Doctor of Philosophy, The Ohio State University, 2007, Integrated Biomedical Science

Organ transplantation is first choice therapy for end stage organ failure. Advances in clinical transplantation have resulted in improved short term transplant survival for most solid organ allografts, yet long term graft survival with preserved organ function is still elusive. Current therapies effectively control conventional immune pathways contributing to transplant rejection, however unconventional rejection pathways have emerged, including CD4-independent, CD8 +T cell mediated rejection, antibody-mediated acute rejection, and memory T cell-mediated rejection. These immune pathways have all been described as significant barriers to establishing long term survival of transplanted organs. A murine model of hepatocellular transplantation activates conventional as well as unconventional T cell-dependent rejection pathways. This prompted studies to investigate the activation, effector mechanisms, and peripheral phenotypes of responding rejection pathways, with emphasis on unconventional rejection. The hypothesis was that allogeneic hepatocytes promote activation of recipient CD8 +T cells through exclusive expression of MHC class I (independent of recipient immune factors) which is manifest by a dominant CD8 +T cell-mediated cytotoxic phenotype of rejection; only in the absence of recipient CD8 +T cells, rejection deviates to a CD4 +T cell-mediated immune pathway and a distinct effector mechanism. The studies showed, in contrast to the hypothesis, recipient immune factors, such as the location of hepatocyte engraftment or the presence of CD4 +T helper cells, significantly influenced CD8 +T cell activation, effector function and mechanism, memory responses, and susceptibility to immune therapy. The data support the modified hypothesis that CD8 +T cells mature along distinct effector pathways depending on the presence of CD4 +T cell help during priming with alloantigen. In the absence of recipient CD8 +T cells, hepatocellular allografts initiate CD4-mediated rejection, (open full item for complete abstract)

Committee: Ginny Bumgardner (Advisor) Subjects: Health Sciences, Immunology

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Sciences

CD4+ T cells are a subset of the adaptive immune response that are classically defined as “helper” type cells, responsible for aiding in the effective recruitment and function of other immune cells. In addition to these traditional helper type roles, however, an additional subset of CD4+ T cells characterized by the ability to perform cytotoxic functions classically attributed to Nature Killer (NK) and CD8+ T cells, has emerged. This additional subset, deemed CD4+ cytotoxic T lymphocytes (CD4-CTLs), produce cytolytic effector molecules and directly kill compromised cells in a major histocompatibility complex class-II (MHC-II) restricted fashion. While initially thought to be a bi-product of in vitro culturing, CD4-CTLs have now been identified in a broad range of both human and murine immune responses. Specifically, CD4-CTLs have been shown to play largely protective roles in numerous antitumor and antiviral responses, including those to influenza and SARS-CoV-2. Conversely, dysregulated CD4-CTLs have been implicated in the pathogenesis of autoimmune diseases, including multiple sclerosis and ulcerative colitis. Despite their documented importance in a wide range of immunological contexts, the mechanisms that underly their differentiation and function remain enigmatic. Here, we first identify the Ikaros Zinc Finger transcription factor Aiolos (encodedby Ikzf3) as a reciprocal regulator of CD4-CTL and T-follicular helper (TFH) cell populations. TFH cells represent a classical CD4+ T helper cell populations that, upon activation and subset specific differentiation, interact with and provide aid to B-cells in the production of high-affinity neutralizing antibodies. In this work, we utilized a murine model of influenza infection, an immunologic context in which both TFH and CD4-CTLs have been shown to play important protective roles. We find that the absence of Aiolos resulted in global disruptions in TFH cell programming, including decreases in key transcription fa (open full item for complete abstract)

Committee: Ken Oestreich (Advisor); Hazem Ghoneim (Committee Chair); Thomas Mace (Committee Member); Murugesan Rajaram (Committee Member); Purnima Dubey (Committee Member) Subjects: Biomedical Research; Immunology

Bachelor of Science (BS), Ohio University, 2023, Biological Sciences

Staphylococcus aureus is a pathogenic Gram-positive bacterium of global importance due to its prevalence and remarkable ability to induce host tissue damage, making treatment of infections difficult. Determining how S. aureus causes cell death in host organisms is of particular interest to biomedical research, as interrupting this pathway could be a major target for treatment without involving traditional antibiotics, and could thus provide new strategies to treat severe and recurring antibiotic resistant infections. Recent research in our laboratory has demonstrated that extracellular vesicles (EVs) produced by S. aureus are capable of inducing macrophage cell death, and that a major component of these EVs is RNA. This project seeks to explore if bacterial-derived RNA, not protected by a vesicle, or “naked RNA,” is capable of inducing cell death in macrophages, and what factors are relevant to this effect.

