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Venator, Edward StephenA Low-cost Mobile Manipulator for Industrial and Research Applications
Master of Engineering, Case Western Reserve University, 2013, EECS - System and Control Engineering
ABBY is a mobile industrial manipulator, a mobile robot equipped with an industrial robotic arm. The goal in creating this robot was to demonstrate that a robust research platform for mobile industrial manipulation can be created quickly at low cost. This goal was achieved by leveraging commercially-available mass produced hardware and open source software. The resulting mobile manipulator incorporates a suite of commercially-available sensors and processing hardware to enable the robot to operate as an intelligent agent alongside humans. The robot demonstrated its abilities by performing simple navigation and manipulation tasks in a laboratory setting, and will soon be employed in research on autonomous kitting in industrial environments.

Committee:

Gregory Lee, PhD (Advisor); Murat Cavusoglu, PhD (Committee Member); Roger Quinn, PhD (Committee Member)

Subjects:

Electrical Engineering; Engineering; Industrial Engineering; Robotics; Systems Design

Keywords:

robot; robotics; industrial robot; mobile manipulator; mobile manipulation; industrial manipulator; robotic arm; industrial robotic arm; kitting; ROS; ROS Industrial

Mouradi, RandWireless Signals and Male Fertility
Doctor of Engineering, Cleveland State University, 2011, Fenn College of Engineering
Rapid advances in wireless technology have increased the number of users of mobile devices. As of 2011, the number of cell phone subscribers have reached 5.3 billion worldwide. Mobile devices have saturated our environment with radio frequency (RF) signals. This situation has created public concern over the effect of such signals on human health. This dissertation focuses on the correlation of RF signals emitted by cell phones with male infertility. A thorough discussion is provided on the effects of RF signals on the development of central nervous system (CNS) neoplasm, the design of these mobile devices, the range of the RF frequencies they emit, the power with which they operate, their specific absorption rate (SAR), the distance between the user and the device while in use, how and where the devices are used, the duration of usage, and the accumulated exposure associated with the use of multiple RF devices. The results of our reviews and experimental in vitro studies show a significant correlation between the usage of mobile phones and human semen parameters, with a decrease in motility and viability, and an increase in the reactive oxygen species (ROS) score. However, in daily usage, a cell phone kept in proximity to the groin is separated from the testes by multiple layers of tissue. To explore this effect, a computational model of scrotal tissues was designed. Our results show that during in vitro experimentation, an effect equivalent to real-life conditions can be obtained by placing the cell phone a few centimeters farther away from the semen sample. The results of our study can be used to calculate the equivalent distance between a radiation source and a semen sample, and to set up in vitro experiments that mimic real-life conditions.

Committee:

Daniel Simon (Advisor); Fuqin Xiong (Committee Member); Rama Gorla (Committee Member); Mekki Bayachou (Committee Member); Taysir Nayfeh (Committee Member)

Subjects:

Biomedical Engineering; Biomedical Research; Electrical Engineering; Electromagnetics; Electromagnetism; Engineering; Health; Public Health; Radiation

Keywords:

Wireless signals effects; cellular phones; mobile phones; RF signals and male infertility; brain tumors; electromegnitic fields; Dosimetry; FDTD; specific absorption rate (SAR); RF radiation; cell phones and ROS; wireless signals and human health.

Patrick-Melin, Amy J.Effect of 7 Days Aerobic Exercise on Insulin Sensitivity, Oxidative Stress, TLR2/TLR4 Cell Surface Expression and Cytokine Secretion in Sedentary Obese Adults
PHD, Kent State University, 2011, College and Graduate School of Education, Health and Human Services / School of Health Sciences

PATRICK-MELIN, AMY J., PH.D., August, 2011 Exercise Physiology

EFFECT OF 7 DAYS AEROBIC EXERCISE ON INSULIN SENSITIVITY, OXIDATIVE STRESS, TLR2/TLR4 CELL SURFACE EXPRESSION AND CYTOKINE SECRETION IN SEDENTARY OBESE ADULTS (218 pp.)

Director of Dissertation: Michael I. Kalinski, Ph.D.

Aerobic exercise training (AEX) has been well documented to improve glucose tolerance and insulin action in patients who are obese (Goodpaster & Kelley, 2003), insulin resistant (Rodgers, M.A., 1988) and in patients with T2DM (Bruce, 2004; Kirwan et al., 1990; Kirwan, Solomon, Wojta, Staten, Holloszy, 2009; Kirwan, Kohrt et al., 2002; Kirwan, et al., 1990) in the absence of weight loss. Mononuclear cells (MNC) are immune mediators of inflammation that are known to be elevated in diseases such as obesity and insulin resistance. Toll like receptors 2 and 4 (TLR2, TLR4) are predominantly found on the plasma membrane of MNCs and play a key role in the innate immune response. However, it is not known whether improvements in glucose tolerance resulting from short term AEX is due to reductions in proinflammatory mediators.

Thirteen sedentary obese (34.38 ± 1.13 kg/m2) adults, age 58.71 ± 3.21 yr, underwent Oral Glucose Tolerance Tests (OGTT's), before and after 7 days of supervised AEX at 70% VO2 max. Insulin sensitivity was assessed via the OGTT, TLR2/TLR4 cell surface expression by flow cytometry, ROS production by chemiluminescense, and MNC derived cytokine (TNF-α, IL-6) secretion by ELISA. Seven days of AEX resulted in improvements in Matsuda insulin sensitivity (p= 0.04) in the absence of weight loss and increased VO2max, (p = 0.004). Metabolic improvements were also noted in CHOOX and FatOX ( p = 0.02, 0.03), Gluc120 and Ins120 responses (p = 0.002, 0.007) and tAUC for Gluc120 and Ins120 (p = 0.04, 0.01). ROS (p= 0.05) and IL-6 production at Gluc120 (p= 0.05) were also significantly reduced. No changes were observed in either CD14 or CD14 CD16 TLR2 or TLR4 response for any time points.

Seven days of aerobic exercise training is sufficient to produce changes in insulin sensitivity in obese adults and these changes are associated with reductions in ROS and improvements in insulin sensitivity of peripheral tissues. However, because there were no changes in innate response, it is likely that a longer training duration greater than seven days may be required to reduce chronic inflammation in obese sedentary humans.

Committee:

Michael I. Kalinski, PhD (Advisor); John P. Kirwan, PhD (Committee Member); Jacob E. Barkley, PhD (Committee Member); Jennifer L. Marcinkiewicz, PhD (Committee Member); Robert E. Stadulis, PhD (Committee Member)

Subjects:

Health Care; Health Sciences; Physiology

Keywords:

Exercise; Toll like Receptors; ROS; obesity; TNF-alpha; IL-6; inflammation

Giedt, Randy JamesReal-Time Acquisition and Analysis of Endothelial Mitochondrial Superoxide Radical Production and Membrane Potential During In Vitro Ischemia/Reperfusion
Master of Science, The Ohio State University, 2009, Biomedical Engineering
The focus of this project was to elucidate the effects of ischemia/reperfusion on mitochondrial superoxide production by cultured endothelial cells in a parallel plate flow chamber in real time. To accomplish this, we used the mitochondria-targeted superoxide-specific fluorescent probe mitoSOX to determine parameters for its most effective use in a system composed of a parallel-plate flow chamber with cultured endothelial cells, a Nikon epifluorescence microscope, and digital image processing software. Based on the literature, the probe’s mitochondrial specificity is linked with the cell mitochondrial membrane potential. This made it necessary to study the effects of ischemia/reperfusion on mitochondrial membrane potential because of the possibility for mitoSOX to leak out of the mitochondria upon loss of mitochondrial membrane potential. The fluorescent probe rhodamine 123 was used in a similar manner as mitoSOX to accomplish this goal. The combination of the novel flow system and mitoSOX enabled us to obtain data comparable to that of a confocal microscope, as verified by the use of a variety of positive controls (both static endpoint images and real time analysis). Experiments using mitoSOX during ischemia/reperfusion showed a higher rate of mitochondrial superoxide production than that of shear alone, while the same experiments using rhodamine 123 showed a loss of mitochondrial membrane potential during ischemia and a partial recovery upon reperfusion. These studies may help us understand the mechanisms of injury on the mitochondria of coronary artery endothelial cells following cardiac ischemia/reperfusion.

