Doctor of Philosophy (Ph.D.), Bowling Green State University, 2019, Photochemical Sciences

Ever since the discovery of the DNA molecular structure, this molecule became a desirable target for the researchers aiming to develop anticancer drugs and therapies. The main emphasis of this dissertation was placed on the design, development and studies of photochemical properties of a novel photo activated DNA damaging agent. Upon light irradiation of compound the release of highly reactive 9,10-phenanthrenequinone was observed. Studies of phenanthrene dihydrodioxin interaction with DNA demonstrated the intercalative mode of binding and slightly preferential binding affinity to the AT-rich DNA sequences. The synthesized agent induced a partial transition from B to Z form of DNA. The phenanthrene dihydrodioxin compound proved to be an efficient single-strand DNA photocleaver upon visible light irradiation. This further demonstrates the potential of photomasked ortho-quinones as efficient DNA damaging agents. This work also describes an unusual transformation from pyridinium substituted methane to the corresponding gem-diol in the mild conditions in the presence of air. Proposed mechanism of the reaction involves the formation of reactive oxygen species (ROS). ROS are known to be highly toxic to biomolecules, including DNA. It is demonstrated in this work that the production of ROS intermediates in the reaction of the gem-diol formation can lead to DNA damage in the dark. In the continuous interest of our research group, a series of bis(pyridinium) and bis(3-carboxamidepyridinium) alkane salts were synthesized in order to study through-space electron delocalization and formation of dimer radical cation species upon electrochemical and photoreduction. Investigation of photoinduced charge separation is important for the development of artificial photosynthetic systems and molecular electronics. Studies of unsubstituted and meta-substituted pyridines connected by the alkyl linker of different length provided additional information on the efficiency of the pyridi (open full item for complete abstract)

Committee: R. Marshall Wilson Prof. (Advisor); Farida Selim Prof. (Other); Pavel Anzenbacher Prof. (Committee Member); Alexander Tarnovsky Prof. (Committee Member) Subjects: Biochemistry; Chemistry; Organic Chemistry; Physical Chemistry

Doctor of Philosophy (PhD), Ohio University, 2018, Molecular and Cellular Biology (Arts and Sciences)

Human cytomegalovirus (HCMV) is primarily an opportunistic pathogen in human, causing significant disease in immunocompromised individuals. A large, double-stranded DNA genome (~230 kilobases) provides the coding capacity for over 200 genes, of which only 25% are required for viral replication in cell culture. The viral UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are essential for replication, and viruses lacking the proper expression of pUL34 cannot replicate in cell culture. Interactions of pUL34 with DNA binding sites (US3 and US9?) represses transcription of (these) two viral immune evasion genes that are dispensable for replication in cell culture. There are 12 additional predicted pUL34-binding sites present in the HCMV genome (strain AD169), with three of them concentrated near the HCMV origin of lytic replication (oriLyt). Analysis of 47 clinical isolates of HCMV confirmed that the predicted UL34-binding sites were highly conserved. Protein-DNA interactions were analyzed during infection with ChIP-seq and confirmed that pUL34 binds to the human and viral genome during infection, including at the three predicted UL34-binding sites in the oriLyt region. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated, oriLyt-dependent DNA replication. Subsequently, mutagenesis of these same sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with 3 virus proteins that are essential for viral DNA replication - IE2, UL44, and UL84, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Lastly, mutagenesis of the predicted UL34-binding site in the third exon of another essential viral gene, UL37, demonstrated that some UL34-binding sites are not important for viral replication.

Committee: Bonita Biegalke (Advisor); Calvin James (Committee Member); Mark Berryman (Committee Member); Justin Holub (Committee Chair) Subjects: Genetics; Molecular Biology; Virology