Committee: Soichi Tanda (Advisor); Ronan Carroll (Advisor) Subjects: Microbiology

Doctor of Philosophy, Case Western Reserve University, 2023, Pathology

Advancements in therapy have improved remission and prolonged survival for patients with B cell malignancies. Unfortunately, for many challenging cancer subtypes such as mantle cell lymphoma, most patients will still relapse and die from the cancer. Chimeric antigen receptor (CAR)-T cell therapy uses genetically modified T cells expressing CAR protein to recognize and kill cancers expressing a specific antigen, such as CD19. Although CD19 CAR-T therapy has been very effective against relapsed/refractory B cell cancers, antigen escape and relapse still occur in up to 40% of patients treated with CD19 CAR-T. This work describes the creation and validation of a novel, BAFF ligand-based CAR that aims to prevent antigen escape by being able to bind multiple BAFF receptors expressed by the cancer cell, rather than one antigen alone. Using cytotoxicity assays, we demonstrate the functionality and specificity of BAFF CAR-T cells in killing several types of B cell malignancies, and we further confirm their efficacy in several mouse models of cancer. These results have helped bring BAFF CAR-T therapy to Phase I clinical trials, and we hope they prove efficacious for patients with relapsed/refractory B cell malignancies. Meanwhile, interest has rapidly increased in natural killer (NK) cell-based immunotherapies. NK cells have important roles in cancer immunosurveillance and offer advantages over T cells in clinical safety and time-to-treatment. However, NK therapies face challenges in efficacy and persistence, often due to the presence of TGF-β, a powerful immunosuppressive cytokine that is frequently elevated in cancer patients and a primary cause of NK cell dysfunction. In this work, we characterize an undiscovered role of cyclin dependent kinase 5 (Cdk5) and its coactivator p35 in NK cell cytotoxicity. Using genetic tools to modulate Cdk5/p35 kinase activity in human NK cells, as well as using NK cells from p35 knockout mice, we show that Cdk5/p35 negatively regulates NK (open full item for complete abstract)

Committee: Reshmi Parameswaran (Advisor); Alex Huang (Committee Chair); John Letterio (Committee Member); Clive Hamlin (Committee Member) Subjects: Biology; Biomedical Research; Immunology; Medicine; Molecular Biology; Oncology

Master of Science (MS), Wright State University, 2020, Physiology and Neuroscience

Humans are exposed daily to a variety of metals that can be harmful to our immune system. Although certain divalent metal cations are essential for numerous cellular functions and are critical trace elements in humans, the uptake mechanisms of these ions remain mostly unknown. Transient receptor potential melastatin 7 (TRPM7), which is expressed in a variety of human cell types, including lymphocytes and macrophages, conducts many divalent metal cations. TRPM7 channels are largely inactive under normal physiological conditions due to cytoplasmic magnesium acting as a channel inhibitor. Magnesium is a cofactor for many biochemical reactions. Low serum levels of magnesium, hypomagnesemia, can occur from increased magnesium loss from renal or gastrointestinal systems, redistribution of magnesium across the cell membranes, and decreased magnesium intake. Magnesium depletion allows both physiological and non-physiological divalent metal cations to enter through TRPM7, which is highly expressed in T-lymphocytes. Alterations to TRPM7 channel activity by channel blockers were found to affect the cell viability sequence. Through the use of Jurkat, a leukemic T-lymphocyte cell line which expresses high levels of TRPM7, HAP1 cells, and a TRPM7 kinase-dead mouse model, the entry of both physiological and non-physiological cations can be quantitated by measuring cell toxicity. A cell toxicity/viability assessment in Jurkat T-lymphocytes provided the sequence of Cd2+ > Zn2+ > Co2+ > Ni2+ > Mn2+ >> Sr2+ ≈ Ba2+ ≈ Ca2+ ≈ Mg2. Homeostatic mechanisms alter the effects of divalent metal cation entry and viability of T-lymphocytes, suggesting that TRPM7 in part contributes to metal ion entry.