Committee:

Barbara R. Alevriadou, PhD (Advisor); Juan Crestanello, MD (Committee Member)

Subjects:

Cellular Biology; Mechanical Engineering; Molecular Biology

Keywords:

Superoxide; MitoSOX; endothelial cells; mitochondria; membrane potential; ROS; Reactive Oxygen Species; Free Radicals; Ischemia; Ischemia/Reperfusion Injury

DelloStritto, Daniel JustinTRPing up the Balance of Oxidative Stress - Transient Receptor Potential Vanilloid 1’s Role in Diabetic Microvascular Disease
Doctor of Philosophy, Northeast Ohio Medical University, 2016, Integrated Pharmaceutical Medicine
Diabetes is a growing epidemic worldwide leading to an increase in cardiovascular morbidity and mortality. The maintenance of coronary blood flow (CBF) is crucial to supplying energy and oxygen to the working myocardium and this has been demonstrated to be disrupted in diabetes. Specifically, our lab has illustrated the ion channel Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is a key regulator in CBF control, but it’s contribution to CBF regulation is attenuated in diabetes. A crucial mechanism behind pathologies seen in diabetes is the imbalance in oxidative stress (OS). Herein, we determined the role for oxidative stress, specifically H2O2, to regulate TRPV1-mediated CBF—demonstrating H2O2 differentially regulates TRPV1. Acutely, H2O2 increases CBF through a TRPV1-dependent signaling; however, exposure to prolonged OS blunts TRPV1-mediated endothelial signaling resulting in uncoupling of CBF. Similarly, this altered OS environment is known to lead to an increase lipid peroxidation (LPO). The LPO product 4-hydroxynonenal (4-HNE) increases oxidative post translational modification (PTM) resulting in altered protein function. As such, we established the ability of 4-HNE to modify TRPV1 resulting in blunting of TRPV1-dependent CBF regulation. Further elucidation determined this process occurs via a carbonylation reaction on Cysteine 621 of TRPV1. Overall, this dissertation demonstrated a role for TRPV1 to sense the oxidative environment and subsequently regulate CBF; however, in diabetes this perfusion is perturbed through the oxidative modification of TRPV1 leading to potential avenues for future therapeutic targeting.

Committee:

Ian Bratz, Ph.D. (Advisor); Altaf Darvesh , Ph.D. (Committee Member); William Chilian , Ph.D. (Committee Member); Marc Penn , M.D., Ph.D. (Committee Member); Derek Damron , Ph.D. (Committee Member)

Subjects:

Biology; Physiology

Keywords:

TRPV1, Diabetes, ROS, 4-HNE, Hydrogen Peroxide, Differential Regulation

Bell-Horwath, Tiffany R.Derivation of Hydroquinone to Produce Selective, Oxidatively Activated Chemotherapeutic Agents
PhD, University of Cincinnati, 2014, Arts and Sciences: Chemistry
DNA modifying agents are stalwarts of chemotherapeutic cancer treatments, but require vital design improvements to improve selectivity, lower side effects, and continue their widespread use. A key problem for DNA modifying agents is lack of specificity. To address this issue, our lab designs novel molecular scaffolds which are activated by a hallmark of some cancers: increased oxidative stress cause by reactive oxygen species (ROS). Oxidative stress, as measured by levels of ROS, oxidized biomolecules, and enzyme activity, is a hallmark of certain cancer cells. My work focuses on a potential path forward in the design of DNA modifying agents by exploiting the increased ROS into a pro-drug approach. Elevation of ROS has been linked to oncogenesis and has been found in several aggressive cancers, including renal cell carcinoma, melanoma, and leukemia. ROS occurs in four major endogenous forms within the cell: superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radical. ROS occur in cells via two discrete mechanisms; first, as a byproduct of metabolism. For example, mitochondria generate superoxide via complex I and III during oxidative phosphorylation. Secondly, ROS are known to derive from several enzymes. Especially important are NADPH oxidases that regulate the function of several tyrosine kinases involved in cell growth and survival. Amplified ROS results in increased DNA damage and mutation, tumor heterogeneity, the ability to self-replicate, and angiogenesis. In turn, these mutations cause enhanced activation of oncogenes. Consequently, it is no surprise that levels of ROS-induced DNA damage correlates with cancer prognosis. We utilize a design strategy wherein the pro-drug is stable, but upon ROS activation a reactive molecule is formed. This leads to more reactive forms of the molecule being present in cancer cells. Thus reactivity, and not uptake, is controlled to induce cytotoxicity more specifically in cancer cells and lower off-target reactions.

Committee:

Edward Merino, Ph.D. (Committee Chair); David Smithrud, Ph.D. (Committee Member); Pearl Tsang, Ph.D. (Committee Member)

Subjects:

Biochemistry

Keywords:

Reactive Oxygen Species;AML;ROS;Anti-Cancer Agent;Drug Design;Acute Myeloid Leukemia

Waker, Christopher A.Metabolic Characterization of MPNST Cell Lines
Master of Science (MS), Wright State University, 2015, Physiology and Neuroscience
Malignant transformation is the process by which cells develop cancer properties. While many causes for malignant transformation are known (i.e. common genetic mutations and/or exposure to toxins or viruses), the basic requirements that allow a cell to stay alive with altered nutrient and energy requirements are just now being studied. In some tumor types malignant cells undergo changes that result in metabolic differences compared to normal cells. These can include defects in mitophagy resulting in an accumulation of dysfunctional mitochondria and/or a metabolic switch resulting in increased glycolysis, termed the Warburg effect. Increased tumor growth and metastasis have also been associated with mitochondrial DNA mutations in some tumor types. In this study, we characterized the mitochondrial function of malignant peripheral nerve sheath tumor (MPNST) cell lines commonly used to study malignant transformation in Neurofibromatosis Type I. We identified metabolic differences between NF1-wildtype (STS26T) and NF1-deficient (ST88-14, 90-8, and S462) MPNST cell lines by measuring extracellular acidification and oxygen consumption, mitochondrial respiration protein expression, and ROS levels. Similar to findings from other malignant tumors, all MPNST cell lines were more glycolytic compared to non-tumorigenic normal human Schwann cells and surprisingly NF1-deficiency correlated with lower glycolytic and mitochondrial respiratory rate compared to wildtype MPNST. Mitochondrial respiratory rates and respiratory protein expression were significantly lower in the NF1-deficient MPNST cell lines when compared to NF1-wildtype MPNST cells. These findings demonstrate that neurofibromin affects glycolysis and mitochondrial respiration in malignant cells.