Doctor of Philosophy, The Ohio State University, 2016, Physics

The eukaryotic genome is organized into a structural polymer called chromatin. Ultimately, all access to genetic information is regulated by chromatin including access required for DNA replication, transcription, and repair. The basic repeating unit of chromatin is the nucleosome which is comprised of ~147 bp of DNA tightly wrapped around a protein histone octamer core. The histone octamer is made up of eight proteins: two each of histones H2A, H2B, H3, and H4. Many mechanisms exist to regulate access to DNA but one of pivotal importance is the creation of unique nucleosomes through i) integration of histone variants and ii) deposition of post translational modifications (PTMs). These modifications help comprise the epigenome of a cell. Classically, the two mechanisms by which they function have been through a direct regulation of nucleosome dynamics, or through third party proteins which are able to recognize the variants or PTMs and facilitate work. The library of potential PTMs therefore forms a sort of histone code which regulates access to DNA. This thesis investigates the intersection of these mechanisms to determine whether the act of recognizing epigenetic information alters DNA accessibility. The primary method used to determine changes in DNA accessibility is though observing the effective binding affinity of a transcription factor to its target site buried within a recombinantly prepared nucleosome which has been modified to carry a PTM and to report on its wrapping state. We find different regulation depending both on the PTM we investigate and the specific PTM-binding protein. We first investigate the H3K36me3-binding protein PHF1 and find that while the PTM it recognizes, H3K36me3, does not alter DNA accessibility, the binding of its recognition domain and N-terminal domain can illicit a change of DNA accessibilty of 8 ± 2-fold. This means that 8 times less DNA binding protein is required to occupy its target site if the nucleosome is bound by PH (open full item for complete abstract)

Committee: Michael Poirier PhD (Advisor); Ralf Bundschuh PhD (Committee Member); Comert Kural PhD (Committee Member); Fengyuan Yang PhD (Committee Member); Michael Barton PhD (Committee Member) Subjects: Biochemistry; Biology; Biophysics; Molecular Biology; Physics

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2015, Photochemical Sciences

Control of chemical reactions becomes especially challenging when chemical processes have to work within the complexity of biological environments. This is one of the reasons why the ability to design “caged” molecules with structure, reactivity, and biological activity that can be activated externally by light continues to draw significant attention, from both the practical and fundamental points of view. Possible applications of such molecules include design of molecular machines and switches, logic gate mimics, optical sensors, drug delivery systems, etc. Since “caged” molecules are of particular use for processes that occur in biochemical systems and in the environment, interesting light-sensitive systems, anti-cancer drugs, have been developed recently to control DNA cleavage. Caged molecules may interact with or bind with DNA and can be classified by their mechanism of action. Each of these classes of molecules has a different structure and interacts with DNA in a different way, but some molecules can combine several functionalities. The preponderance of caged molecules, anti-cancer drugs, capable of DNA cleavage or their metabolites incorporate Electron Transfer (ET) functionalities, which play important roles in physiological responses. These main groups include quinones (or phenolic precursors), metal complexes, aromatic nitro compounds (or reduced derivatives), and conjugated imines (or iminium species). Redox cycling with oxygen can occur giving rise to Oxidation Stress (OS) through generation of Reactive Oxygen Species (ROS) which can contribute to drug efficacy or can lead to undesirable toxicity. In some cases, ET results in interference with normal electron transport chains. In this work a series of caged molecules-chiral Pyrene Dihydridioxins (PDHD)-DNA chiral DNA intecalators and PDHD-metal complexes bearing masked o-quinone functionality activated through intramolecular ET were synthesized. The o-quinone release and intramolecular ET can be ea (open full item for complete abstract)

Committee: Marshall Wilson (Advisor); Jill Zeilstra-Ryalls (Other); Thomas Kinstle (Committee Member); Alexander Tarmovsky (Committee Member) Subjects: Biochemistry; Biomedical Research; Chemistry; Molecular Chemistry; Molecular Physics; Molecules; Organic Chemistry; Pharmaceuticals; Pharmacy Sciences