Committee: Juliusz Ashot Kozak Ph.D. (Advisor); Christopher N. Wyatt Ph.D. (Committee Member); David R. Ladle Ph.D. (Committee Member) Subjects: Cellular Biology; Immunology; Pharmacology; Physiology

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

Zinc is an essential element for life, and its vital benefit for human health is well known in the field of food and nutrition. Due to their low toxicity and high biosafety, zinc compounds have been used very early by ancient people to cure wounds and eye sores. Furthermore, zinc as a promising antimicrobial agent has been confirmed by a series of recent reports. Thus far, most studies of its antimicrobial activity have been focused on ZnO NPs prepared by the conventional bottom-up methods such as solution preparation, hydrothermal synthesis, to name but a few. As the result, the reported minimal inhibitory concentration (MIC) values of these NPs range from μM to mM. The general mechanism of metal-based NPs is widely debated in the literature. Generally, ZnO-based NPs can produce reactive oxygen species (ROS) that lead to the damage of protein, membrane and DNA of bacteria. Another path of antimicrobial activity is that ZnO-based NPs can release metal ions to disturb the electron transport chain and regulate the bacterial metabolic process. The Zn2+ ion is an essential ion for many microorganisms due to its involvement in various cellular reactions, but at higher concentration it becomes cytotoxic. Owing to their size and varied morphological features, nanoparticles can facilitate their interactions with biomolecules. Based on the previous mentioned concepts, we have successfully synthesized and investigated the antibacterial activity of four types of nanoparticles based on different zinc compounds using the top-down method to prepare dispersions of polymer-coated different Zn-based NPs with the aid of ultrasound energy. We have successfully developed the broad-spectrum Zn-based NPs against both the Gram-negative bacteria and Gram-positive bacteria. We have found that this novel top-down method for preparing Zn NPs using sonication can produce ultrafine Zn-based NPs with the reduced MIC values. Such NPs can easily rupture the cellular wall. Particularly notable is t (open full item for complete abstract)

Committee: Songping Huang (Advisor) Subjects: Chemistry

PHD, Kent State University, 2018, College of Arts and Sciences / Department of Chemistry and Biochemistry

Iron plays an irreplaceable role in the cell and is found in numerous proteins that use iron as a biological catalyst to perform cell maintenance, growth and cell division. In tumor cells, as well as in normal cellular proliferation, there is a dependence on iron and its availability. With the recognized need to create and test new anti-tumor agents, utilizing the fact that cancer cells disproportionately tend to take up greater amounts of iron than do normal cells, this allows a pathway to be exploited using other metals such as gallium that can interfere in iron metabolism. Gallium ion possesses unique medicinal properties due to its resemblance to iron and is a known mimic of this essential metal. Therefore showing promise for treatment of a variety of diseases and disorders, particularly cancer and bacterial infection due to its antitumorigenic properties and antimicrobial activity. Use of simple soluble inorganic gallium salts, represented by Ganite® for treating a variety of diseases have already reached clinical applications. However, there are still several drawbacks of using gallium salts to deliver gallium at the cellular level, including (1) low-transport capacity of gallium because of the limited plasma concentrations of apo-Tf available for Ga(III)-binding in the blood stream; (2) slow kinetics due to the need to recycle the Tf after the Ga(III) ion is delivered inside the cell; and (3) hydrolysis of Ga(III) ions is a concentration-limiting factor and the origin of renal toxicity of drugs based on soluble gallium salts. To circumvent these limitations of the transferrin-receptor mediated uptake. Gallium nanoparticles with pH sensitivity are synthesized on a PVP (polyvinylpyrrolidone) template for cell culture viability studies on various cancer cell lines. Three gallium-based nanoparticle systems are synthesized and investigated here which confirm cellular uptake in tumorigenic cells and/or cytotoxicity to tumor cells. Effectively bypassing the l (open full item for complete abstract)

Committee: Songping Huang (Advisor); Bansidhar Datta (Committee Member); Mietek Jaroniec (Committee Member); Robin Selinger (Committee Co-Chair); Qi-Huo Wei (Committee Member) Subjects: Biochemistry; Cellular Biology; Chemical Engineering; Chemistry

