disease progresses to chronic conditions. Assessing the underlying mechanism for the development of these headaches could contribute to designing better therapeutic interventions. We attempted to study the effects of nitro-oxidative stress on the pathophysiology of migraine with aura. Our nanosensors allowed us to detect the real-time changes in NO and ONOO- and provided a valuable tool in the analysis of these two species that are involved in a number of physiological and pathological processes. Concurrent EEG measurements were undertaken to decipher the changes in the neuronal activity. The computation of [NO]/[ONOO-] provided a quantitative value for nitro-oxidative stress while spectral analysis of EEG reflected the changes in the synaptic transmission. We found an inverse correlation between fast neuronal oscillations and [NO]/[ONOO-]. Together, they showed a relationship between nitro-oxidative stress and neuronal hyperexcitability where both NO and ONOO- influenced the spiking profile of neurons. Different analgesics showed changes in the [NO]/[ONOO-] and mediated.
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    Committee: Tadeusz Malinski PhD (Advisor); Michael Held PhD (Committee Member); Hao Chen PhD (Committee Member); Sergio Ulloa PhD (Committee Member) Subjects: Biochemistry; Molecular Chemistry; Neurosciences

  • 8. Alhumaid, Haidar Nanoanalytical Studies of Bacterial Adhesion to the Membrane of Endothelial Cells

    Master of Science (MS), Ohio University, 2016, Chemistry and Biochemistry (Arts and Sciences)

    Adhesion of bacteria to human cells is the first step of bacterial infection. It is suspected that the endothelium is where bacterial infection takes place. Endocarditis and meningitis are examples of bacterial infection to endothelial cells. Despite the numerous studies of the infection of endothelial cells, adhesion of bacteria to endothelium is not fully understood. In this study, we investigated the role of cytoprotective NO and cytotoxic ONOO- on the adhesion of E.coli bacteria to human umbilical vein endothelial cells (HUVECs). The collision of bacteria with endothelial cells induces the release of NO and subsequently ONOO-. By using electrochemical nanosensors, we directly measured the concentrations of NO and ONOO- at different concentrations of bacteria (1×107 - 1×109 bacteria/ml). The concentration of NO increased linearly with increasing bacterial concentration. The concentration of ONOO- increased at low concentrations of bacteria from 1×107 to 1×108 bacteria/ml and decreased at high bacterial concentration at 5×108 and 1×109 bacteria/ml. The ratio of [NO]/[ONOO-] at 1×107, 5×107 and 1×108 bacteria/ml indicates that the attachment of bacteria leads to eNOS uncoupling. Fluorescence spectroscopy was also used to determine the adhesion of bacteria at various bacterial concentrations and proved our hypothesis – the adherence of bacteria increases as the number of bacteria increases. Among several eNOS modulators studied here, sepiapterin showed a significant effect on restoring the endothelium and reducing the adhesion of bacteria. The adhesion of bacteria to endothelial cells is most likely to occur when the level of both NO and ONOO- is significantly reduced.
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    Committee: Tadeusz Malinski (Advisor) Subjects: Chemistry

  • 9. Salim, Heba MODIFIED ELECTRODES WITH GRAFTED DNA AND OLIGONUCLEOTIDES FOR DETECTION AND QUANTIFICATION OF PEROXYNITRITE

    Master of Science in Chemistry, Cleveland State University, 2016, College of Sciences and Health Professions

