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Rajanahalli Krishnamurthy, PavanToxicity Of Silver Nanoparticles In Mouse Embryonic Stem Cells And Chemical Based Reprogramming Of Somatic Cells To Sphere Cells
Doctor of Philosophy (Ph.D.), University of Dayton, 2011, Biology

1: Silver nanoparticles (Ag Np’s) have an interesting surface chemistry and unique plasmonic properties. They are used in a wide variety of applications ranging from consumer products like socks, medical dressing, computer chips and it is also shown to have antimicrobial, anti bacterial activity and wound healing. Ag Np toxicity studies have been limited to date which needs to be critically addressed due to its wide applications. Mouse embryonic stem (MES) cells represent a unique cell population with the ability to undergo both self renewal and differentiation. They exhibit very stringent and tightly regulated mechanisms to circumvent DNA damage and stress response. We used 10 nm coated (polysaccharide) and uncoated Ag Np’s to test its toxic effects on MES cells. MES cells and embryoid bodies (EB’s) were treated with two concentrations of Ag Np’s: 5 µg/ml and 50 ug/ml and exposed for 24, 48 and 72 hours. Increased cell death, ROS production and loss of mitochondrial membrane potential and alkaline phosphatase (AP) occur in a time and a concentration dependant manner. Due to increased cell death, there is a progressive increase in Annexin V (apoptosis) and Propidium Iodide (PI) staining (necrosis). Oct4 and Nanog undergo ubiquitination and dephosphorylation post-translational modifications in MES cells thereby altering gene expression of pluripotency factors and differentiation of EB’s into all the three embryonic germ layers with specific growth factors were also inhibited after Ag Np exposure. Flow cytometry analysis revealed Ag Np’s treated cells had altered cell cycle phases correlating with altered self renewal capacity. Our results suggest that Ag Np’s effect MES cell self renewal, pluripotency and differentiation and serves as a perfect model system for studying toxicity induced by engineered Ag Np’s.

ABSTRACT 2: The reprogramming of fibroblasts to pluripotent stem cells and the direct conversion of fibroblasts to functional neurons has been successfully manipulated by ectopic expression of defined factors. We demonstrate that mouse fibroblasts can be converted into sphere cells by detaching fibroblast cells by proteases and then using AlbuMAX I-containing culture medium without genetic alteration. AlbuMAX I is a lipid-rich albumin. Albumin-associated lipids arachidonic acid (AA) and pluronic F-68 were responsible for this effect. The converted colonies were positive for both alkaline phosphatase and stage specific embryonic antigen-1 (SSEA-1) staining. Global gene expression analysis indicated that the sphere cells were in an intermediate state compared with MES cells and MEF cells. The sphere cells were able to differentiate into tissues representing all three embryonic germ layers following retinoic acid treatment, and also differentiated into smooth muscle cells following treatment with vascular endothelial growth factor (VEGF). The study presented a potential novel approach to transdifferentiate mouse fibroblast cells into other cell lineages mediated by AlbuMAX I-containing culture medium.

Committee:

Hong Yiling (Advisor); Shirley J. Wright (Committee Member); Mark G. Nielsen (Committee Member); Tsonis A. Panagiotis (Committee Member); Shawn M. Swavey (Committee Member)

Subjects:

Biology

Keywords:

Silver Nanoparticles; Silver Nanotoxicity; Mouse Embryonic Stem Cells; Induced Pluripotent Stem Cells; Chemical Based Reprogramming; Small Molecules; Zinc Oxide Nanoparticles; Zinc Oxide Nanotoxicity