Master of Science (MS), Wright State University, 2017, Microbiology and Immunology

In these experiments, SIM-A9 microglia were exposed to Staphylococcus aureus cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN) at a concentration of 5 ug/mL for six, twelve, eighteen, and twenty-four hours and the cytokine expression, cytoskeleton organization, and cell viability of the cells were observed. Following LTA and PGN exposure, TNF-a; secretion increased at each time interval and was highest observed at 24 hours. No significant IL-10 secretion was detected. Over the 24 hour period, cell viability and cytotoxicity of LTA and PGN treatment groups were not significantly different from the control, indicating the observed inflammatory cytokine response was not due to cell death. These data suggest that LTA and PGN play a predominantly inflammatory role in the first twenty-four hours of S. aureus CNS infection.

Committee: Nancy Bigley Ph.D. (Advisor); Barbara Hull Ph.D. (Committee Member); Dawn Wooley Ph.D. (Committee Member) Subjects: Immunology; Microbiology

Doctor of Philosophy, The Ohio State University, 1987, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

PhD, University of Cincinnati, 2016, Arts and Sciences: Chemistry

Cancer is a major public health problem in the worldwide and second leading cause of death in the United States. The major problem in treatment of cancer is the off target cytotoxicity. Therapeutic selectivity is therefore essential. Evidence suggests that, compared to normal cells, many types of cancer cells have increased level of ROS, a condition known as oxidative stress. Acute myeloid leukemia (AML) is known to possess of elevated levels of ROS, due to the increase expression of NADPH oxidases. Therefore to enhance the selectivity towards cancer we designed and synthesized novel anti-cancer agents that are converted to reactive molecules upon oxidation by ROS. In this study I explored the possible use of reactive oxygen-activated DNA modifying (RAC) agents against AML. A key amine on the lead agent was investigated via cytotoxicity assays and was found necessary for potency. The two best compounds were screened via the NCI-60 cell panel. These two compounds had potency between 200 and 800 nM against many of the leukemia cancer cell types. Subsequent experiments explored activity against a transformed AML model that mimics the molecular signatures identified in primary AML patient samples.

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

Master of Science, The Ohio State University, 2015, Comparative and Veterinary Medicine

Porcine reproductive and respiratory syndrome virus (PRRSV) is widespread globally, causing huge economic loss to pig farmers. In infected pigs, PRRSV dysregulates the host innate immunity by inhibition of type I Interferon (IFN-alpha) production and NK cell-cytotoxicity followed by down-regulating the adaptive immunity. Recently, PRRSV non-structural proteins (Nsps) are found to be playing important roles in suppressing the host innate immune response, especially through the viral Nsp1ß protein. In vitro studies have demonstrated rescue in the IFN-alpha production in cells infected with mutant Nsp1ß PRRSV. Therefore, our goal was to evaluate whether in mutant Nsp1ß PRRSV infected pigs there will be a rescue in innate immune response compared to wild-type virus. Our results showed that, in mutant Nsp1ß PRRSV infected pigs the NK cell-cytotoxicity and IFN-alpha production were significantly rescued, which partially increased the production of a vital adaptive immune response inducing anti-viral cytokine IFN-gamma. Furthermore, improvement in adaptive immune responses was supported by substantial increase in the frequency of cytotoxic T cell and memory T cell responses in pigs. In summary, our findings have provided better understanding of the function of PRRSV Nsp1ß protein in pigs, which could help in developing new PRRSV vaccine candidates.

Committee: Gourapura Renukaradhya (Advisor); Lee Chang-Won (Committee Member); Torrelles Jordi (Committee Member) Subjects: Virology

Master of Science (MS), Wright State University, 2011, Pharmacology and Toxicology