Committee:

Debra Mayes, Ph.D. (Advisor); Robert Putnam, Ph.D. (Committee Member); Christopher Wyatt, Ph.D. (Committee Member)

Subjects:

Biomedical Research; Cellular Biology; Neurosciences

Keywords:

Neurofibromatosis Type 1; malignant peripheral nerve sheath tumor; metabolism; glycolysis; oxidative phosphorylation; ROS; mitochondria; proton leak; NF1; Ras; Electron transport chain; Extracellular Flux

Mohan Kumar, DipuInsights into the Host Cell Entry of Ehrlichia chaffeensis: Roles of the Bacterial Outer Membrane Protein EtpE
Doctor of Philosophy, The Ohio State University, 2014, Comparative and Veterinary Medicine
Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis (HME), is an obligatory intracellular rickettsial pathogen that replicates in the membrane-bound compartment (inclusion) in monocytes/macrophages. Entry into mammalian host cells is essential for an obligatory intracellular pathogen to sustain its life and consequently to manifest disease associated with the infection. Although some information regarding the host cell invasion of E. chaffeensis have been known, crucial information about the identity of its invasive ligand or cognate host cell receptor used for binding and entry was unknown. In this study, we report, a novel entry triggering protein of Ehrlichia named EtpE that functions as an invasin. Antibody against EtpE (the C-terminal fragment, EtpE-C) greatly inhibited E. chaffeensis binding, entry, and infection; EtpE-C-immunization of mice significantly inhibited E. chaffeensis infection. EtpE-C-coated beads entered both phagocytes and non-phagocytes, and the entry was blocked by compounds that block E. chaffeensis entry. Furthermore, yeast two-hybrid screening revealed that DNase X, a glycosylphosphatidyl inositol-anchored mammalian cell-surface protein binds EtpE-C. Antibody against DNase X or reduction of DNase X by small interfering RNA impaired E. chaffeensis binding, entry, and infection. In experimentally infected DNase X-/- mice, E. chaffeensis load in peripheral blood was significantly lower than in wild-type mice. Using affinity pull-down of host cell lysates with rEtpE-C followed by LC-MS/MS analysis, we identified two additional interacting mammalian proteins: a transmembrane glycoprotein CD147 and a cytosolic protein hnRNP-K. Far-Western blot and immunofluorescence analysis showed that CD147 interacts with rEtpE-C and is recruited to E. chaffeensis entry foci. Functional neutralization of surface-exposed CD147 with monoclonal antibodies or knock-down of CD147 using shRNA inhibited E. chaffeensis internalization and infection, but not binding. Functional ablation of cytoplasmic hnRNP-K significantly hampered E. chaffeensis entry and subsequent infection. We found that chemical inhibitors of actin dynamics drastically inhibited E. chaffeensis entry and infection of host cells. Actin and N-WASP was seen directly recruited to E. chaffeensis entry foci. By affinity column-based pull down and co-immunoprecipitation N-WASP was shown to be present in the protein complex, formed during E. chaffeensis invasion of host cells, along with EtpE, DNase X and CD147. EtpE-C was able to polymerize actin in an in vitro pyrenyl-actin polymerization assay. This activity was specific for EtpE-C but not for EtpE-N and was specifically mediated by DNase X in an N-WASP dependent manner. The phagosomes with rEtpE-C coated beads inhibited assembly of NADPH oxidase components on its phagosomal membrane and rEtpE-C coating of beads inhibited ROS generation by BMDM cells where as non-coated cells or rEtpE-N coated beads induced significant response. Thus, DNase X mediated entry pathway seemed to be evolutionarily selected by E. chaffeensis entry since this route of entry provided definitive survival advantage for the pathogen. This study is the first to demonstrate the invasin, its mammalian receptor and their in vivo relevance in any ehrlichial species, a novel upstream mode of pathogen-induced potential actin cytoskeletal mobilization, establishes the unique advantage of this infectious entry route and multifunctional role of EtpE in E. chaffeensis entry and thus in HME pathogenesis.

Committee:

Yasuko Rikihisa, PhD (Advisor); Michael Oglesbee, DVM, PhD (Committee Member); Xin Li, PhD (Committee Member); Mingqun Lin, PhD (Committee Member)

Subjects:

Immunology; Medicine; Microbiology

Keywords:

Ehrlichia chaffeensis; Human monocytic ehrlichiosis; EtpE; DNase X; GPI-1anchored protein; receptor, ligand; invasin; receptor-mediated endocytosis; CD147; hnRNP-K; actin; cytoskeletal mobilization; vaccine; N-WASP; ROS; NADPH Oxidase; lysosomes; vaccine

Ni, YingGenes Encoding Succinate Dehydrogenase as Susceptibility Factors in Cowden and Cowden-Like Syndrome
Doctor of Philosophy, Case Western Reserve University, 2012, Molecular Medicine

Cowden syndrome (CS), a Mendelian autosomal-dominant disorder, predisposes to breast, thyroid, and other cancers. Because of the complex clinical features of CS, such that patients may present with just subtle skin lesions, and the fact that each of the features could also occur in general population, this syndrome is difficult to recognize and remains under-diagnosed. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a ubiquitous tumor suppressor that plays a role in both heritable and sporadic neoplasias. Germline mutations in PTEN have been reported in 25% of a large-series of community accrued classic CS individuals and only 5% of patients with features reminiscent of CS but not fully meeting diagnostic criteria (CS-like). We hypothesized that there are other susceptibility genes for CS/CS-like phenotypes.

Mutation analysis revealed that germline variations in succinate dehydrogenase (SDH) genes occur in 8% (65/830) of PTEN mutation-negative CS/CS-like individuals (SDHvar+). None of these SDH variants were found in 350 population controls (p<0.001). Patients with SDH variants have elevated breast, thyroid, and renal cancer risks compared to PTEN mutation carriers. SDH variants also occur in 7% (33/473) of PTEN mutation-positive (PTENmut+) CS/CS-like individuals. Presence of SDH variants appears to further modify PTEN mutation cancer risks over those of PTEN mutation in isolation. Patient-derived SDHvar+ lymphoblastoid cells had elevated cellular reactive oxygen species (ROS) and lipid peroxidation levels, highest in cells with both PTEN mutation and SDH variant (PTENmut+/SDHvar+), correlating with increased apoptosis resistance in these cells. In addition to stabilized and hyperactivated HIF1¿¿¿¿ signaling, we also observed the loss of steady-state p53 in the majority of SDHvar+ cells. This loss of p53 may be regulated by MDM2-independent NQO1-mediated protein degradation, likely due to the imbalance of metabolites FAD and NAD+/NADH in SDHvar+ cells. Interestingly, ¿¿¿¿-tocopherol (vitamin E) treatment, but not other antioxidants, protected SDHvar+ cells from oxidative damage and rescued SDHvar+ cells from apoptosis resistance.

Together, our findings suggest SDH could be candidate predisposing and modifier genes for malignancies in CS/CS-like. SDH variants may disrupt mitochondrial metabolic equilibrium and impact multiple signaling pathways. Understanding SDH-related tumorigenic mechanism may guide future tailored preventive or therapeutic approaches.

Committee:

Charis Eng, MD, PhD (Advisor); George Stark, PhD (Committee Chair); Jonathan Smith, PhD (Committee Member); Mira Milas, MD (Committee Member); Micheala Aldred, PhD (Committee Member)

Subjects:

Biomedical Research; Genetics; Molecular Biology

Keywords:

SDH; Cowden syndrome; PTEN; ROS; vitamin E; p53; mitochondrial metabolism

Fish, Jesse O.Robotic Tour Guide Platform
Master of Sciences (Engineering), Case Western Reserve University, 2013, EECS - Computer Engineering
This thesis describes the development of a mobile robot research platform (Roberto) designed for use in developing LIDAR, vision, and other sensor-based mapping and localization systems, path planning and steering systems, and emotive human-robot interaction systems. The platform has been designed to allow for flexibility and extensibility while assuring safety and stability. The ROS open source library has been utilized to leverage middleware solutions common to many problems in robotics. This platform has been demonstrated to have the capability to operate safely around humans in a dynamic and unmodified environment, giving tours of the Glennan building at Case Western Reserve University. This platform allows for teleoperation as well as full autonomous functionality.