Kathayat, DipakIdentification of Novel Small Molecule Growth Inhibitors Specific to Avian Pathogenic Escherichia coli
Master of Science, The Ohio State University, 2017, Comparative and Veterinary Medicine
Avian Pathogenic E. coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), is one of the most common bacterial pathogens affecting chickens, turkeys, and other avian species. It causes multiple extra-intestinal infections which subsequently lead to high morbidity and mortality, production losses, and increased slaughter condemnation resulting severe economic loss to the global poultry industry. Antimicrobial medication is the major approach currently employed to reduce the incidence and mortality associated with this infection. However, multi-drug resistant (MDR) APEC strains are reported nowadays worldwide. Furthermore, vaccination which is used as a subsidiary approach to prevent infection frequency is not sufficient to provide protection against diverse heterologous APEC serotypes. Therefore, the objective of this study is to identify novel small molecule (SM) growth inhibitors of APEC and to evaluate toxicity and efficacy of identified SMs, in vitro and in vivo. Here, using a Tecan Sunrise™ absorbance plate reader, a pre-selected enriched SM library containing 4,182 SMs was screened at 100 µM concentration against a predominant field APEC serotype, APEC O78, grown in minimal M63 media. Of the total 4,182 SMs, 41 SMs inhibited APEC O78 growth. The majority of growth inhibitory SMs were found belonging to chemical groups; quinolines, piperidines, pyrrolidinyls, and imidazoles. Among 41 SMs, 30 SMs exhibited bacteriostatic activity, while remaining 11 SMs displayed bactericidal activity and were selected for further studies. Dose-response analysis of these selected SMs revealed their dose-dependent activity with minimal inhibitory concentration (MIC) ranging 12.5 µM to 200 µM. These selected SMs were found broadly effective against various APEC serotypes such as O1, O2, O8, O15, O18, O35, O109, and O115. Six of these SMs exhibited narrow-spectral activity affecting 1-3 tested commensal bacteria. Except SM11, other SMs were least toxic to Caco-2 epithelial (<10%) and HD11 macrophage cells (<10%) at 200 µM concentration. Seven of these SMs were least hemolytic (<10%) to avian and sheep red blood cells, whereas remaining four SMs were more hemolytic (>20%) at 200 µM concentration. All of the selected SMs were found effective in significant (P<0.01) reduction of intracellular APEC O78, O1, and O2 survival in infected Caco-2, HD11 and THP-1 cells at varying concentrations ranging from 1X to 2X of MIC. No resistance was observed to any of these selected SMs when APEC O78 was treated at lethal (2X MBC) or sub-lethal (0.75X MIC) concentration of SMs. In vivo evaluation of these SMs using greater wax moth (Galleria mellonella) revealed their low toxicities (<10%), except for SM1. Furthermore, treatment with these SMs significantly (P<0.0001) extended the survival of infected larvae, except for SM8, and significantly (P<0.05) reduced the APEC load inside the larva, except for SM8 and SM9. In summary, we have identified 11 novel anti-APEC SMs. Our future studies will focus on investigating; effects of these SMs on APEC preformed biofilms, interactions with antimicrobials that are currently being used, effects in infected chickens, and elucidation of their mechanism of actions. We expect that these studies will enable the development of novel narrow-spectrum SM antimicrobials for the control of APEC infections.

Committee:

Gireesh Rajashekara (Advisor); Chang-Won Lee (Committee Member); Anastasia Vlasova (Committee Member)

Subjects:

Animal Diseases; Microbiology; Therapy; Veterinary Services

Keywords:

Small molecules, novel antimicrobials, avian pathogenic Escherichia coli, antimicrobials resistance

Vazquez, Ana CarolinaIdentification and Characterization of Compounds with Antiviral Activity against Influenza Viruses
PHD, Kent State University, 2008, College of Arts and Sciences / School of Biomedical Sciences

Prophylactic and therapeutic antiviral drugs for influenza are available as an adjunct to vaccination. However, their effectiveness is already being limited because of the rapid emergence of drug resistant isolates due to the vast use of these drugs and the recombination potential of the viral genome. Moreover, an immediate pandemic threat (influenza H5N1) raises issues like the lack of surge capacity and availability of drugs as well as potential drug resistance.

Given the need for additional antiviral agents, our study intended to identify new compounds with potential anti-influenza activity. This was done by screening a library of 34,000 small molecules by means of a cell-based system that assayed the inhibition of virus-induced cell death. As a result, 330 primary hits were identified from which two lead compounds (QMV-13 and QMV-15) and three of their analogs (QMV-13B, QMV-15A, QMV-15B) were described as potential candidates for further characterization. First, we evaluated their cytotoxicity in a variety of primary and immortalized cell lines. Next, their antiviral activity against influenza A/WSN/33 was confirmed using different methods. Furthermore, replication of a cohort of influenza A and B laboratory-adapted, clinical isolates, and drug resistant strains was quantified in the presence of the leads, resulting in low IC50 values. Next, we tested the specificity of their antiviral activity against other RNA viruses such as YFV, WNV, hPIV3, and HIV, eliminating the possibility of a broad spectrum of action. Time-of-drug-addition assays, viral protein expression, and virus growth kinetics experiments characterized our compounds as early inhibitors of virus replication. The lack of inhibitory response against virus neuraminidases helped eliminating that enzyme as their target. Finally, serial passages of the virus in the presence of the lead compounds were done to generate drug resistant variants that could pinpoint more accurately their target.