Nowadays, 24% of the nanomaterial-based consumer products contain silver nanoparticles (AgNPs) and exploit the well-known antimicrobial properties of silver. Although AgNPs have a wide range of biomedical research and industrial applications, very little is known about their toxicity. The main goal of this study is to synthesize, characterize and manipulate Creighton colloidal AgNPs for future cytotoxicity studies. These “naked” AgNPs were free from chemically aggressive capping/stabilizing agents, reaction byproducts or organic solvents. To achieve this goal, colloidal AgNPs were successfully: a) synthesized in large volumes (5L) using a slightly modified Creighton method by the reduction of silver nitrate with sodium borohydride, b) characterized using UV-Vis absorption spectrophotometry, flame atomic absorption spectroscopy, Raman spectroscopy and transmission electron microscopy (spherical AgNPs of 1-100 nm in diameter, moderately aggregated, free of impurities and having a surface plasmon resonance at 396 nm), and c) size-selected (mostly AgNPs of 1-20 nm in diameter) and highly concentrated (5 L of 14.98 ppm down to 4 mL of 7,461.65 ppm) in a small volume of water with minimal aggregation using tangential flow ultrafiltration. These homogenous and highly concentrated, “naked” AgNPs will be used in future cytotoxicity studies that will establish the median lethal concentration (LC50) of AgNPs and will identify AgNPs suitable for surface-enhanced Raman spectroscopy-based biosensing applications in our laboratories.

Committee: Ioana Sizemore PhD (Advisor); David Cool PhD (Committee Member); Thomas Lockwood PhD (Committee Member) Subjects: Chemistry; Nanoscience; Toxicology

Doctor of Philosophy (PhD), Wright State University, 2011, Biomedical Sciences PhD

Silver nanoparticles (AgNPs) have received tremendous attention for their antimicrobial properties; however, many gaps in knowledge exist. To address these issues, three research objectives were examined. The first objective hypothesized AgNPs can be size selected and concentrated via tangential flow ultrafiltration. The second objective hypothesized a high-throughput method could be developed to screen nanoparticle antiviral-activity and cytotoxicity simultaneously. The third objective hypothesized AgNPs inhibit viruses by preventing viral entry. For objective one, a tangential flow ultrafiltration scheme was tested on AgNPs synthesized via the Creighton Colloid method. AgNPs were analyzed via transmission electron microscopy. In objective two, an HIV-1 vector was adapted to 96-well format and modified to utilize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for simultaneous cytotoxicity and antiviral measurement. The third objective was investigated using Vaccinia virus (VACV). AgNP effects on VACV entry were monitored through β-galactosidase reporter assay and confocal microscopy. Western blots detected AgNP/VACV interaction. Plaque assays monitored AgNP inhibition over the entire VACV replication cycle. MTT and trypan blue exclusion measured AgNP cytotoxicity. In objective one, tangential flow ultrafiltration demonstrated size selection and concentration of AgNPs. Filtered AgNPs were uniform and unaggregated with average 11 nm diameters. A high-throughput, standardized assay was developed in objective two. AgNPs had antiviral-activity at non-cytotoxic concentrations (IC50 = 16 μg mL-1). In the third objective, AgNPs prevented VACV entry in both cytoprotective and virucidal capacities at non-cytotoxic concentrations (IC50 = 48 μg mL-1). In the absence of macropinocytosis, AgNPs retained virucidal-activity but not cytoprotective effects. AgNPs bound to viral entry proteins. Plaque assays demonstrated that AgNPs inhibited the entire VACV r (open full item for complete abstract)

Committee: Dawn Wooley PhD (Advisor); Gerald Alter PhD (Committee Member); Steven Higgins PhD (Committee Member); Barbara Hull PhD (Committee Member); Mill Miller PhD (Committee Member) Subjects: Nanoscience; Nanotechnology; Virology

MS, University of Cincinnati, 2012, Medicine: Molecular Genetics, Biochemistry, and Microbiology