    Peroxynitrite (ONOO-) is a strong oxidizing and nitrating agent, and its formation has been correlated with many pathological conditions. It is generated in-vivo through the diffusion-controlled reaction between nitric oxide and superoxide. Peroxynitrite has been linked to nitration of protein and DNA as well as to DNA strand breaks. Accumulation of mutations and/or other kinds of DNA damage represent a carcinogenic risk. The accurate measurement of peroxynitrite concentration has been a challenge since this analyte is very unstable and reacts with many cellular targets. Development of analytical techniques capable of rapid and sensitive detection of this fast-reacting and damaging agent is an important research target to determine the chemical damage by this oxidant both at the tissue and the cellular levels. In this work, we develop DNA films as sensitive sensing platforms to detect and quantify ONOO- DNA damage. We have used two methods for DNA immobilization on the electrodes surfaces: (1) electrochemical grafting and (2) layer-by-layer (LBL) deposition methods. In the first method, we generate carboxylic acid groups on the electrode surface via electrochemical reduction of trans-4cinnamic acid diazonium tetrafluoroborate, followed by coupling of pre-activated carboxylic groups with amino terminated oligonucleotide. In the LBL deposition method, we construct films of alternate layers of posittively charged poly(diallyl dimethyl ammonium) and the target DNA as a negatively charged counterpart on the surface of the graphite electrode. On both platforms (grafted oligos and DNA films), we assess the effect of defined exogenous levels peroxynitrite metabolite on the electrochemical response of the DNA interface. Particularly for the grafted DNA oligonucleotides, we focused on detecting the differential response of complementary strands versus DNA helices with a single base mismatch. We show in the current work that electrodes modified with DNA oligonucleotides show (open full item for complete abstract)
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    Committee: Mekki Bayachou PhD (Committee Chair); Aimin Zhou PhD (Committee Member); Yana Sandlers PhD (Committee Member) Subjects: Chemistry

  • 10. Hua, Jiangzhou Patterns of Low Density Lipoprotein are Determinants in the Induction of Nitroxidative Stress in Cardiovascular System

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

    Clinical research suggests that small and dense low density lipoprotein (LDL) may be correlated with increased cardiovascular risk. However, the effect of LDL with different patterns in the cardiovascular system, especially in endothelial cells, remains vague. In this study, we used nanosensors to measure real-time production of nitric oxide (NO) and peroxynitrite (ONOO-) and investigated their role in the regulation of cell adhesion and noxious effects on human umbilical vein endothelial cells (HUVECs). Direct injection of LDL with different patterns (pattern A: 1.016-1.019 g/mL, pattern I: 1.024-1.029 g/mL, and pattern B: 1.034-1.053 g/mL) to HUVECs immediately generated ONOO- and NO. The [NO] to [ONOO-] ratio was 2.7±0.4, 0.5±0.1 and 0.9±0.1 for pattern A, B and I, respectively. A ratio below 1.0 indicates a serious imbalance between cytoprotective NO and cytotoxic ONOO-. LDL (50% B and 50% I) stimulated endothelial cells to produce the highest concentration of ONOO- (293±14 nmol/L) and lowest concentration of NO (166±10 nmol/L), whereas LDL composed of 60% A, 20% B and 20% I produced the lowest concentration of ONOO- (77±8 nmol/L) and highest concentration of NO (436±28 nmol/L). All patterns of LDL upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1). Monocyte adhesion increased about 575%, 276%, and 194% (vs. control) in the presence of 400 µg/mL of patterns B, I, or A LDL, respectively. Our results suggest that pattern B can stimulate endothelial cells to produce the highest level of ONOO- and decrease NO to the lowest level, resulting an imbalance between NO and ONOO- , followed by endothelial dysfunction. Our results also suggest that endothelial cells of African Americans are more susceptible to LDL stimulation than endothelial cells of Caucasian Americans. Therefore, imbalance between NO and ONOO- in the presence of LDL, followed by endothelial dysfunction, and upregulation of ICAM-1 and (open full item for complete abstract)
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    Committee: Tadeusz Malinski (Advisor); Hao Chen (Committee Member); Michael Held (Committee Member); Tiao J. Chang (Committee Member) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology

  • 11. Khan, Alamzeb 1α,25-Dihydroxyvitamin D3 Reverses Nitric Oxide and Peroxynitrite Imbalance in Dysfunctional Endothelium: A Nanomedical Approach

    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)
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    Committee: Tadeusz Malinski (Advisor) Subjects: Biochemistry

  • 12. Tang, Yuanyuan Nitric Oxide/Peroxynitrite Imbalance Induces Adhesion of Cancer Cells to Lymphatic Endothelium - Clinical Implications for Cancer Metastasis