Master of Science (M.S.), University of Dayton, 2014, Bioengineering

A novel di-runthenated porphyrin has been synthesized that is capable of photocleaving plasmid DNA within the photodynamic therapy window of 600-800nm. The desired porphyrin was synthesized through reaction of 4-pyridine carboxaldehyde, 4-trifuorlomethyl benzaldehyde and pyrrole under reflux in propionic acid and isolated through column chromatography using methylene chloride/methanol as the eluent. Coordination of cis-Ru(bipy)2Cl2 moieties was achieved through reflux under nitrogen in glacial acetic acid to give the bis-Ru(bipy)2Cl]2[5,15-(4-pyridyl)-10,20-(p-trifluoromethylphenyl)-porphyrin. UV-Vis spectra of the porphyrin and its ruthenated analog revealed an intense Soret band at 410 nm and Q-bands at 500 and 650nm. Cyclic voltammetry was used to determine the oxidative and reductive characteristics of the porphyrin and its ruthenated analog. DNA titrations using buffered solutions of the ruthenated porphyrin and calf thymus DNA were performed spectrophotometrically. The binding constant of the ruthenated porphyrin was determined to be 1.30 x 106 M-1. The ability of the ruthenated porphyrin to photocleave DNA was evaluated by irradiating aqueous samples of plasmid DNA and the complex at a ratio of 5 base pairs to 1 complex using a mercury arc lamp with a 500nm filter. Use of the 500nm filter allowed for observation of the photodynamic therapy window of 600-800nm. Samples were taken at 5 minute intervals and compared using gel electrophoresis to confirm the formation of the photocleaved nicked form of the plasmid DNA.

Committee: Shawn Swavey (Advisor); Doug Dudis (Committee Member); Robert Wilkens (Committee Member); Donald Comfort (Committee Member) Subjects: Chemistry; Inorganic Chemistry

Master of Science, University of Toledo, 2004, Chemistry

In the Mueser laboratory, we study how DNA replication and repair proteins recognize DNA in a structure-specific manner. Bacteriophage T4 is used as a model system to study DNA replication as it encodes all ten proteins required for DNA replication. Much is known about how the individual proteins function in replication but not much is known about the structural aspects of the protein-protein or protein-DNA interactions at the replication fork. The goal of our research is to study how these replication proteins interact with each other and with DNA. We work towards achieving this goal by crystallizing the protein-protein and protein-DNA complexes and then solving their structures, using macromolecular crystallography techniques. We then use the structural information gathered to analyze the interactions. The overall goal of this master's thesis project was to learn many of the techniques involved in protein chemistry and protein crystallization. My research was tailored to protein expression, purification and crystallization so I could learn an array of techniques and become familiar with various pieces of instrumentation. I wanted to be able to use this knowledge in future research positions. My work was focused on two of the replication proteins from Bacteriophage T4: T4 gene 59 helicase assembly protein and T4 gene 32 single-stranded binding protein. These two proteins interact in the absence of DNA and form a complex at the replication fork. I was responsible for expressing mutated and truncated forms of the native proteins on a large scale and developing purification protocols in order to prepare pure protein for crystal screening. After my research with the T4 helicase assembly protein began, I also started working on a similar helicase assembly protein from a related system – bacteriophage KVP40 59 protein. I was also responsible for developing a purification protocol for single-stranded DNA substrates that were used to prepare forked substrates for the cryst (open full item for complete abstract)

Committee: Timothy Mueser (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, The Ohio State University, 2011, Physics

We investigate the use of physical modeling to extract mechanistic details from quantitative biological data, with a focus on the physical properties of nucleic acids. It is well understood that DNA stores genetic information, RNA acts as a carrier of this information, and that both must interact with a wide array of protein complexes in order to perform these functions. However, the physical mechanisms by which these interactions occur are much less clear. For example, Protein-bound duplex DNA is often bent or kinked. Yet, quantification of intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA cyclization experiments have indicated that DNA may form sharp bends more easily than predicted by the established worm-like chain (WLC) model. One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges. We test this model for three sequences at temperatures from 23C to 65C. We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures. There are also many important proteins which bind single-stranded nucleic acids, such as the nucleocapsid protein in HIV and the RecA DNA repair protein in bacteria. The presence of such proteins can strongly alter the secondary structure of the nucleic acid molecules. Therefore, accurate modeling of the interaction between single-stranded nucleic acids and such proteins is essential to fully understanding many biological processes. We develop a model for predicting nucleic acid secondary structure in the presence of single stranded binding proteins, and implement it as an extension of the Vienna RNA Package. Using this model we are able to predict the probability of the protein binding at any position in the nucleic acid sequence, the impact of the protein on nucleic acid base pairing, the end-to-end distance distribution for the nucleic acid, and FRET distributions for (open full item for complete abstract)