In the past two decades Acinetobacter baumannii (Ab) has grown from relative obscurity to one of the most important global nosocomial infections. Ab is classified as a low grade pathogen, infecting only the immunocompromised. Overtime, Ab has evolved the capability to incorporate extraneous DNA into its own genome which has led to a dramatic increase in resistance to conventional antibiotics. To compound the problem, Ab can form biofilms which provide an even greater level of resistance against antibiotics and environmental pressures including biocides and desiccation. Adherent properties of Ab biofilms enable the organism to grow on a variety of surfaces and medical devices composed of glass, plastic or steel and instruments including stethoscopes and ventilator tubing. A previous study was conducted using High-Throughput Screening (HTS) to identify novel compounds that are capable of killing and/or inhibiting biofilm formation of Pseudomonas aeruginosa (Pa) or Staphylococcus epidermidis (Se) and Acinetobacter baumannii (Ab) biofilms. The novel compounds were divided into 5 groups based on the HTS results against one or more of the microorganisms. The first group of compounds (designated 3, 7, 8 and 11) were effective against Pa, Se and Ab biofilms. Group 2 compounds (1, 2, 9, 10, 12, and 13) were effective against only Pa and Ab biofilms. Group 3 compounds (14, 15 and 16) were effective only against Pa biofilms. Group 4 compounds (Se1 to Se16) were effective only against Se biofilm. Finally, group 5 compounds (17 to 34) were effective only against Ab biofilm. The biofilms were challenged in two phases. The first phase determined the compounds' ability to inhibit the initial formation of a biofilm. The second phase determined the compounds' ability to kill a mature biofilm. Confocal Laser Scanning Microscopy (CLSM) was used to study the effects of the compounds on initial biofilm formation and killing of mature biofilm. Of the compounds examined for inhibiting in (open full item for complete abstract)

Committee: Daniel Hassett PhD (Committee Chair); Thomas Lamkin PhD (Committee Member); Edmund Choi PhD (Committee Member) Subjects: Microbiology

PhD, University of Cincinnati, 2009, Engineering : Electrical Engineering

Toxic heavy metals are a growing threat to global environmental health. Heavy metals may enter the human body in food, water, or air, or by absorption through the skin. Once in the body, they compete with and displace essential minerals and interfere with organ system function, thus causing a variety of diseases. To respond to these growing problems, there has been a large demand for the development of devices for environmental health monitoring, which are capable of fast, reliable, and accurate measurement of heavy metals in a portable platform.In this research, a microfabricated electrochemical sensor with planar bismuth electrode for heavy metals measurement using anodic stripping voltammetry has been designed, fabricated, characterized, and applied to environmental health monitoring. By developing as a microfluidic polymer lab-on-a-chip (LOC), which is incorporated into a portable analyzer, on-site continuous sampling and monitoring of the contamination of heavy metals can be realized. The sensor has also been applied for on-chip monitoring of cadmium (Cd) concentration in an integrated cells-on-a-chip platform to perform Cd cytotoxicity test. Compared with previous work, this research is mainly focused on using standard BioMEMS fabrication technology to develop an environmentally-friendly, ready-to-use solid-state sensor that can perform rapid heavy metals measurement in natural- or biological-environments without disturbing the sensing environment and media. Moreover, this on-chip sensor can be easily adapted into different LOC platforms or incorporated with analyzers for carrying out heavy metals measurement to meet various demands in environmental health monitoring. In this research, multiple parameters that can affect on the sensor's performance have been explored and fully investigated. The sensor has been characterized and optimized based on experimental results, and applied to environmental monitoring applications. To perform automatic and continuous sa (open full item for complete abstract)

Committee: Chong H. Ahn PhD (Committee Chair); Joseph H. Nevin PhD (Committee Member); Jason C. Heikenfeld PhD (Committee Member); Paul L. Bishop PhD (Committee Member); William R. Heineman PhD (Committee Member); Am Jang PhD (Committee Member) Subjects: Electrical Engineering; Environmental Engineering

Doctor of Philosophy, The Ohio State University, 2012, Chemical and Biomolecular Engineering