Although the exact mechanism by which compounds QMV-13, QMV-13B, QMV-15, QMV-15A, and QMV-15B exert their anti-influenza activity requires further investigation, we have repeatedly characterized them as early inhibitors of viral infection, finding that has significantly reduced the number of possible targets and may increase their value as novel therapeutics due to their potential applicability as prophylactic agents.

Committee:

Miguel E. Quinones-Mateu, PhD (Committee Chair); Robert T. Heath, PhD (Committee Member); Philip E. Pellett, PhD (Committee Member); Oscar Rocha, PhD (Committee Member); Kenneth S. Rosenthal, PhD (Committee Member); Christopher J. Woolverton, PhD (Committee Member)

Subjects:

Biomedical Research; Cellular Biology; Molecular Biology; Virology

Keywords:

antiviral compounds; influenza virus; high throughput screening; library of small molecules; characterization; cytotoxicity; antiviral activity; hits; leads

El-Khoury, Patrick Z.Femtosecond Dynamics of Small Polyatomic Molecules in Solution: A Combined Experimental and Computational Approach
Doctor of Philosophy (Ph.D.), Bowling Green State University, 2010, Photochemical Sciences
A detailed understanding of condensed-phase ultrafast photo-induced chemical reaction dynamics is still sought after. This is because of the intrinsic complexity of liquid-phase photophysical and photochemical phenomena arising from competing intra- and intermolecular processes. Such processes often take place on a timescale of a few femtoseconds to several tens of picoseconds. In this work, the model photochemical processes used to investigate ultrafast photo-induced reaction dynamics in solution are bond-breaking and bond making reactions. The model compounds are di- and poly-halogenated methanes. The gas-phase photochemistry of these small molecules is thoroughly investigated, which enables to draw a direct comparison to the photophysical and photochemical dynamics in solution. Moreover, in contrast to the thoroughly investigated di- and triatomic molecular systems, more vibrational degrees of freedom are available both to the model parent molecules and nascent polyatomic radical species. Thus, a detailed mapping of the photochemical reaction paths of these systems develops into a comparative advantage, revealing different couplings between the reactive modes and other dark states in a far-from-equilibrium situation. The complexity of the encountered ultrafast phenomena requires the use of several experimental and computational approaches. Results of femtosecond transient absorption, picosecond transient resonance Raman, and matrix isolation experiments in concert with ground and excited state ab initio calculations are discussed in this context. The findings from this work illustrate the power of a solvent environment in (i) altering the topology of ground and excited state potential energy surfaces, and (ii) leading to different photoproducts through intermediates otherwise absent from gas-phase studies.

Committee:

Alexander Tarnovsky, PhD (Committee Chair); Massimo Olivucci, PhD (Committee Co-Chair); R. Marshall Wilson, PhD (Committee Member); H. Peter Lu, PhD (Committee Member); Rex Lowe, PhD (Other)

Subjects:

Chemistry

Keywords:

Photochemistry; Ultrafast; Small Molecules; Pump-Probe

Behnke, Shelby LeeResonance Raman Investigations of [NiFe] Hydrogenase Models
Master of Science, The Ohio State University, 2016, Chemistry
Hydrogenase (H2ases) enzymes carry out bidirectional hydrogen production and oxidation reactions. To better understand the mechanism of hydrogen conversion, spectroscopic studies on small molecule mimics provide important metrics to correlate structure and function of the native enzymes. In this work, a series of molecular complexes that mimic the [NiFe] hydrogenase have been synthesized with different phosphine ligand substituents and analyzed using multiple analytical techniques, including nuclear magnetic resonance, Fourier transform infrared, and resonance Raman spectroscopies. Three model compounds have been selected as the focus of this investigation into the vibrational structure of the [NiFe] active site: [Ni(dppe)(µ-pdt)(µ-H)Fe(CO)3][BF4], [Ni(dcpe)(µ-pdt)(µ-H)Fe(CO)3][BF4], and [Ni(dppbz)(µ-pdt)(µ-H)Fe(CO)3][BF4]. (dppe= diphenylphosphinoethane, dcpe= dicyclohexylphosphinoethane, and dppbz= diphenylphosphinobenzene). These compounds have been previously synthesized and shown to exhibit high activity for proton reduction but have not been fully characterized spectroscopically to assess the solution-phase structure of the metal-hydride core. (Barton et al., 2010, JACS. 132, 14877-14885.) Specifically, resonance Raman (RR) spectroscopy was used to study the vibrational bands of each of the molecules, which were then assigned to specific normal modes of the molecule. The [Ni(dppe)(µ-pdt)(µ-H)Fe(CO)3]BF4 compound has been previously studied in great detail by RR, and this molecule was used as a template from which to understand the structures of the series. It was found that perturbations of ligands on the metal center alter the molecular vibrations, particularly of the metal-hydride core. This work provides important metrics for comparison between synthetic and the natural enzyme systems, with the intention of better understanding the mechanisms of native hydrogenases and informing next-generation catalyst design.

Committee:

Hannah Shafaat, Dr. (Advisor); Nagib David , Dr. (Committee Member)

Subjects:

Chemistry

Keywords:

Resonance Raman; Spectroscopy; Hydrogenase; small molecules

Anupam, RajaneeshCharacterization of binding of tRNA and ligands to T box antiterminator
Doctor of Philosophy (PhD), Ohio University, 2007, Molecular and Cellular Biology (Arts and Sciences)

Bacillus subtilis tyrS is an aminoacyl-tRNA synthetase gene that is a member of the T box family of genes, which are found in many Gram-positive bacteria. This family includes aminoacyl-tRNA synthetase, amino acid biosynthetic and amino acid transport genes, and these genes are characterized by a highly-conserved sequence of nucleotides known as the T box sequence. Expression of these genes is regulated by the interaction of uncharged cognate tRNA with the 5' untranslated region of the nascent mRNA. One feature of this interaction is the base pairing of the tRNA acceptor end with four bases of the antiterminator, a conserved mRNA structural element that contains a seven-nucleotide bulge flanked by two helices. The tRNA-antiterminator base pairing prevents the formation of an alternative terminator element and results in complete transcription of the gene. This research investigates binding of tRNA and small molecules to the T box antiterminator using two antiterminator models, AM1A and AM1A(C11U) which represent the wild type aniterminator and a reduced function variant, respectively. From chemo-enzymatic probing and fluorescence studies data, the structural changes occurring in AM1A upon the binding of the acceptor end tRNA to only the first four nucleotides of the bulge and the greater flexibility of AM1A(C11U) compared to AM1A indicate that binding of tRNA occurs via tertiary structure capture and induced fit. Native gel mobility shift, fluorescence studies, and nucleotide analog interference mapping (NAIM) indicated that the integrity of the A2 helix and Mg2+ concentration play an important role in binding of tRNA to antiterminator. The functional groups probed by NAIM of absolutely conserved A10 (A223 of B. subtilis tyrS) were not essential for the binding of tRNA, but the presence of a methyl group on the amino functionality of A10 may sterically hinder binding. Using fluorescence resonance energy transfer and enzymatic probing, neomycin B and two oxazolidinones were found to bind selectively to AM1A with high affinity at the 5' end of the bulge. All three ligands competitively inhibited the binding of tRNA to AM1A. In addition, there appeared to be formation of a new binding pocket for neomycin in the AM1A•tRNA complex.

Committee:

Jennifer Hines (Advisor)

Keywords:

Antitermination; Small molecules; Chemo-enzymatic probing; Nucleotide analog interference mapping (NAIM); FRET and Gel shifts