Committee:

Wyatt Newman (Advisor); Francis Merat (Committee Member); Greg Lee (Committee Member)

Subjects:

Computer Engineering; Computer Science; Robotics; Robots

Keywords:

ROS; robot; tour guide;

Eduafo, Augusta K.Mechanisms of Hyperglycemia-Induced ROS Production in Osmotically Swollen Glial Cells
Master of Science (MS), Wright State University, 2015, Anatomy
Cerebral edema is a potentially fatal complication of diabetes. There is evidence for increased Reactive Oxidative Species (ROS) production during cerebral edema in diabetics. This can lead to oxidative stress, thought to contribute to the onset and progression of diabetes and can cause cell injury and cell death. ROS, in moderate amounts, are involved in physiological processes within the body that produce favorable cellular responses. Unfortunately, high levels of ROS can lead to cellular damage of lipids, membranes, proteins and DNA. Hyperglycemia can cause an increase in ROS production due to activation of NADPH oxidases. The purpose of this study is to determine if there is increased ROS production in swollen hyperglycemic cells thus leading to cell injury and also to determine the mechanism by which this increased ROS production occurs. C6 rat glioma cells were grown in normal glucose medium or were exposed to high glucose medium for 24 hours to simulate diabetic conditions. Cells were then perfused with isoosmotic and hypoosmotic PBS and ROS production was measured over a 30-minute experiment. There was no change in ROS production when cells grown in normal glucose (normoglycemic) medium were osmotically swollen. Cells grown in high glucose (hyperglycemic) medium had increased basal ROS production compared with cells grown in normal glucose medium. These cells further increased their ROS production when osmotically swollen. Blocking Nox2 and Nox4 enzyme activity decreased basal ROS production of cells grown either in normoglycemic or hyperglycemic conditions. Blocking Nox2 and Nox4 also decreased ROS production of hyperglycemic cells during hypoosmotic exposure. However, cells grown in normal glucose medium increased their production of ROS when osmotically swollen in the presence of Nox2 and Nox4 inhibitors. Western blot analysis was also done to determine expression of eNos in hyperglycemic cells as compared to normoglycemic cells. There was no difference in expression of eNos in hyperglycemic cells compared to normoglycemic cells. Normoglycemic cells did not increase their ROS production in hypoosmotic conditions. Hyperglycemic cells had increased ROS production in isoosmotic conditions. This increase in ROS production is even greater when hyperglycemic cells are swollen. The increase in ROS production in hyperglycemic cells may be due to Nox, but it’s not due to eNos. Swelling hyperglycemic cells in normoglycemic conditions causes mild cell injury. Hyperglycemia, a major risk factor of diabetes, causes cellular injury but even more so when there is cellular edema.

Committee:

James Olson, Ph.D. (Advisor); Debra Mayes, Ph.D. (Committee Member); Adrian Corbett, Ph.D. (Committee Member)

Subjects:

Biomedical Research; Health Care; Neurosciences

Keywords:

ROS; hyperglycemia; glial; DHE; DPI; NADPH oxidase; Nox2; Nox4; swollen;

Chakraborty, RikhiaHomeostatic Regulation of Interleukin-4-Mediated Cell Signaling
Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2009, College of Science
Cytokines are intracellular messengers that activate multiple signaling pathways and regulate cell survival, differentiation, migration, apoptosis and immune responses. We are investigating the homeostatic control of cytokine-mediated cell signaling using interleukin-4 (IL-4) as a model cytokine. Protein tyrosine phosphorylation is essential for cytokine-dependent signal transduction through the Janus Kinase-Signal Transducer and Activator of Transcription (Jak-STAT) pathway. Therefore, protein tyrosine dephosphorylation is a key mechanism providing the homeostatic control of the pathway. Here, we have shown for the first time that immediately following ligand-dependent activation, IL-4 receptor induces an intracellular calcium flux via insulin receptor substrate (IRS)-phosphoinositide 3-kinase (PI3K)-phospholipase C (PLC)-gamma pathway which, in turn, induces protein kinase C (PKC)-dependent activation of NAD(P)H oxidase (NOX)5 that generates reactive oxygen species (ROS). IL-4 also induces NOX1-mediated ROS production via IRS-PI3K-Ras-related C3 botulinum toxin substrate (RAC) 1 pathway. Moreover, we have also demonstrated that IL-4-generated ROS, in turn, promote IL-4 receptor activation by oxidatively inactivating protein tyrosine phosphatase 1B (PTP1B) that physically associates with and deactivates IL-4 receptor. In addition, we have shown that antioxidant enzymes peroxiredoxin II, IV and VI (Prx II, IV, VI) physically interact with the IL-4 receptor and become oxidized by IL-4 generated ROS, thereby neutralizing IL-4 generated ROS and compromising ROS-mediated amplification of IL-4 signaling. Cytokine-activated Jak-STAT pathway is believed to operate from cell surface to the nucleus via DNA-protein and protein-protein interactions without involving any second messengers. Here, we demonstrate, for the first time to our knowledge, a role for second messengers (ROS) in the amplification of Jak-STAT signal transduction in IL-4-stimulated cells. Further, our data suggests that redox status of cells controls the magnitude of IL-4 receptor activation and signal transduction.

Committee:

Saikh J. Haque, Ph.D. (Advisor); Anton Komar, Ph.D. (Committee Member); Barsanjit Mazumder, Ph.D. (Committee Member); Dennis J Stuehr, Ph.D. (Committee Member); Xiaoxia Li, Ph.D. (Committee Member)

Subjects:

Biochemistry; Biology; Biomedical Research; Cellular Biology; Immunology; Molecular Biology

Keywords:

IL-4; ROS; NOX; Jak-STAT; Peroxiredoxins

Chen, LiangIkappaB Kinase beta in the Regulation of Cell Migration, Senescence and Fibrosis
PhD, University of Cincinnati, 2012, Medicine: Toxicology (Environmental Health)
IKKβ is a protein kinase critical for the transmission of inflammatory cytokine signals, such as TNFα, to activation of transcription factor NF-κB and regulation of gene expression. Genetic studies in mice, however, show that IKKβ is not only involved in inflammation, but also plays a pivotal role in tissue homeostasis and stress responses. Our previous studies show that IKKβ is essential for maintaining redox homeostasis, but the underlying mechanisms and biological consequences have not been understood. The central theme of this thesis is to investigate how IKKβ modulates homeostasis and what roles it plays in biological processes. The thesis is composed of three main components. First, mouse fibroblasts were used to investigate the molecular signatures and functions of IKKβ. I showed that genetic knockout of Ikkβ gene in fibroblasts reduced the expression of anti-oxidants thus increased the intracellular ROS level. The increased ROS in turn activated AP-1, leading to transcriptional activation of the TGFβ gene promoter and its expression, while increased TGFβ can act through NOX4 to further stimulate ROS production. Removal of IKKβ therefore triggered the autocrine amplification of the ROS-TGFβ loop that led to progressive ROS accumulation. The IKKβ-deficient fibroblasts ultimately displayed SMAD activation, leading to the overproduction of extracellular matrices and remodeling enzymes, accelerated cell motility, and enhanced markers for myofibroblast transformation and cellular senescence. These results suggested that IKKβ safeguarded the homeostasis of fibroblasts by repressing a ROS-AP-1-TGFβ autocrine regulatory loop. Second, the in vivo roles of IKKβ were assessed in keratocytes, the fibroblasts in corneal stoma of the eye. I generated mice with Ikkβ ablation specifically in corneal keratocytes. While the knockout mice had normal corneal appearance, suggesting that IKKβ was dispensable for corneal keratocytes development and maintenance, they exhibited aggravated infiltration of inflammatory cells, cellular senescence and severe scar formation compared to normal mice in response to alkali burn injury. The wounded corneas of the knockout mice also exhibited enhanced activation of stress response and fibrogenic pathways, including JNK/AP-1 and TGFβ/SMAD cascade. Hence, similar to fibroblasts, keratocytes in corneal stroma utilize IKKβ for maintaining homeostasis and repressing ROS and TGFβ during injury responses Finally, the roles of IKKβ in epithelial cells of the cornea were investigated by generating and using the corneal epithelial-specific Ikkβ knockout mice. These mice had normal cornea development and maintenance, but significantly delayed healing of corneal epithelium wounds. Furthermore, delayed wound healing in the knockout cornea was not due to reduced cell proliferation or increased apoptosis, but was correlated to a slower epithelial cell migration. Taken together, results presented in this thesis reveal that IKKβ has highly cell type-specific functions apart from its well-recognized roles in inflammatory responses. While IKKβ promotes corneal epithelial cell migration at the surface of ocular tissues, it prevents senescence, fibrosis and scar tissue formation in corneal stromal fibroblasts in response to injuries. Correspondingly, IKKβ represses a ROS-TGFβ regulatory loop in fibroblasts, thereby preventing ROS, cell migration, senescence and fibrosis.