Drug screening is a long and costly process confronted with low productivity and challenges in using animals. 3-D cell-based high-throughput platforms have been developed to improve drug-screening efficiency and minimize animal testing. However, online monitoring of cell proliferation, pH, and dissolved oxygen (DO) has been a challenge in 3-D cell-based assays. In this work, a 40 micro-well plate bioreactor (40-MBR) system was developed as a high-throughput platform for 3-D cell cultures. This 40-MBR has similar dimensions of a 384-well plate (384-MWP) can provide real-time and noninvasive monitoring of cell proliferation, pH, and DO in 3-D microenvironments. A colon cancer cell line expressing enhanced green fluorescent protein (EGFP) under the control of a constitutive CMV promoter was tested with two potential cancer drugs using the 40-MBR and 384-MWP. Compared to the 384-MWP, the 40-MBR gave more reliable and highly reproducible growth kinetic data with reduced experimental errors. This study demonstrated the potential application of the 40-MBR as a high-throughput platform for screening potential cancer drugs and evaluating their cytotoxic effects in the early-stage drug discovery. Cytotoxicity and embryotoxicity of chemicals were also investigated in the 40-MBR using EGFP-expressing embryonic stem cells (ESCs). Embryonic stem cell test (EST) has been used as an in vitro model for assessing embryotoxicity. However, the current EST can only provide end-point data and cannot predict embryotoxicity of chemicals affecting organs such as bone. In this study, a novel high-throughput embryotoxicity assay was developed using EGFP-expressing ESCs under the control of a survivin promoter. Survivin expression is closely associated with embryo development and cell differentiation. For control, ESCs expressing EGFP under the control of a CMV promoter were used to monitor cytotoxicity of chemicals. Using survivin as a diagnostic marker for predicting embryotoxicity was fi (open full item for complete abstract)

Committee: Shang-Tian Yang (Advisor); Jeffery Chalmers (Committee Member); Andre Palmer (Committee Member) Subjects: Chemical Engineering

Doctor of Philosophy, The Ohio State University, 2010, Oral Biology

Fabrication of a successful maxillofacial prosthesis with appearance match to the patient's skin is often a significant challenge. An accurate method for collecting appearance information and the application of advanced colorant formulation theories would make possible a pleasing appearance match of a maxillofacial prosthesis to the patient's skin. Dental and maxillofacial applications of a non-contact spectral reflectance system with its potential to provide more reliable measurements recently initiated. A study on accuracy and precision of this measuring system is conducted by comparing with four commonly used contact color measuring instruments, which provides more evidence for its further application on color matching of maxillofacial prosthesis. A newly-developed laser light diffusing method, which correlates laser light diffusing area (LLDA) to translucency parameter (TP), is investigated on maxillofacial elastomeric specimens by comparing a previously proposed method, color difference due to edge loss (CDEL). The translucency of human skin and translucent maxillofacial materials can be described quantitatively with this non-destructive, non-contact method, which might be further applied on application of reproduction of facial morphology and appearance. Pigments and dyes play a key role in pigmentation and coloration of maxillofacial prosthetic elastomers. Addition of pigments and dyes into the base material might produce complicated reactions or interactions which in turn may cause various effects on optical properties, biocompatibility and thermal properties of the material. Optical properties of a base maxillofacial prosthetic material are significantly affected when pigmentation by pigments and dyes. Kubelka-Munk (K-M) theory has been widely used on pigmentation and coloration of maxillofacial materials. Accuracy of the K-M theory with three different interfacial reflection corrections (IRC) on maxillofacial elastomers is evaluated, and the IRC va (open full item for complete abstract)

Committee: William Johnston (Advisor); William Brantley (Committee Member); Robert Seghi (Committee Member); John Walters (Committee Member) Subjects: Biomedical Research; Dental Care; Health Care; Materials Science; Physics; Polymers

Doctor of Philosophy, The Ohio State University, 2010, Pharmacy

Natural products have played an important role in anticancer drug development for many decades. A recent review analyzing clinically approved anticancer drugs in Western countries and Japan during a twenty-five year period from 1981 to 2006 showed that, under the class of “non-biologicals/vaccines”, 63 of 81 (77.8%) anticancer drugs were either natural products or their derivatives, or synthesized molecules based on natural product pharmacophores. As part of a collaborative, multi-disciplinary approach to the discovery of new naturally occurring anticancer drugs, two medicinal plants, namely, Hyptis brevipes and Vitex quinata, collected in Indonesia, were selected for further investigation. The chloroform-soluble extract of a sample of the entire plant of H. brevipes showed activity against the MCF-7 human breast cancer cell line. Bioassay-guided fractionation and purification of the CHCl3-soluble extract of H. brevipes led to the isolation of six new 5,6-dihydropyrone derivatives, namely, brevipolides A-F (342-346), together with seven known compounds. Brevipolides A-F (342-346), and a previously known 5,6-dihydropyrone derivative (347), were assigned with the absolute configuration, 5R, 6S, 7S, and 9S, as elucidated by analysis of data obtained from their CD spectra and by Mosher ester reactions. Brevipolides B and D, as well as compound 347 exhibited ED50 values of 6.1, 6.7 and 3.6 µM against MCF-7 cells. Brevipolides A, B, and F, and compound 349 (the known 5,6,3′-trihydroxy-3,7,4′-trimethoxyflavone) gave ED50 values of 5.8, 6.1 7.5, and 3.6 µM against HT-29 cells, respectively. However, no significant cytotoxicity was found against Lu1 cells for any of the compounds isolated. When these compounds were subjected to evaluation in a panel of mechanism-based in vitro assays, compound 347 were found to be active in an enzyme-based ELISA NF-κB p50 assay, with an ED50 value of 15.3 µM. In a mitochondrial transmembrane potential assay, brevioplide C, compounds 348 and (open full item for complete abstract)