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

    Adhesion of cancer cells to endothelial cells is a key step in metastasis. However, the molecular mechanisms mediating cancer cell/endothelial cell interaction and adhesion process are not fully understood. Nitric oxide (NO) has been found to play an important role in the regulation of local blood flow and may be involved in lymph node metastasis. It has also been shown that the effects of NO are difficult to separate from the effects of its byproduct, peroxynitrite (ONOO-). In this study, we used a nanomedical approach to examine the imbalance between cytoprotective NO and cytotoxic ONOO- in lymphatic endothelial cells, a key mediator of cancer cell adhesion. By using electrochemical nanosensors, we monitored in vitro NO and ONOO- release from human lymphatic endothelial cells (HLECs) stimulated by cancer cells. Our study showed that at low [NO]/[ONOO-] ratio (lower than 1.5), the adhesion of CACO-2 or HT1080 cancer cells to HLECs increased. An increase level of [NO]/[ONOO-] with agents that facilitate NO formation (e.g., SNAP, PEG-SOD, MnTBAP, VAS2870 and L-arginine) suppressed lymphatic metastasis of CACO-2 human colon cancer cell line and the highly invasive HT1080 human fibrosarcoma cell line through inhibition of the surface expression of intercellular adhesion molecule 1(ICAM-1) and vascular cell adhesion molecule 1(VCAM-1) on HLECs. Collectively, this study demonstrates for first time the crucial role of the [NO]/[ONOO-] balance in cancer cell-lymphatic endothelial cell interaction, which may be exploited clinically to prevent lymphatic metastasis.
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    Committee: Tadeusz Malinski (Advisor) Subjects: Analytical Chemistry; Biochemistry; Chemistry

  • 13. Jiang, Lu-Lin The Pivotal Role of Nitric Oxide and Peroxynitrite Imbalance in Epileptic Seizures

    Doctor of Philosophy (PhD), Ohio University, 2014, Chemistry and Biochemistry (Arts and Sciences)

    Epilepsy is one of the most severe neurological disorders. However, the detailed molecular mechanism in triggering epileptic seizures is still unclear. Nitric oxide (NO) is a versatile neurotransmitter in the brain; it acts as a messenger and antiplatelet aggregation agent in the cerebral vasculature. Peroxynitrite (ONOO-), a cytotoxic compound, can be easily produced by the diffusion-controlled reaction between NO and superoxide anion (O2·-). This study used a nanomedical approach to elucidate the role of NO and ONOO- in epileptic seizures. The nanomedical approach involving a system of nanosensors (diameter 200~300nm) has been used to measure directly in vivo release of NO and ONOO- in the brains of Sprague-Dawley (SD) rats during the process of pilocarpine-induced epileptic seizure events. Seizure events were simultaneously monitored by electroencephalography (EEG). Pilocarpine stimulated both NO and ONOO- production in the brain. The ratio of NO to ONOO- concentration ([NO]/ [ONOO-]) that reflected the balance between NO and ONOO- shifted with time. Epileptic seizures were observed only at the relatively low ratio of [NO]/ [ONOO-]. The latency, duration, and frequency of seizure events have also been influenced by the balance between NO and ONOO-.
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    Committee: Tadeusz Malinski (Advisor) Subjects: Biochemistry

  • 14. Awad, Salah The Role of Nitric Oxide/Peroxynitrite Imbalance in Diabetes and Salt-Induced Hypertension

    Doctor of Philosophy (PhD), Ohio University, 2013, Chemistry and Biochemistry (Arts and Sciences)