Sargeant, Aaron MatthewPreclinical Efficacy and Safety Evaluation of Novel Small-Molecule Targeted Agents for the Prevention and Treatment of Prostate Cancer
Doctor of Philosophy, The Ohio State University, 2009, Veterinary Biosciences
Prostate cancer is the most commonly diagnosed noncutaneous cancer and the second leading cause of cancer death in men. To combat the assortment of genomic and cellular aberrations that occur with the progression of this disease, we have developed novel classes of histone deacetylase (HDAC) inhibitors, 3-phosphoinositide dependent protein kinase-1 (PDK1)/Akt inhibitors, and indole-3-carbinol analogs using phenylbutyrate, celecoxib, and indole-3-carbinol, respectively, as scaffolds. Here, we assess both the efficacy and safety of the lead compounds of these classes (OSU-HDAC42, OSU-03012, and OSU-A9) administered orally in a series of preclinical studies carried out, in part, in preparation for prevention and regression trials in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Following the acquisition of relevant in vitro and dose-ranging data, the tumor-suppressive efficacy of selected doses was evaluated in PC-3 xenograft ± TRAMP-C2 syngeneic mouse models. The oral drug formulation found to achieve the most promising benefit-risk ratio was incorporated into a diet and administered to TRAMP mice for the assessment of its morphologic and molecular effects on the development of prostatic intraepithelial neoplasia (PIN) and carcinoma. Toxicity was evaluated by histopathologic, hematologic, and body and organ weight analysis. OSU-HDAC42 achieved the most potent blockade of prostate tumorigenesis reported in the TRAMP model, suppressing the absolute and relative weights of the urogenital tracts by 86% and 85%, respectively, in association with intraprostatic modulation of biomarkers indicative of HDAC inhibition, increased apoptosis and differentiation, and decreased proliferation. This compound, while sparing body weight, caused reversible testicular degeneration and hematologic alterations. In addition to its prostate chemopreventive effects, OSU-03012 was found to induce the hepatic biotransformation enzymatic system and caused phenotypic changes partially linked to sustained PDK1/Akt inhibition through inactivation of the downstream regulator of glycogen synthesis, glycogen synthase. OSU-A9 was compared vis-à-vis two slightly modified agents, of which the methylated derivative OSU-A9M was selected for continued lead optimization. Collectively, this work suggests therapeutic value in incorporating these novel compounds into the treatment regimen of patients with PIN, and reveals other biological parameters that should be considered for monitoring in the future preclinical and clinical use of these and similarly-targeted agents.

Committee:

Ching-Shih Chen, PhD (Advisor); Robert Brueggemeier, PhD (Committee Member); Steven Clinton, MD, PhD (Committee Member); Thomas Rosol, DVM, PhD (Committee Member)

Subjects:

Animals; Biochemistry; Health Care; Molecules; Oncology; Pathology; Pharmaceuticals; Pharmacology; Therapy; Toxicology; Veterinary Services

Keywords:

prostate cancer; chemoprevention; anticancer therapy; mouse models; small molecules; veterinary pathology; toxicologic pathology; TRAMP; xenograft; risk assessment; toxicity; HDAC inhibitors; testicular degeneration; cytochrome p450; indole-3-carbinol; OS

Butaeva, EvgeniiaUltrafast Photochemistry of Polyhalogenated Methanes and Non-Metals
Doctor of Philosophy (Ph.D.), Bowling Green State University, 2015, Photochemical Sciences
A molecular level understanding of photodynamics in condensed media is one of the recent challenges to chemical physics. This is because of the intrinsic complexity of liquid-phase photophysical and photochemical singularities arising from competing intra- and intermolecular processes. Such processes often take place on a timescale of a few femtoseconds (10-15 s) to several tens of picoseconds (10-12 s). In this work, the model photochemical processes used to investigate ultrafast photo-induced reaction dynamics in solution. The model compounds are non-metal/metal polyhalogenated small molecules. The gas-phase photochemistry of these small molecules is thoroughly examined, which also enables to establish the connection between liquid and gas phase dynamics. Furthermore, contrary to the scrupulously investigated di- and triatomic molecular systems, more vibrational degrees of freedom are accessible both for the model parent molecules, nascent polyatomic radical species, and isomer photoproducts. Therefore, a detailed mapping of the photochemical reaction paths of these molecular systems can possibly reveal different couplings between the reactive modes and other dark states in a far-from-equilibrium situation. The complexity of the encountered ultrafast events requires the utilization of several experimental and computational approaches. Results of femtosecond transient absorption, picosecond transient resonance Raman, excited state ab initio calculations are discussed in this context.

Committee:

Alexander Tarnovsky, Dr. (Advisor); Haowen Xi, Dr. (Other); Marshall Wilson , Dr. (Committee Chair); Alexey Zayak, Dr. (Committee Chair)

Subjects:

Chemistry; Physical Chemistry

Keywords:

Ultrafast transient absorption spectroscopy; Ab initio calculations; Small molecules