Committee:

Ying Xia, PhD (Committee Chair); Winston Whei Yang Kao, PhD (Committee Member); Richard Lang, PhD (Committee Member); Chia-Yang Liu, PhD (Committee Member); Alvaro Puga, PhD (Committee Member)

Subjects:

Environmental Health

Keywords:

IKK&946;;TGF&946;;ROS;cornea;wound healing;fibrosis;

Wang, XinglongImpaired Balance of Mitochondria Fission and Fusion in Alzheimer Disease
Doctor of Philosophy, Case Western Reserve University, 2009, Pathology
Alzheimer's disease (AD) is the leading cause of dementia in the elderly, characterized by neurofibrillary tangles, senile plaques and a progressive loss of neuronal cells in selective brain regions. Mitochondrial dysfunction is a prominent and early feature of the disease, although the reason for this is unclear. Nonetheless, emerging evidence suggest that mitochondrial function is dependent on the dynamic balance of fission and fusion events which are regulated by mitochondria fission and fusion proteins (i.e. DLP1/OPA1/Mfn1/Mfn2/Fis1). While an impaired balance of mitochondria fission and fusion is being increasingly implicated in neurodegenerative diseases, few studies have examined this aspect in AD. To address this issue, in this study, we investigated mitochondria morphology and distribution in primary fibroblasts and neurons from normal subjects and those from AD patients. We found disease-related changes in mitochondrial morphology and distribution as well as changes in expression levels and distribution of mitochondrial fission and fusion proteins. To understand the underlying mechanisms of these mitochondria alterations in AD, we overexpressed or knocked down functional mitochondrial fission and fusion proteins in primary human fibroblasts, M17 cells (derived from a human neuroblastoma) and rat E18 primary hippocampal neurons. Interestingly, functional protein changes in AD fibroblasts or neurons mimicking that found in AD, are correlated with similar changes in mitochondrial morphology and distribution to that observed in AD fibroblasts or neurons. We further demonstrated that ROS or amyloid-β are likely the potential pathogenic factor that causes an impaired balance of mitochondrial fission/fusion, mitochondria dysfunction and even synaptic abnormalities. Taken together, this is the first study to show that ROS or amyloid-β might induce mitochondria dynamic abnormalities in mitochondria, mitochondrial dysfunction and further neuronal dysfunction through their different effect on mitochondrial fission and fusion proteins.

Committee:

Mark A. Smith, PhD (Committee Chair); Xiongwei Zhu, PhD (Advisor); Robert B. Petersen, PhD (Committee Member); Shigemi Matsuyama, PhD (Committee Member); Clive R. Hamlin, PhD (Committee Member); George Perry, PhD (Committee Member)

Subjects:

Pathology

Keywords:

Alzheimer disease (AD), Mitochondrial dysfunction, amyloid-&946;, DLP1, OPA1, Mfn1, Mfn2, Fis1, mitochondria fission and fusion, mitochondrial morphology and distribution, ROS

Perko, Eric MichaelPrecision Navigation for Indoor Mobile Robots
Master of Sciences, Case Western Reserve University, 2013, EECS - Computer and Information Sciences
This thesis describes a precision navigation system for indoor mobile robots, developed to address deficiencies in the ROS navigation stack when used for precision navigation. It includes a precision localization subsystem, based on a planar laser scanner, wheel encoders, a gyroscope and an a priori map, and a precision path execution system made up of a steering algorithm, a trajectory generator and a simplistic path planner. A 3D octree costmap based on the OctoMap library was also developed for collision detection. Geometric parameterizations for path segments were developed for use by those components. The precision navigation system was evaluated using a physical robot, CWRU's HARLIE, as well as in a Gazebo simulation. This precision navigation system allowed HARLIE to precisely navigate indoors, following paths made up of straight lines, constant curvature arcs and spin-in-place segments with as little as just over three centimeters of RMS lateral offset error.

Committee:

Wyatt Newman, PhD (Advisor); Gregory Lee, PhD (Committee Member); Francis Merat, PhD (Committee Member)

Subjects:

Robotics; Robots

Keywords:

precision navigation; mobile robot; ROS; localization; steering; trajectory generation; OctoMap

Kizhakkekkara Vadukoot, AnishTargeting the Stress Response to ROS: Design and Development of Novel and Selective Anti-cancer Agents
PhD, University of Cincinnati, 2016, Arts and Sciences: Chemistry
Cancer is defined as abnormal growth of cells which tend to proliferate in an uncontrolled way and, in some cases, to metastasize. Cancer can be initiated by various factors working alone or in combination. Some cancers are caused by external factors such as tobacco, diet, certain chemicals, radiation, and viruses while other cancers are caused by internal factors such as hormones, immune conditions, and inherited genetic mutations. Damage to DNA by reactive oxygen species (ROS) is widely accepted as a major cause of cancer. ROS are chemically reactive molecules formed as a natural byproduct of metabolism in cells. These species play an important role in cell signaling and homeostasis. ROS levels are kept under control by cellular antioxidant systems. Whenever there is an imbalance in ROS generation ad its regulation, it leads to oxidative stress. Elevated levels of ROS have been detected in different types of cancer cells where they are known to promote tumor progression and survival including renal cell carcinoma, melanoma, and leukemia. These ROS levels are higher when compared to normal cells. This high ROS levels in cancer cells makes them susceptible to oxidative stress induced cell death and can be exploited for development of selective anticancer therapy. Many chemotherapeutic strategies are designed to increase cellular ROS levels to induce irreparable damages to cell leading to apoptosis. This can be achieved by using compounds that inhibit antioxidant systems or through inhibition of specific signaling pathways that upregulate antioxidants in cancer cells. The resulting increase in ROS may induce tumor cell death either through damaging functions of ROS like DNA oxidation, lipid peroxidation or by specific initiation of apoptosis via death signaling pathways. As basal ROS levels in normal cells are comparatively low, these cells are not significantly affected leading to selectivity in chemotherapy. My work focuses on design and development of novel anticancer agents that are ROS activated. These are stable prodrugs which are activated only in the presence of ROS. Upon activation they tend to form a potent electrophile which can react with cellular nucleophiles such as DNA or cellular antioxidants causing their inactivation leading to ROS imbalance and ultimately triggering apoptosis.