    Both diabetes and hypertension are risk factors for cardiovascular diseases. Diabetes mellitus and high blood pressure complications are associated with dysfunctional endothelium, where the molecular mechanism is still unclear. Endothelial dysfunction refers to the loss of nitric oxide (NO). The objective of this research was to study the molecular mechanisms of NO, peroxynitrite (ONOO-) (using electrochemical nanosensors) and L-arginine transport in type 2 diabetes and salt-induced hypertension in human umbilical vein endothelial cells (HUVECs). The influence of various concentrations of glucose, sodium chloride (NaCl), sodium (Na+) and chloride (Cl-) on NO, ONOO- release and L-arginine transport was measured in human endothelial cells. Exposure of HUVECs to elevated concentrations of glucose, NaCl, Na+ and Cl- reduced significantly NO release and increased nitroxidative stress levels (elevated ONOO-). L-arginine transport was significantly inhibited under hyperglycemic and hypertensive conditions. NO bioavailability ameliorated under physiological and elevated glucose and salt concentration after the administration of L-arginine. In contrast, ONOO- concentrations decreased significantly after the supplementation of L-arginine. Similar study on African American HUVECs was performed to elucidate the influence of glucose and salt on NO, ONOO- and L-arginine transport. This study showed a decrease in NO and L-arginine transport and an increase in oxidative stress under elevated glucose and NaCl levels. African American HUVECs also produced a lower NO concentration and higher ONOO- level than White American HUVECs. A low ratio of NO to ONOO- [NO]/[ONOO-] in HUVECs under hyperglycemic and hypertensive conditions clearly imply a dysfunctional endothelium. Similarly, the effect of glucose on NO, ONOO-, L-arginine transport and insulin secretion was studied in hamster pancreatic beta cells (HIT-T15) under various glucose concentrations. Elevated glucose concentrations (open full item for complete abstract)
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    Committee: Tadeusz Malinski Dr. (Advisor) Subjects: Biochemistry

  • 15. Allen, Rebecca Macrophage Microbicidal Activity is Enhanced by Stressor-Exposure

    Doctor of Philosophy, The Ohio State University, 2012, Integrated Biomedical Science Graduate Program

    Exposure to social stressors is known to prime the innate immune system for enhanced reactivity to inflammatory stimuli, but the mechanisms by which stressor exposure can enhance immune activity are not well-defined. In mice, exposure to a social stressor called social disruption (SDR) increases circulating cytokines and primes splenic macrophages for an enhanced capacity to kill Escherichia coli, primarily through an increased production of the highly microbicidal compound peroxynitrite. Previous results demonstrate that the intestinal microbiota are in part responsible for the SDR-induced increase in circulating cytokines; reducing the microbiota through the use of a broad spectrum antibiotic cocktail prevented the SDR-induced increase in IL-6 and MCP-1. These studies tested the hypothesis that intestinal microbiota also contribute to the stressor-induced increase in the ability of splenic macrophages to kill E.coli. To test this hypothesis with SDR, groups of co-housed male mice were repeatedly defeated through direct interactions between the resident mice and an aggressive intruder. Following stress, E. coli were co-cultured with the splenic macrophages, and the number of bacteria within the macrophages was enumerated at 20 and 90 min (to determine the number of bacteria phagocytosed and then killed, respectively). We also measured changes in iNOS and pro-inflammatory cytokine expression as well as production of both superoxide anion and its reaction product with nitric oxide, peroxynitrite. When endogenous bacterial populations were eliminated through the use of germ free mice or reduced in mice treated with an antibiotic cocktail were stressed with SDR, we failed to observe the characteristic increases in pro-inflammatory cytokine and iNOS expression, superoxide anion, and peroxynitrite production. This lack of stressor-induced changes in splenic macrophage activity was associated with the failure of the stressor to enhance bacterial killing typically associ (open full item for complete abstract)
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    Committee: Michael Bailey PhD (Advisor); John Sheridan PhD (Committee Chair); William Lafuse PhD (Committee Member); John Walters PhD (Committee Member) Subjects: Biomedical Research; Immunology

  • 16. Kohr, Mark Modulation of Cardiac Contraction by Reactive Nitrogen Species

    Doctor of Philosophy, The Ohio State University, 2009, Integrated Biomedical Sciences