Committee: Ralf Bundschuh (Advisor); Michael Poirier (Advisor); Mohit Randeria (Committee Member); Samir Mathur (Committee Member); David Symer (Committee Member) Subjects: Biophysics

Doctor of Philosophy, Miami University, 2007, Microbiology

Eukaryotic cells possess mechanisms that monitor breaks in genomic DNA and repair them. The effectors of double strand break repair (DSBR) comprise a variety of proteins, including the Mre11/Rad50/Nbs1 (MRN) complex and the mediator of DNA damage checkpoint protein 1 (Mdc1). The Adenovirus (Ad) genome is linear, double stranded DNA that can be a substrate for “repair” by host DSBR proteins that link genomes end-to-end forming concatemers. The virus defends itself against this repair by producing regulatory proteins that interfere with DSBR. Early region 4 (E4) orf3-11kDa protein relocalizes the cellular MRN complex to nuclear track-like structures. The E4-orf6 34kDa/E1b-55kDa complex targets MRN for proteasome mediated degradation. Mutants that lack the E4-11kDa and 34kDa proteins activate the cellular damage response and are severely defective for DNA replication. We have investigated roles for the MRN complex and Mdc1 as sensors and effectors of damage signaling in an Ad-E4 mutant infection, particularly related to the onset of an efficient viral DNA replication. Briefly, we find that the MRN complex regulates the re-localization of Mdc1 early in the infection with an E4 mutant virus, consistent with its role as a sensor of DNA damage. Mdc1 is re-localized in response to viral infection and binds E4 mutant viral DNA, but does not appear to have a role in the regulation of viral DNA replication. The MRN complex, however, is relocalized to E4 mutant viral DNA replication centers in an Nbs1 dependent manner and binds viral DNA. The Nbs1 dependent binding of the complex to E4 mutant viral DNA inhibits the efficient onset of viral DNA replication consistent with the role of the MRN complex as an effector in the damage response and repair pathway. Investigating the ability of host DSBR proteins to interfere with E4 mutant DNA synthesis provides a model for understanding the mechanism by which these proteins sense and respond to the presence of aberrant or damaged DNA.

Committee: Eileen Bridge (Advisor) Subjects:

Master of Science, The Ohio State University, 2006, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2021, Chemistry

The structures adopted by biological macromolecules and macromolecular complexes are directly tied to their function. By better understanding the relationship between structure and function of biomolecules, lifesaving and life-changing interventions can be designed such as small molecule inhibitors of viral enzymes. Characterization of macromolecules with high-resolution structural techniques has greatly improved our fundamental understanding of how structures dictate function. High-resolution structural techniques including x-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance spectroscopy, however, have many challenges and so they require complementary techniques. Native mass spectrometry is one such technique that can be used to interrogate macromolecular assemblies and accurately determine molecular weight(s), oligomeric state(s), ligand-binding, and topology of the assembly. Native mass spectrometry has the advantage of not being limited by some of the common issues encountered by high-resolution techniques like molecular flexibility and sample heterogeneity that can limit resolution attainable or prevent structure determination altogether. This attribute is particularly valuable for the characterization of protein-nucleic acid complexes that have proven to be some of the more challenging complexes for high-resolution techniques. Throughout this work, native mass spectrometry is emphasized as a clear approach for examining differences in macromolecular assemblies that highlight the structural diversity of macromolecular systems which would otherwise not be evident. Chapter 3 describes identification of the physiological protein interface of a plant protein, BX1.This approach demonstrates the use of native mass spectrometry and covalent cross-linking mass spectrometry to solve a common issue with X-ray crystallography, namely artificial protein contacts formed during the crystallization process. Chapter 4 describes the investigation of (open full item for complete abstract)

Committee: Vicki Wysocki (Advisor); Venkat Gopalan (Committee Member); Karin Musier-Forsyth (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, The Ohio State University, 2021, Biochemistry