Committee:

Edward Merino, Ph.D. (Committee Chair); Michael Baldwin, Ph.D. (Committee Member); James Mack, Ph.D. (Committee Member)

Subjects:

Organic Chemistry

Keywords:

ROS;Cancer;Chemotherapy;Prodrugs;DNA alkylating agents;Acute Myeloid Leukemia

Ricker, Justin TInhibition of OV2008 Cancer Cell Proliferation in the Presence of Oleoylethanolamide, JW480 and Chlorpyrifos-oxon
BS, Kent State University, 2015, College and Graduate School of Education, Health and Human Services / School of Health Sciences
Acylethanolamide oleoylethanolamide (OEA) and its metabolically stable analog AM3102 (N-[(1R)-2-hydroxy-1-methylethyl-9Z-octadecenamide) induces apoptosis in OV2008 ovarian adenocarcinoma cells independent of PPAR-a receptor signaling pathway (ASCB 2012, Abstract #888). This cytotoxicity is reversed in the presence of a-tocopherol, indicative of reactive oxygen species (ROS) involvement in cell death. Also, palmityl trifluoromethyl ketone (PTK), independent of its inhibitory effect on phospholipase A2, enhances the toxicity of OEA (ASCB 2012, Abstract #888). The enzyme neutral cholesterol ester hydrolase 1 (NCEH1) has also been shown to be a target of trifluoromethylketones (Nat Biotechnol 21:687, 2003). Inhibition of NCEH1 leads to reduced migration of SKOV3 ovarian cancer cells in vitro and their growth in vivo (Chem Biol 13: 1041, 2006). Therefore, in this study, using Chlorpyrifos-Oxon (CPO) and JW480, potent inhibitors of the enzyme NCEH1, I explored the relationship between OEA and NCEH1 in the OV2008 cancer cell line. Cytotoxicity was observed in response to both OEA (IC50: 14-21 µM) and CPO (IC50: 48-50 µM). When the compounds were administered together, cytotoxicity was enhanced (OEA IC50: 8-9 µM; CPO IC50: 15-19 µM) with evidence of weak synergism. Western blot analysis and immunofluorescent localization of OEA, CPO, and JW480 treated cells showed that CPO appears to increase the expression of NCEH1. Furthermore, Western blot analysis of the antioxidant enzymes GST-µ, GPX4, Mn-SOD and Zn/Cu-SOD, and the autophagic marker, light chain 3 A/B shows that OEA, CPO, or JW480 do not appear to affect the expression of these proteins. Live cell labeling with dihydroethidium showed that these compounds have no statistically significant effect on overall superoxide production. Live cell fluorescence microscopy using MitoSOX Red, however, revealed an increase in superoxide anion in the mitochondria in the presence of OEA. The results of this study suggest that one mechanism by which OEA induces cytotoxicity in OV2008 cells is via superoxide generation. It is possible that OEA-mediated increase in ROS makes the OV2008 cells further susceptible to CPO and JW480. I also demonstrate an uncharacterized exaggerated pH gradient across the cellular membrane of cells treated with OEA and JW480, with the effects being more pronounced after incubation in JW480. The cause of this exaggerated gradient remains to be elucidated.

Committee:

Paul Bagavandoss (Advisor); Matthew Lehnert (Committee Member); Julie Cremeans-Smith (Committee Member); Leslie Heaphy (Committee Member)

Subjects:

Biochemistry; Oncology

Keywords:

OEA; oleoylethanolamide; CPO; Chlorpyrifos-oxon; JW480; NCEH-1; OV2008; KIAA1363; ROS; reactive oxygen species; pH

Willis, William LYB-1 Stress-Response Protein Conformation Implicated in Post-transcriptional Control of Myofibroblast Differentiation
Doctor of Philosophy, The Ohio State University, 2013, Integrated Biomedical Science Graduate Program
Vascular smooth muscle alpha-actin (SMA) is an indicator of myofibroblast differentiation, as well as one of several fetal contractile protein isoforms re-expressed in adult cardiomyocytes in response to mechanical stress-injury. The stress-response protein, Y-box binding protein-1 (YB-1) binds SMA mRNA and regulates its translational activity. Our central hypothesis is that YB-1 drives maladaptive SMA expression in injury-activated myofibroblasts by modulating the packaging, delivery, and translational efficiency of its cognate mRNA. In a mouse model for cardiac fibrosis, we observed that accumulation of fetal SMA protein in cardiac sarcomeres was associated with accumulation of punctate YB-1 deposits which localized to perinuclear regions as well as polyribosome-enriched cytosol proximal to cardiac intercalated discs. Samples from both fibrotic mouse hearts as well as SMA positive endomyocardial biopsies from human heart transplant patients were enriched with high molecular weight, heat-denaturing resistant YB-1 oligomers migrating in the range of 100-250 kDa during reducing SDS-PAGE. Based on these intriguing observations, which suggested that YB-1 oligomer formation may be associated with the packaging and translation control of SMA mRNA, we examined the regulatory aspects of YB-1 oligomerization using a model system based on isolated human pulmonary fibroblasts (hPFBs). Activation of SMA gene expression in hPFBs by TGFbeta1 was associated with formation of denaturation-resistant YB-1 oligomers with selective affinity for a SMA exon-3 translation-silencer sequence. We discovered that YB-1 is a substrate for the protein-crosslinking enzyme transglutaminase 2 (TG2) that catalyzes calcium-dependent formation of covalent gamma-glutamyl-isopeptide linkages in response to reactive oxygen signaling. TG2 transamidation reactions using intact cells, cell lysates, and recombinant YB-1 revealed covalent crosslinking of the 50 kDa YB-1 polypeptide into protein oligomers distributed during SDS-PAGE over a 75 kDa to 250 kDa size range. In vitro YB-1 transamidation required nanomolar levels of calcium and was enhanced by the presence of SMA mRNA. YB-1 crosslinking was reversible as a function of free-calcium concentration and TG2 enzyme availability. Metabolic stress incurred during tissue injury may also promote conversion of fibroblasts to the myofibroblast phenotype, as temporary loss of oxygen perfusion promotes a hypoxic, energy-deficient pro-oxidative cellular microenvironment. Stimulation of AMP-activated kinase (AMPK) activity with 5-Aminoimidazole-4-carboxyamide ribonucleoside (AICAR) activated TG2 transamidation and induced the formation of high molecular weight YB-1 oligomers with enhanced affinity for the SMA mRNA exon-3 translation-silencer sequence. AMPK and peroxide administration differentially regulated phosphorylation of the YB-1 cold-shock domain (CSD), which modulates YB-1 subcellular localization and SMA mRNA binding efficiency. AICAR suppressed YB-1 phosphorylation, which prevented nuclear translocation and activated SMA mRNA binding. In contrast, peroxide stimulation activated Erk/MAPK dependent phosphorylation of the YB-1 CSD and dispersed YB-1: SMA mRNA complexes. Thus, we propose that coordinated AMPK activation and ROS production during myofibroblast differentiation regulates SMA expression at the post-transcriptional level, by coordinating the respective packaging and deployment/translation activation of SMA mRNA from YB-1 ribonucleoprotein complexes. In summary, intracellular calcium accumulation and increased ROS levels incurred during metabolic and biomechanical stress may govern SMA mRNA translational activity during wound healing and cardiopulmonary stress responses via TG2-mediated crosslinking of the YB-1 mRNA-binding protein.