    Reactive nitrogen species, including peroxynitrite and nitroxyl, have been demonstrated to affect myocardial contractility. Many of these reactive nitrogen species are produced endogenously within cardiac myocytes and serve as critical regulators of myocyte function. Alterations in the production of these reactive species may be responsible for part of the dysfunction that is observed during many pathophysiological conditions of the myocardium. Although studies have partially documented the contractile effects of many different reactive nitrogen species, few have examined the specific mechanism of action. Therefore, in order to address deficiencies in regards to the physiological and pathophysiological regulation of myocardial function by reactive nitrogen species, we set out to characterize the contractile effects and specific mechanism of action for two distinct reactive nitrogen species, peroxynitrite and nitroxyl. We first characterized the effects of both high and low concentrations of peroxynitrite on cardiac myocyte function during three different contractile states (basal, submaximal and maximal beta-adrenergic stimulation). High peroxynitrite produced a negative effect on myocyte function that became more pronounced as the contractile state in the cardiac myocyte was increased. Conversely, low peroxynitrite produced a positive effect on myocyte function that was greatest during basal contraction and diminished as the contractile state in the cardiac myocyte was increased. Interestingly, the effects of both high and low peroxynitrite were completely absent during all contractile states in phospholamban knockout myocytes. These results indicate that high and low peroxynitrite both exert effects on cardiac myocyte contraction by targeting the critical excitation-contraction coupling phosphoprotein, phospholamban. In order to elucidate the specific signaling pathway of high peroxynitrite, we examined phospholamban phosphorylation at the cAMP-dependent kinase s (open full item for complete abstract)
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    Committee: Mark Ziolo PhD (Advisor); Cynthia Carnes PharmD, PhD (Committee Member); Jonathan Davis PhD (Committee Member); Paul Janssen PhD (Committee Member); Jay Zweier PhD (Committee Member) Subjects: Biomedical Research

  • 17. Jacoby, Adam The Role of Nitric Oxide and Nitroxidative Stress in Amyotrophic Lateral Sclerosis

    Doctor of Philosophy (PhD), Ohio University, 2010, Chemistry and Biochemistry (Arts and Sciences)

    Amyotrophic lateral sclerosis (ALS) has been studied for many years, yet there have been few innovations in its therapy. This debilitating and fatal disease has proven elusive to all but a few treatments. These treatments have only extended life for short periods of time in ALS patients and have not succeeded in arresting the progression of ALS. Our approach to understanding the oxidative stress behind the disease was studied using nanosensors that directly measure nitric oxide and peroxynitrite concentrations in vivo. Nitric oxide (NO) was thought of as a nuisance in ALS patients, but new studies show that nitric oxide removal proves ineffective at preventing ALS progression. We determined that NO performs a much more vital role in ALS than previously understood. Our conclusions show that after ALS onset, the enzyme responsible for NO production, nitric oxide synthase (NOS), remains in a dysfunctional state. This proved that NO in ALS neurons was already depleted and that peroxynitrite was prevalent, in terms of global concentration, within the neurons. Dysfunctional NOS accounted for increased production of superoxide, which is a common pathological symptom in most ALS cases. We determined that one main malignant affector in ALS is the peroxynitrite produced from dysfunctional NOS. As a corollary, the restoration of coupled NOS and NO production would prove beneficial. The NOS cofactor and co-substrate levels were proven to be in a diminished state, failing to provide sufficient production of nitric oxide with respect to superoxide. The concentrations of the co-substrate L-arginine and the cofactor tetrahydrobiopterin were main targets for treatment in this study. The subsequent restoration of coupled NOS demonstrated that cofactor and co-substrate concentrations play an important role in ALS. Continued production of NO at a normal concentration was important to maintain the neuron function. The treatments with L-arginine and sepiapterin were beneficial and impede (open full item for complete abstract)
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    Committee: Tadeusz Malinski (Advisor) Subjects: Biochemistry

  • 18. Huang, Xiaoyan Nitric Oxide/Peroxynitrite Balance in Kidney – Effect of Diabetes and Obesity

    Doctor of Philosophy (PhD), Ohio University, 2008, Chemistry and Biochemistry (Arts and Sciences)