Understanding the structure and function of macromolecules and their performative complexes is indispensable when developing novel treatments for disease. Nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) provide powerful insights into macromolecular structure and dynamics and thereby harbor the potential for aiding the development of much needed therapeutics. The studies presented here describe the application of NMR spectroscopy and cryo-EM to characterize the structure and/or dynamics of three distinct biochemical entities with fundamental biological implications. These targets include cyclic peptides being developed as cancer therapies, a homo-oligomeric ring protein that serves as a model system for understanding allosteric regulation, and a recombinase that selectively binds and becomes activated to site-specifically cleave DNA. Cyclic peptides are capable of binding to challenging molecular targets (e.g., proteins involved in protein-protein interactions) with high affinity and specificity, but generally cannot gain access to the intracellular environment because of poor membrane permeability. In chapter 2, I describe my work to characterize a pair of conformationally constrained cyclic cell-penetrating peptides (CPP) containing a D-Pro-L-Pro motif, beginning with cyclo(AFΦrpPRRFQ) (where Φ is L-naphthylalanine, r is D-arginine, and p is D-proline). The structural constraints provided by cyclization and the D-Pro-L-Pro motif permitted the rational design of cell-permeable cyclic peptides of large ring sizes (up to 16 amino acids). This strategy was applied by my collaborators to design a potent, cell-permeable, and biologically active cyclic peptidyl inhibitor, cyclo(YpVNFΦrpPRR) (where Yp is L-phosphotyrosine), against the Grb2 SH2 domain, a key mediator in Ras activation. Multidimensional NMR spectroscopic and circular dichroism analyses revealed that the initial cyclic CPP as well as the Grb2 SH2 inhibitor assume a pr (open full item for complete abstract)

Committee: Mark Foster (Advisor); Thomas Magliery (Committee Member); Charles Bell (Committee Member) Subjects: Biochemistry

PhD, University of Cincinnati, 2020, Medicine: Molecular and Developmental Biology

Rapid growth of the embryonic brain requires accurate cell specification and sufficient proliferation to generate a diversity of neuronal subtypes. Neurogenesis in the developing mammalian forebrain is controlled via precise spatial and temporal expression of key transcription factors. The homeodomain transcription factor, Gsx2, is important for three key functions during the development of the mouse forebrain: 1) Dorso-Ventral patterning of neural progenitors by establishing the pallio-subpallial boundary; 2) Balancing the maintenance of proliferative progenitors with neurogenesis in the Lateral Ganglionic Eminence (LGE); and 3) Defining progenitor identity to specify distinct neuronal subtypes. Despite its clear functional importance, little is known about the mechanisms by which Gsx2 performs these functions during forebrain development. Here, I will describe two new molecular functions of the Gsx2 protein. First, we found a novel direct interaction between Gsx2 and the proneural basic helix-loop-helix (bHLH) transcription factor Ascl1, which promotes neurogenesis during forebrain development. We show that physical interactions between Gsx2 and Ascl1 within dividing neuronal progenitors prevents Ascl1 from forming homodimers or heterodimers with other bHLHs, and thereby inhibits Ascl1 binding to DNA. Based on our data, we propose a model in which Gsx2 induces Ascl1 gene expression, but limits Ascl1's ability to promote neuronal differentiation through direct Gsx2-Ascl1 protein-protein interactions that inhibit the activation of neurogenic target genes. Therefore, Gsx2-Ascl1 co-expressing cells are primed for neurogenesis, but will not differentiate until Gsx2 is down-regulated. Second, we have identified two types of Gsx2 DNA binding sites within LGE enhancers: monomer sites (M-sites) that independently bind Gsx2 and dimer sites (D-sites) that cooperatively bind Gsx2. We define the DNA binding site features required for cooperative DNA binding and demonstrate it (open full item for complete abstract)

Committee: Brian Gebelein Ph.D. (Committee Chair); Kenneth Campbell Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Masato Nakafuku M.D. (Committee Member); James Wells Ph.D. (Committee Member) Subjects: Developmental Biology

