Master of Science in Biological Sciences, Youngstown State University, 2015, Department of Biological Sciences and Chemistry

Recombineering, also known as recombination-mediated genetic engineering, is a molecular genetics technique that utilizes homologous recombination to modify the genome of prokaryotes and eukaryotes in vivo. One recombination system is the lambda Red recombination system that is controlled by the lambda bacteriophage, which contains the red genes that encode the Exo, Beta, and Gam proteins. The Exo, Beta, and Gam proteins are involved in the process of double strand break repair and are responsible for homologous recombination. This method of recombination has replaced the more conventional and time-consuming genome modification technique using restriction endonuclease enzymes and DNA ligase. I hypothesize that the lambda Red recombination system will be successful in Enterobacter sp. YSU because it was developed in Escherichia coli and has been proven to be successful in Enterobacter cloacae, Enterobacter aerogenes and Saccaromyces cerevisiae. pKD46 plasmid was PCR amplified to remove the ampicillin resistance gene because Enterobacter sp. YSU is already resistant to ampicillin which is the selectable marker for pKD46. A chloramphenicol and kanamycin resistance gene was PCR amplified and mixed with the pKD46 PCR product without the ampR gene, treated with T4 Polynucleotide Kinase, and ligated to construct two new plasmids, pKD46-cm and pKD46-kan. pKD46-cm and pKD46-kan were used for recombination with pBR322 plasmid to replace the ampR gene with chloramphenicol or kanamycin resistance. AmpR was successfully replaced with kanamycin resistance using pKD46-cm and with chloramphenicol resistance using pKD46-kan in E. coli. Recombination in Enterobacter sp. YSU via transformation was attempted using pKD46-kan to replace cmR with kanamycin resistance because it was successful in E. coli. However, it was not successful because cmR from pACYCY184 does not appear to be expressed well in YSU. This recombination system can be useful in helping understand gene function by c (open full item for complete abstract)

Committee: Jonathan Caguiat Ph.D. (Advisor); David Asch Ph.D. (Committee Member); Xiangjia Min Ph.D. (Committee Member) Subjects: Biology; Genetics; Microbiology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2015, Biochemistry Program, Ohio State

Bacteriophage lambda encodes a two-component Synaptase-Exonuclease (SynExo) system used for generating end-to-end concatemers of the viral genome before packaging. Lambda exonuclease (exo) is a processive 5'-3' exonuclease that degrades linear dsDNA into mononucleotides, generating a 3' single-stranded overhang. Redß is a single-strand annealing protein (SSAP) that binds to the resulting overhang and anneals it to a complementary strand. Redß serves as a model to study the conserved single-strand annealing (SSA) DNA repair pathway, yet its manner of oligomerization, DNA binding and annealing remain elusive due to difficulty in obtaining a high-resolution structure. Here, we quantitatively compare the characteristics of the N-terminal DNA binding domain of Redß (Redß177) with that of the full-length protein by measuring their DNA binding, in vivo activity, and interaction with exo. RedßFL and Redß177 have unique DNA binding preferences to ssDNA and annealed duplex product, and display significant differences in recombination activity, revealing that the C-terminus of Redß is required for full in vivo activity and a robust interaction with its intrinsic partner, exo. The C-terminal residues 182-261 (RedßCT) fold into a monomeric, alpha-helical structure and are sufficient in forming an interaction with exo, yet are unable to bind ssDNA or annealed duplex. Additionally, we show the ability of Redß177 to form oligomeric structures similar to those of RedßFL, alone and in the presence of DNA. Redß oligomerization is a highly dynamic and concentration-dependent process, and the presence of DNA substrates stabilizes the oligomer. The Redß-exo synaptosome complex also appears to be highly dynamic, and we propose a unique model for Redß-exo processing of dsDNA ends. As we continue to pursue a 3D crystal structure for Redß, data presented here supports a modular domain structure for Redß: an N-terminal domain that contains the determinants for ssDNA binding and oligomerizatio (open full item for complete abstract)

Committee: Charles Bell (Advisor) Subjects: Biochemistry

Doctor of Philosophy, The Ohio State University, 2011, Neuroscience Graduate Studies Program

Drug dependence is a persistent problem throughout the world. Once addicted to a drug, many users have difficulty quitting use, even when they desire to stop using. This substance dependence is both psychological as well as neurophysiological. In 2008 the U.S. Department of Health classified 22 million Americans as having a significant degree of drug dependence. Additionally, the U.S. Office of National Drug Control Policy stated that the health care costs in 2004 for substance abuse was estimated to be comparable to cancer. There is therefore undoubtedly both an individual and societal need for therapeutic interventions in drug dependence. While a great deal is known about the molecular action of these addictive substances, very little is understood about the underlying neurocircuitry of addiction. The process of addiction is a learned behavior and is not solely driven by homeostatic adaptations to the drug itself. Psychoactive drugs overrun the natural reward circuitry of the brain, forcing maladaptive learning of drug associated rewards which then become overvalued in comparison to natural rewards. This dissertation approaches the study of drug addiction using two separate techniques: one regionally specific to the nucleus accumbens, and one cell type specific to dopaminergic and GABAergic neurotransmitter systems of the central nervous system. All drugs of abuse increase striatal dopamine release despite their widely varied mechanisms of action. The ventral striatum primarily consists of the Nucleus Accumbens (NAc), which has long been thought of as the origin of addiction-like behaviors. As such, the NAc has been proposed to be one of the primary reward centers of the brain. Utilizing viral delivery of short-hairpin RNA (shRNA) into the NAc, knockdown of specific subtypes of receptors can be achieved. In this manner, behavioral testing can investigate changes in rodent addiction-like behavior following region specific knock down of dopamine receptor isoforms. (open full item for complete abstract)

Committee: Howard Gu PhD (Advisor); Wolfgang Sadee PhD (Committee Member); Rene Anand PhD (Committee Member); John Oberdick PhD (Committee Member); Susan Cole PhD (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology; Neurobiology; Neurosciences; Pharmacology