Committee: A. Douglas Kinghorn PhD (Advisor); Esperanza Carcache de Blanco PhD (Committee Member); James R. Fuchs PhD (Committee Member); Pui-Kai Li PhD (Committee Member) Subjects: Pharmaceuticals

Doctor of Philosophy, The Ohio State University, 2006, Oral Biology

Activation of neuroendocrine responses by restraint stress (RST) suppresses natural killer cell (NK) activity during an experimental influenza A/PR8 viral infection. RST-induced activation of the HPA axis upregulates glucocorticoids (GC) and endogenous opioids which may be responsible for the observed suppression of NK activity. GC receptor antagonism modulates trafficking and splenic cellularity. However, the specific effects on NK activity remain unclear. Opioid receptor antagonism with naltrexone (NTX) abrogates RST-induced suppression of splenic NK cytotoxicity suggesting endogenous opioids modulate NK cytotoxicity. However, the specific subtype of opioid receptors involved remains unclear. These studies examined the effects of GC and specific opioid receptor subtype antagonism on splenic NK cellularity and cytotoxicity. Additionally, these studies examined whether the opioid signaling mechanism involved a direct effect on NK cells or a possible modulation in NK-stimulatory cytokine mRNA expression. C57BL/6 mice were treated daily with the GC receptor antagonist RU486, the opioid receptor antagonist NTX, or i-, a-, or e-opioid receptor specific antagonists. Mice were infected intra-nasally with A/PR8 virus and underwent daily RST. Splenocytes were obtained three days post infection. For in vitro studies, splenocytes were incubated with NTX or morphine (MOR) and tested for cytolytic activity at 24 and 48 hours. NK cytotoxicity was assessed using a standard chromium release assay. FACS analysis was used to detect the number of CD3-DX5+ NK cells. Cytokine mRNA expression was assessed using realtime PCR. RST-induced suppression of NK activity was a result of decreases in number of splenic NK cells and cytotoxicity. GC Receptor antagonism restored cellularity and i-opioid receptor antagonism restored NK cytolytic activity. Splenic NK cells incubated with morphine or NTX for up to 48 hours showed no change in NK cytotoxicity. Preliminary data suggested that antagonism (open full item for complete abstract)

Committee: John Sheridan (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2005, Chemistry

Advances in carbohydrate synthesis and analysis have led to a greater understanding of the roles of these molecules in biological processes. This knowledge in turn has led to a greater ability to utilize carbohydrate-based pharmaceuticals in manipulating these processes. We have undertaken both synthetic and biological studies to further knowledge in the area of carbohydrate-based pharmaceuticals. These studies include novel syntheses of L-daunosamine and L-ristosamine, as well as intermediates of these sugars that are suitable for glycosylation with diverse acceptors via the benzenesulfinyl piperidine/trifluoromethanesulfonic anhydride (BSP/Tf2O) glycosylation method. Further, we have developed a simplified system by which the roles of carbohydrates in drugs that bind to DNA can be studied. This system makes use of a facile Sonogashira coupling to link propargyl glycosides to aromatic intercalator moieties. These molecules were then evaluated for DNA binding by viscometry, and were assayed for cytotoxicity against MCF-7 and MDA-MB-231 breast cancer cell lines. Clear differences in activities among library compounds were evident, and some compounds evidenced IC50 values of < 20 µg/mL. This system shows potential for use in many other studies.

Committee: Robert Coleman (Advisor) Subjects: Chemistry, Organic