Doctor of Philosophy, The Ohio State University, 2019, Molecular Genetics

Zinc is the second most abundant transition metal in humans and is essential for life. Zinc is required for the activity of more than 300 enzymes spanning the six major classes of enzymes. Also, the coordination of zinc ions confers proper structural conformation to numerous proteins. Given the importance of zinc to cellular metabolism, zinc deficiency is harmful to health. Zinc, in excess, is also toxic to cells. Therefore, all organisms have evolved zinc homeostasis mechanisms to maintain an optimum range of intracellular zinc levels. Zinc-responsive transcriptional regulation is one of the most common means to achieve zinc homeostasis. Transcriptional factors can regulate the expression of zinc transporters, zinc-sequestering proteins, and abundant zinc-binding proteins according to cellular zinc status. Most of our knowledge about the zinc-responsive transcriptional regulation in eukaryotes is derived from Zap1 from Saccharomyces cerevisiae and MTF-1 from vertebrates, which activate gene expression in response to zinc deficiency and excess zinc, respectively. However, there are several questions remaining to be answered, including the regulation of mammalian zinc-regulated genes independent of MTF-1 and the properties that enable proteins to sense intracellular zinc levels. The fission yeast Schizosaccharomyces pombe lacks a Zap1 homolog, and yet regulates gene expression in response to zinc. It was later discovered that Loz1 was required for this zinc-responsive transcriptional regulation of gene expression. In this study, I have investigated the role of Loz1 in the zinc-responsive regulatory pathway. The results from this study show that Loz1 is a transcriptional factor that represses the expression of target genes by binding to their promoters under zinc-replete conditions. For the Loz1-mediated regulation in vivo, a Loz1 Response Element (LRE) with the sequence 5'-CGNMRATCNTY-3' is necessary and sufficient. By combining information about the Loz1 binding (open full item for complete abstract)

Committee: Amanda Bird (Advisor); Craig Burd (Committee Member); Guramrit Singh (Committee Member); Jian-Qiu Wu (Committee Member) Subjects: Genetics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2018, Physics

DNA/RNA-protein interactions are crucial to the maintenance, processing, and regulation of the genetic information contained within a cell. Sets of particular DNA/RNA-protein interactions can confer specificity to a protein's binding behavior, which makes the consideration of DNA and RNA with modifications interesting in how they alter the landscape of interactions. High-throughput sequencing (HTS) techniques of DNA and RNA can offer unmatched genome-wide characterizations of where and how proteins interact with DNA and RNA by generating libraries of millions of reads that can capture the whole distribution of these interaction events. In this dissertation we used HTS analysis techniques to probe the location and state of two nucleic acid modifications on a genome-wide scale: The methylation of Cytosines in the CpG dinucleotide (mCpG) in the human genome, and the nucleotide addition by 3'-5' polymerases to the 5'-ends of RNAs. In the case of DNA methylation, the sequencing data represents DNA fragments enriched in mCpGs by way of interaction with the methyl-CpG-binding domain (MBD) protein MBD2. In the case of 5'-end nucleotide addition, the sequencing data is a snapshot of non-coding RNAs whose 5'-ends may have been extended by a Thg1-like protein (TLP). In both sets of studies, the information about the modification in turn conveys information on the relevant DNA-protein and RNA-protein interactions, and what sequences or structures are preferred by the proteins reading or depositing the modifications. In Chapter 2, a deep sample of methylated genomic DNA allows us to characterize the sequence, spacing, and mCpG density biases introduced through MBD2 interaction during a protein pulldown experiment. In Chapter 3, we use the characterizations of those interactions -- the 3 bp minimum separation between mCpGs, the nonlinear relationship between mCpG number and pulldown efficiency -- to build a model to predict expected pulldown from a SssI Control sample, which is t (open full item for complete abstract)

Committee: Ralf Bundschuh (Advisor); Michael Poirier (Committee Member); Jane Jackman (Committee Member); Andrew Heckler (Committee Member) Subjects: Biophysics; Physics