    Nitric oxide (NO) is a vasoprotective signaling molecule. Peroxynitrite (ONOO-), the reaction product of the superoxide (O2-) with the nitric oxide, is the main cytotoxic component of nitroxidative stress. This work elucidated the [NO]/[ONOO-] imbalance in the kidney of rats with type I and type II diabetes. Obesity factor has also been studied by induced high fat diet to rats before or after type II diabetes was established. The releases of NO and ONOO- were monitored in situ by placing NO and ONOO- nanosensors near the surface (5 ± 2 µm) of medulla part of kidney slices. Intracellular L-arginine was measured by HPLC-fluorescence and eNOS protein levels were examined by immunological Western blotting analysis. These studies clearly indicate that the [NO]/[ONOO-] imbalance in the type I and type II diabetes is caused by uncoupled eNOS. The deficiency of intracellular L-argnine and tetrahydrobiopterin may be linked to eNOS uncoupling. At early stage of diabetes, the [NO]/[ONOO-] balance could be restored after treatments with L-arginine and sepiapterin. Obesity, itself, showed inhibitory effect on eNOS activity and NO release. However, obesity and type II diabetes increased hyperglycemia, hyperlipidaemia and eNOS uncoupling at the late stage. All of these factors lead to a severe [NO]/[ONOO-] imbalance and form high oxidative/nitroxidative stress in kidney. Different herbal drug treatments improved the [NO]/[ONOO-] balance by different mechanisms. Corn silk, a hypercholesterol inhibitor, improved the [NO]/[ONOO-] balance more significantly than ginseng at the early stage of type II diabetes. However, ginseng showed better effect than corn silk on the improvement of the [NO]/[ONOO-] balance at the late stage. Our overall results indicate that the function of endothelial nitric oxide synthase can be severely uncoupled in diabetic kidney. The uncoupled eNOS is the main generator of cytotoxic ONOO-. An overproduction of ONOO- and diminished generation of NO sh (open full item for complete abstract)
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    Committee: Tadeusz Malinski (Advisor); Howard D. Dewald (Committee Member); Glen P. Jackson (Committee Member); Shiyong Wu (Committee Member); Tiao J. Chang (Committee Chair) Subjects: Analytical Chemistry; Biochemistry; Biomedical Research

  • 19. Choi, Ji Yeon The Effects of Nitric Oxide and Peroxynitrite on Cancer Cells

    Master of Science (MS), Ohio University, 2008, Chemistry (Arts and Sciences)

    Cancer is a group of diseases characterized by uncontrolled growth and this spread of abnormal cells is caused by various external and internal reasons. This study elucidated the relationship between the growth of cancer cells and concentration of NO and ONOO¯. Nanosensors were applied to measure nitric oxide and peroxynitrite concentration in cancer and normal cells. The ratio between NO/ONOO¯ concentrations was used as an indicator of oxidative stress in cancer cells. ONOO¯ concentration measured from HT1080 cancer cells (fibrosacoma) and MRC-5 normal cells (fibroblasts) was higher than NO concentration. ONOO¯ released from HT1080 cancer and MRC-5 normal cells was similar, while NO concentration in HT1080 cancer cells was significantly higher than in MRC-5 normal cells. Enzyme levels were measured by using Western blotting and correlated with NO and ONOO¯ levels in normal and cancer cells treated with 3-morpholinosydnonimine hydrochloride (SIN-1) (ONOO¯ donor) and sodium nitroprusside (SNP) (NO donor), respectively. After the preincubation with SNP and SIN-1 in MRC-5 cells, a significant decrease in eNOS expression was observed compared to HT1080 cancer cells. This indicates that MRC-5 normal cells were more sensitive to the changes of exogenous NO and ONOO¯. It indicated that exogenous NO or ONOO¯ downregulate eNOS expression in both cancer and normal cells. A treatment of CACO-2 cancer cells with SNP decreased eNOS expression more significantly that a treatment with SIN-1. In this study, we found that NO not only affected eNOS expression but also cell growths. The effect of NO and ONOO¯ on cell growth was investigated by preincubating HT1080 cancer and MRC-5 normal cells with L-arginine, NO substrate and L-NAME, a NOS inhibitor. L-arginine treatment promoted HT1080 cancer cell growth and increased the number of viable cells. HT1080 cancer cells preincubated with L-arginine promoted cell growth and increased the cells number. NO had a greater in promoting effec (open full item for complete abstract)
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    Committee: Tadeusz Malinski PhD (Advisor); Hao Chen PhD (Committee Member); Marcia Kieliszewski PhD (Committee Member) Subjects: Chemistry

  • 20. Madajka, Maria Nitroxidative Stress Induced Neurodegeneration In Intracerebral Hemorrhagic Stroke-a Nanomedical Approach

    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.
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    Committee: Tadeusz Malinski (Advisor) Subjects:
Release 3.2.12