Committee:

Arthur Strauch, PhD (Advisor); Denis Guttridge, PhD (Committee Member); Lai-Chu Wu, PhD (Committee Member); Mark Ziolo, PhD (Committee Member)

Subjects:

Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology

Keywords:

YB-1; transglutaminase 2; translational control; RNA-binding protein; myofibroblast; AMPK; smooth muscle alpha-actin; cardiac remodeling; cardiac intercalated disc; metabolic stress; fibrosis; ROS; TGFbeta1

Stuckey, Crystal ElaineOxidative Stress and Cell Death in Osmotically Swollen Glial Cells
Master of Science (MS), Wright State University, 2008, Anatomy
Regulation of normal volume is an important aspect of cell homeostasis. Possible mechanisms which signal volume regulation are increasing the rate of reactive oxygen species (ROS) production and release of ATP for interaction with purinergic receptors. We examined whether an increase in ROS production during cell swelling also led to cell injury of C6 glioma cells. Cells were loaded with 5,6-carboxy-2,7-dihydrofluorscein diacetate (DCFDA) to fluoroscopically measure the rate of cellular ROS production and were perfused with phosphate-buffered saline solutions (PBS) containing 100 μM carbenoxolone to inhibit dye efflux. Cell death was determined cytometrically and by measuring the release of lactate dehydrogenase (LDH) into the culture medium 24 hr after exposing cells for 60 min to isoosmotic or hypoosmotic PBS. Immediately after changing the perfusion solution from isoosmotic to hypoosmotic PBS, the production of ROS increased by 60.2% + 19.6, but returned to baseline after 5 min. Increased efflux of ATP was not observed in hypoosmotic conditions. ROS production was not directly activated by endogenously applied extracellular ATP, but ATP increased ROS production in swollen cells. Cells swollen by hypoosmotic solutions had a slight increase in the probability of necrotic cell death. Our data suggest that increased ROS produced by cells swollen during hypoosmotic stress does not lead to significant cell injury in cultured C6 cells.

Committee:

James Olson, PhD (Advisor); Robert Putnam, PhD (Committee Member); Thomas Brown, PhD (Committee Member)

Subjects:

Cellular Biology; Microbiology; Neurology

Keywords:

C6; glia; DCFDA; hypoosmotic; ROS; ATP; cell death; NADPH oxidase; mitochondrial ETC; DPI; oligomycin; rotenone

Zhu, LinBiocompatibility of Carbon Nanomaterials: Materials Characterization and Cytotoxicity Evaluation
Doctor of Philosophy (Ph.D.), University of Dayton, 2012, Engineering

The objective of this research is to investigate the biocompatibility of carbon nanomaterial. It was found that the cytotoxicity of multiwalled carbon nanotubes (MWNTs) depend on their concentration, size, and surface chemical groups (e.g., -COOH). MWNTs and MWNT-COOH could accumulate in human lung macrophage cells (U937) to different degrees, and they did not produce overt cell toxicity within the concentration range of 5-50 µg/ml up to 24 h. However, there were morphological alterations at low doses of MWNT-COOH and significant reactive oxygen species (ROS) generation for MWNTs at higher doses, indicating a distinguished possible cellular stress response and DNA damage from both materials.

In the second part of this study, reduced graphene oxide (rGO) was demonstrated to show the concentration-dependent and cell-specific cytotoxicity. Specifically, rGO was found to stimulate cell proliferation of human skin fibroblast cells at relatively low concentrations (< 5 µg/mL), but inhibit human skin fibroblast cells proliferation at high concentrations. rGO-induced concentration-dependent and cell-specific generation of ROS and activation of NF-κB transcription factors were also observed, indicating an oxidative stress mechanism. Furthermore, rGO was showed to be more biocompatibility to human skin fibroblast cells with respect to mouse embryonic fibroblast cells (NIH-3T3 cells).

In the third part of this study, the soft lithography technique was used to build PDMS microfluidic devices for monitoring cells viability and performing dynamic study of the carbon nanomaterial biocompatibility. Compared to the traditional in vitro technique, this research opens up a new approach to biocompatibility evaluation of nanomaterials with a reduced usage of animal in toxicity study. By using the newly-developed microfluidic devices, the biocompatibility of MWNTs and rGO were investigated. It was found that both particles could enter into the circulation system in the microfluidic devices. Possible damage to bovine aortic endothelium cells (BAECs) caused by carbon nanomaterials was investigated. The interaction of MWNTs (1D) and rGO sheets (2D) with BAECs in both static (cell culture) and dynamic (microfluidic) environments indicated that both nanoparticles reduced the mitochondrial function and lipoprotein (LDL) uptake. These results were concentration and morphology depended. MWNTs showed a better biocompatibility than rGO in both static and dynamic environments, while the microfluidic tests exhibited better biocompatibilities than those in cell culture dishes for both nanoparticles.

Committee:

Daniel Eylon, PhD (Committee Chair); Liming Dai, PhD (Advisor); Harihara Baskaran, PhD (Committee Member); Donald Klostermen, PhD (Committee Member); P. Terrence Murray, PhD (Committee Member); Gerald Shaughnessy, MS (Committee Member); James Snide, PhD (Committee Member)

Subjects:

Chemical Engineering

Keywords:

carbon nanotube; MWNT; SWNT; rGO; graphene; DNA damage; cytotoxicity; microarray; U937; NIH-3T3 cell; BAEC; Human fibroblast cell; microfluidic; ROS; MTT

Adams, Gregory NicholasProlylcarboxypeptidase protects from vascular dysfunction and promotes vascular repair
Doctor of Philosophy, Case Western Reserve University, 2013, Pathology
Prolylcarboxypeptidase (PRCP) is a cell surface protease that degrades peptides (including angiotensin II, des-Arg9-bradykinin and alpha-melanocyte stimulating hormone) and activates plasma prekallikrein. The enzyme is highly expressed by endothelial cells where it activates prekallikrein to liberate the vasoactive peptide bradykinin. The following studies characterize the vascular phenotype of the PRCP-deficient (PRCPgt/gt) mouse. Compared to wildtype animals, PRCPgt/gt are hypertensive and demonstrate faster times to induced arterial thrombotic occlusion. PRCPgt/gt have increased levels of vascular reactive oxygen species (ROS) and the hypertensive, prothrombotic phenotypes are corrected by oral antioxidant treatment. The vascular dysfunction induced by PRCP depletion was observed in vitro by siRNA treatment of endothelial cells. Additionally, PRCPgt/gt mice show signs of defective angiogenesis and vascular repair. The animals have decreased presence of vessels in sub-cutaneous injections of matrigel and decreased endothelial sprouting from excised aortic segments. Full thickness skin wounds from PRCPgt/gt heal slower than wildtype mice at the visual level and have decreased invasive blood vessel repair. PRCPgt/gt exhibit defective repair of large vessels as demonstrated by slow recovery from hind limb ischemia and thickened neo-intima formation following wire injury of the femoral artery. In sum, this work suggests PRCP protects from the pathological manifestations of vascular dysfunction and promotes angiogenesis and large vessel repair.

Committee:

Alvin Schmaier, MD (Advisor); Keith McCrae, MD (Committee Chair); Mukesh Jain, MD (Committee Member); Roy Silverstein, MD (Committee Member); Clive Hamlin, Ph.D. (Committee Member)

Subjects:

Biomedical Research; Pathology

Keywords:

hypertension; thrombosis; reactive oxygen species; ROS; endothelial; angiogenesis

Downing, TrevorTHE RELATIONSHIP BETWEEN LACTIC ACID, REACTIVE OXYGEN SPECIES AND THE HYPOXIA-INDUCED ACIDIFICATION SEEN IN CHEMOSENSITIVE NEURONS OF THE NUCLEUS TRACTUS SOLITARIUS (NTS)
Master of Science (MS), Wright State University, 2006, Anatomy
The NTS is one of many sites of chemoreception meaning that it responds to changes in CO2 and pH. NTS neurons also acidify approximately 0.13 pH units in response to hypoxia. Experiments involved brainstem slices exposed to a control hypoxic bout followed by hypoxia in conjunction with different drugs. The drugs included fluorocitrate, 4-hydroxycinnamate, oxygen-glucose deprivation and iodoacetate. Iodoacetate produced the strongest blunting of the hypoxia-induced acidification and decreased the response by ~53%. Results from the studies using the superoxide probe DHE showed an increase in the levels of ROS during the hypoxic exposure prior to reoxygenation. Two ROS scavengers melatonin and manganese(III)tetrakis(1-methylpyridyl) porphyrin pentachloride both reduced the increase in ROS by ~30%. In conclusion, the hypoxia-induced acidification seems to be due to lactate production which is proposed to come from neuronal sources as the glial source of lactate was ruled out with the fluorocitrate and 4-hydroxycinnamate studies.