Master of Science, University of Toledo, 2018, Medicinal Chemistry

Cancer is the second leading cause of death in the United States of America and the entire world and the disease burden is exacerbated due to resistance to drugs that are currently in clinical use. Histone deacetylase (HDAC) enzymes are highly expressed in cancer cells and HDACs are considered viable targets for drug intervention. HDACs cleave the acetyl groups from acetylated lysine side chains of proteins and modulate crucial cellular processes including gene expression. Four HDAC inhibitors (HDACi) have been approved by the US FDA as anticancer drugs for clinical use, but these drugs have numerous side effects due to low selectivity. This study presents an attempt to develop selective HDAC inhibitors using 1-(1H-imidazol-2-yl) ethan-1-one moiety as a novel metal-binding group as possible alternative to current cancer drug treatment options. In this study, molecular modeling studies were carried out using crystal models of two different HDAC isoforms and HDLP to formulate novel HDAC inhibitors that have high selectivity. The designed analogs, 14a-g were synthesized and evaluated for biological activity. The compounds were tested for anti-proliferative activity in the NCI 60 cell lines assay. Compound 14a showed significant cell growth inhibition at 10 µM concentration with 87% mean cell growth inhibition. This compound was further tested in the dose response assay. It showed anti-proliferative activity in micro molar range against most of the cell lines while four leukemia cell lines were sensitive at sub-micro molar concentration of compound. Data from dose response assay showed that the activity improved significantly when a trifluoromethyl group is installed at the para-position of the phenyl ring cap group and with N-methylation of the imidazole ring According to docking studies of all seven compounds 14a-g on HDLP, compounds with N-methyl imidazole rings 14a-d showed flipping of the molecule in the HDAC active site, the acetamide oxygen binding to zinc ion (open full item for complete abstract)

Committee: Viranga Tillekeratne Ph.D. (Committee Chair); James Slama Ph.D. (Committee Member); Zahoor Shah Ph.D. (Committee Member) Subjects: Chemistry; Pharmaceuticals; Pharmacy Sciences

PhD, University of Cincinnati, 2017, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Telomeres are the protective DNA-protein complexes at the ends of chromosomes. Mammalian telomeres are composed of double-stranded DNA with a repetitive sequence (TTAGGG) followed by a short single-stranded overhang. The DNA is bound by a series of proteins that prevent chromosome fusions and protect the DNA terminus from being sensed as damage. These telomere proteins also aid in telomere replication. Shelterin, the primary telomere protein complex, binds to both the double- and single-strand regions of the telomere. Shelterin is important for protecting telomeres from being sensed as damage. It also brings in telomerase for telomere extension. The other major telomere protein complex is CST (CTC1-STN1-TEN1). CST functions in telomere replication first by aiding passage of the replication machinery through the telomere duplex and then enabling fill-in synthesis of the telomeric C-strand following telomerase action. CST also has genome-wide roles in the resolution of replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Here we address CST mechanism by using STN1 OB-fold mutant (STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. In vitro binding studies show that STN1 directly engages both short and long ssDNA, however STN1-OBM preferentially destabilizes binding to short substrates. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST bindin (open full item for complete abstract)

Committee: Carolyn Price Ph.D. (Committee Chair); Iain Cartwright Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Anil Menon Ph.D. (Committee Member); Satoshi Namekawa Ph.D. (Committee Member) Subjects: Biogeochemistry

Master of Science, University of Akron, 2017, Physics

Fluorescence correlation spectroscopy (FCS) is a useful experimental technique, that uses statistical analysis of fluorescence intensity fluctuations and a correlation function. The method permits studying the kinetics and interactions of particles not only on the surface of the solution, but also in the bulk. Another benefit of FCS is that it does not require the selection of specific molecules or time intervals for its measurements. By analyzing biological dynamics, we can learn about concentration, viscosity, individual movement and diffusion of particles as well as interactions and the possibility of binding between them. In this work, we present a brief explanation on the analytic formalism of the governing concepts of FCS, as well as detail the experimental setups. We also discuss protein-vesicle interactions and the kinematics of double-stranded DNA and labeled nucleotides. The brightness and size of molecules, before and after adding vesicles to the protein solution, are calculated. Finally the possibility of vesicle-protein binding is validated.