Committee:

Jay Dean (Advisor)

Keywords:

hypoxia; ACIDIFICATION; ROS; NEURONS; glial; glial cells; NTS

Shakeel, AmlaanService robot for the visually impaired: Providing navigational assistance using Deep Learning
Master of Science, Miami University, 2017, Computational Science and Engineering
Assistive technology helps improve the day to day activities for people with disabilities. One of the methods utilized by assistive technologists employs the use of robots. These are called service robots. This thesis explores the idea of a service robot for the visually impaired to assist with navigation and is inspired by the use of guide dogs. The focus of this thesis is to develop a robot to achieve autonomous indoor navigation using computer vision to identify image based goals in an unfamiliar environment. The method presented in this thesis utilizes a deep learning framework, called Faster R-CNN, to train a computer to classify and localize exit signs in real time. A proof of concept is presented using NVIDIA Jetson, and TurtleBot, a robot kit, which runs a robot software development framework Robot Operating System (ROS). The model is trained successfully using Faster R-CNN and is validated. The model is used for real-time object classification on the prototype robot.

Committee:

Yamuna Rajasekhar (Advisor); John Femiani (Committee Member); Donald Ucci (Committee Member)

Subjects:

Computer Science; Electrical Engineering; Robotics

Keywords:

Assistive technology; Deep learning; Robotics; Indoor navigation; Computer vision; Robot Operating System; ROS; Caffe; Faster R-CNN; Convolutional Neural Networks; CNN; Microsoft Kinect; Service robots; visually impaired; mobility; depth perception

Shy, Brigette M.Student Perceptions of Therapist Credibility Based on Attention to Religious and Spiritual Functioning at Intake
Doctor of Philosophy, University of Akron, 2012, Counseling Psychology

This investigation tested empirically whether college student participants (N = 176) would perceive a counselor during an intake interview as more credible when she asked questions of a religious and spiritual nature versus when she did not. Additionally, because research has suggested clients prefer to discuss religion and spirituality in counseling (Rose, Westefeld, & Ansley, 2001), and that level of religiosity influences perceptions of the counselor in this situation (Guinee & Tracey, 1997), the current study investigated whether preference for discussion of religious and spiritual issues and level of participant religiosity would influence counselor ratings. Results of the current study indicated students did not find the intake counselor who included religious and spiritual items in the intake session more credible than the intake counselor who did not include religious and spiritual items in the intake. Results also demonstrated preferences for discussing religious and spiritual issues in counseling and participant level of religiosity did not moderate the relationship between group assignment (religious and spiritual experimental condition versus non-religious and spiritual experimental condition) and counselor ratings on the CRF-S (Corrigan & Schmidt, 1983).

Analyses comparing participants who had never received counseling before (n = 115) and participants who had previous counseling experience (n = 61), suggested the participants who had no previous counseling experience rated the counselor in both role plays significantly higher overall. Exploratory analyses with a short-answer open-ended opinion question suggested some students (31%) want to discuss religious and spiritual issues in counseling; some students (23%) stated their preference to discuss religious and spiritual issues in counseling depended on other factors; and some students (41%) did not want to discuss religious or spiritual issues in counseling. The fact that student participants rated the counselor who introduced religious and spiritual issues in this brief section of an intake interview similar to when she did not is noteworthy and is discussed in terms of implications for research, training, supervision, and clinical work.

Committee:

Charles Waehler, Dr. (Advisor); Linda Subich, Dr. (Committee Member); Suzette Speight, Dr. (Committee Member); John Queener, Dr. (Committee Member); Linda Perosa, Dr. (Committee Member)

Subjects:

Psychology

Keywords:

credibility; competence; rating; counselor; CRF-S; ROS; CAST; ATSPPH-S: religion; religious; religiosity; level; spiritual; preference; student; client; research; analogue; roleplay; raters; religious and spiritual functioning; cultural sensitivity

Carpenter, Oliver L.Ultraviolet Light-Induced Regulation of Transcription and Translation, COX-2 Expression and Noncanonical NF-κB Activation
Doctor of Philosophy (PhD), Ohio University, 2013, Chemistry and Biochemistry (Arts and Sciences)
NF-κB is a transcription factor that controls expression of genes involved in the immune and inflammatory responses as well a being a key component in the onset of cancers. In this study we provided evidence that MSK1 is responsible for a non-canonical late-phase activation of NF-κB upon UVB irradiation. Our data demonstrated that following UV irradiation, MSK1 is activated via phosphorylation at the 24 h time point coinciding with translocation of NF-κB into the nucleus. Investigations into the signaling pathways upstream of MSK1 through the use of specific inhibitors for mitogen-activated protein kinase and p38 revealed that both kinases are required for full phosphorylation during the late-phase (24 h), while p38 is paramount for phosphorylation during the early-phase (6 h). Electromobilty shift assays (EMSA) showed that inhibition of MSK1 resulted in a marked reduction in NF-κB binding affinity without altering the nuclear translocation of NF-κB. Supershift EMSA implicate that the p65, but not p50, isoform of NF-κB is involved in late-phase activation in response to UVB irradiation. Together, the results of these studies shed light onto a novel pathway of MSK1 mediated late-phase activation of NF-κB in response to UV irradiation. Exposure to UV induces a prolonged expression of COX-2 via transcriptional activation that is due in part to an increase in NF-κB activity. While transcriptional regulation of COX-2 expression has been well studied, the role of translational regulation of COX-2 synthesis upon UV-irradiation is not yet clear. In this study, we show that the phosphorylation of the alpha subunit of eIF2α plays an important role in the regulation of COX-2 expression after UV-irradiation. Our data demonstrates that UV light induces COX-2 expression in wild-type mouse embryo fibroblasts (MEFS/S) and that the inducibility is reduced in MEFA/A cells in which the phosphorylation site, Ser-51 in eIF2α, is replaced with a nonphosphorylatable Ala (S51A). UV light-induced transcription of COX-2 is delayed in MEFA/A cells, which correlates with NF-κB activation as we previously reported (Wu et. Al, J. Bio. Chem, 2004). Additionally, the translational regulation of COX-2 binding protein TIAR expression is reduced in MEFS/S cells but not in MEFA/A cells at 24 hours post-UV. These results suggest that translation initiation plays a role in a complex and dynamic regulation of COX-2 expression. Based on our results we proposed a novel eIF2α phosphorylation-centered network for the regulation of COX-2 expression after UV irradiation.

Committee:

Shiyong Wu, PhD (Advisor)

Subjects:

Biochemistry; Cellular Biology; Molecular Biology; Oncology

Keywords:

Nuclear Factor Kappa B; NF-kB; COX2; MSK1; Ultraviolet; UV; Ultraviolet light; Mitogen and Stress Activated; NF-kB regulation; Nuclear Factor Kappa B regulation; MAPK; MSK1 regulation; UV Light; ROS; Translation and Transcription Regulation

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