Committee: Adam Smith Dr. (Advisor); Ben Hu Dr. (Committee Chair); Jutta Luettmer-Strathmann Dr. (Committee Member) Subjects: Biochemistry; Biophysics; Molecular Biology; Molecular Chemistry; Physical Chemistry; Scientific Imaging

Doctor of Philosophy, The Ohio State University, 1997, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2014, College of Sciences and Health Professions

Subtelomeres consist of sequences adjacent to telomeres and contain genes involved in important cellular functions, as subtelomere instability is associated with several human diseases. Balancing between subtelomere stability and plasticity is particularly important for Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major surface antigen, variant surface glycoprotein (VSG), to evade the host immune response. VSGs are expressed exclusively from subtelomeres in a strictly monoallelic fashion. Telomere proteins are important for protecting chromosome ends from illegitimate DNA processes. However, whether they contribute to subtelomere integrity and stability has not been well studied. We have identified a novel T. brucei telomere protein, T. brucei TRF-Interacting Factor 2 (TbTIF2), as a functional homolog of mammalian TIN2. A transient depletion of TbTIF2 led to an elevated VSG switching frequency and an increased amount of DNA double strand breaks (DSBs) in both active and silent subtelomeric VSG expression sites (BESs). Therefore, TbTIF2 plays an important role in VSG switching regulation and is important for subtelomere integrity and stability. TbTIF2 depletion increased the association of TbRAD51 with the telomeric and subtelomeric chromatin, and TbRAD51 deletion further increased subtelomeric DSBs in TbTIF2-depleted cells, suggesting that TbRAD51-mediated DSB repair is the underlying mechanism of subsequent VSG switching. Surprisingly, significantly more TbRAD51 associated with the active BES than with the silent BESs upon TbTIF2 depletion, and TbRAD51 deletion results in much more DSBs in the active BES than in the silent BESs in TbTIF2-depleted cells, suggesting that TbRAD51 preferentially repairs DSBs in the active BES. Interestingly, depletion of TbTIF2 affects the protein's level of its interacting TTAGGG Repeat binding Factor, TbTRF. In addition, a transient depletion of TbTRF led to a s (open full item for complete abstract)

Committee: Li Bibo Ph.D (Advisor); Boerner Valentin Ph.D (Committee Member); Kondratov Roman Ph.D (Committee Member); Almasan Alexandru Ph.D (Committee Member); Severson Aaron Ph.D (Committee Member); Taylor Derek Ph.D (Committee Member) Subjects: Molecular Biology; Parasitology

PhD, University of Cincinnati, 2008, Arts and Sciences : Chemistry

Many steps are involved in transfection and must be overcome to provide a successful gene delivery agent: DNA binding/compaction, cellular recognition and uptake, endosomal escape, trafficking through the cell and eventual release of cargo. All of these potential hurdles are being investigated by the Reineke group using our polymeric vectors, which are composed of carbohydrate and oligoamine monomers; however, the crucial first step to efficient transfection, DNA binding, is an event rarely dissected quantitatively. The purpose of this dissertation research was to carefully elucidate aspects of the mechanism of interaction between our polymers and plasmid DNA (pDNA) through biophysical techniques, such as microcalorimetry, dynamic light scattering, zeta potential, circular dichroism, and FTIR spectroscopy. The combination of these results revealed the significance of the carbohydrate hydroxyl stereochemistry and amide spacing to the DNA affinity, as a result of hydrogen bonding to the backbone and base pairs. As the amine number increased from 1 to 4, the DNA binding mechanism of trehalose-containing polymers became less dependent upon electrostatics and more on hydrogen bonding. This change is a result of decreasing charge fraction with increasing amine density. To determine the necessity of charge-charge interaction between our polycations and nucleic acids, a new series of polymers, replacing the oligoamine with an analogous oligo(ethylene glycol), was synthesized and its characterization compared to the poly(glycoamidoamine)s previously studied. It was shown that removing the electrostatic component prevented DNA binding. Therefore, we conclude that long-range Coulombic attraction initiates the interaction between our polymeric vectors and nucleic acids, but then hydrogen bonding becomes a dominant contributor. Stabilization of our delivery agents to physiological salt and serum conditions is also critical to their clinical use, so another aspect of this thesi (open full item for complete abstract)

Committee: Theresa M. Reineke PhD (Committee Chair); Thomas L. Beck PhD (Committee Member); H. Brian Halsall PhD (Committee Member); Bruce S. Ault PhD (Committee Member); Matthew L. Lynch PhD (Committee Member) Subjects: Biophysics; Chemistry; Polymers