Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Molecular Virology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

No description available.

Molecular typing of strains among the highly diverse population of Helicobacter pylori (H. pylori) is an important approach for both basic and clinical studies. Genomic DNAs prepared from 18 gastric biopsy specimens, 21 H. pylori clinical isolates obtained from gastric biopsy specimens, and five isolates collected from a single patient at weekly intervals, were subjected to a combined single- and double-stranded conformational polymorphism (SDSCP) assay. The results showed that 19 of 21 isolates tested were discriminated by SDSCP analysis. SDSCP analysis of five H. pylori isolates collected from the same patient at different times resulted in five identical profiles, suggesting the reproducibility of the method. When DNA preparations from 18 gastric biopsy specimens were subjected to SDSCP analysis, 18 unique profiles were generated that matched those of their corresponding cultured H. pylori isolates from each patient. For comparison, polymerase chain reaction (PCR)-based restriction fragment length polymorphism analysis yielded only nine profiles for 20 strains. The data suggest that SDSCP analysis may be an effective and reliable method for differentiation of H. pylori strains directly from gastric biopsy specimens without requiring isolation of the organisms by culture.

Recognition of non-self, pathogen-associated molecular patterns is a central component of host immune response to pathogens like viruses. Intracellular detection of viral nucleic acids leads to the production of type I interferons (IFN-I) and subsequent establishment of an antiviral state in infected and neighboring cells. Viruses have evolved multiple mechanisms to counteract IFN-I responses in infected cells, however, viral nucleic acids released from dying cells can stimulate IFN-I production in surrounding or distal uninfected cells. This thesis examines the mechanisms by which cells recognize extracellular viral nucleic acids and the subsequent downstream antiviral signaling. Class A scavenger receptors (SR-As) internalize extracellular viral double-stranded RNA (dsRNA) to mediate IFN-I responses, but little is known about extracellular viral DNA. We observed that extracellular DNA is recognized and internalized by SR-As in a manner like extracellular dsRNA. Furthermore, we established that SR-A1 is sufficient in mediating extracellular dsRNA-induced cellular responses and other nucleic acid receptors like SR-J1 and DEC-205 are dispensable. Finally, a direct interaction of RNA and DNA species was demonstrated with the coiled-coil collagenous domain of SR-A1, but not the scavenger receptor cysteine rich domain of SR-A6.We elaborated the role of SR-A1 by identifying the cellular processes activated through SR-A1 following uptake of extracellular dsRNA. Cytosolic sensors are essential in mediating an antiviral response to the endocytosed dsRNA, but the mechanism of endoplasmic release and cytoplasmic entry of dsRNA remains an enigma. We demonstrated that the lack of a dsRNA-channel, SIDT2, impaired the ability of the cells to mediate an antiviral response to extracellular dsRNA. Understanding host responses to extracellular viral nucleic acids will enable the development of novel vaccines and antiviral therapeutics against RNA and DNA viruses that efficiently counteract these responses in infected cells. / Thesis / Doctor of Philosophy (PhD) / Viral infections remain a threat to global health as new diseases continue to emerge. To develop effective vaccines and antivirals to combat viruses and alleviate human disease require a deeper understanding of virus-host interactions. Host cells identify virus-associated molecules to detect viruses and eliminate them whereas, viruses employ tactics to prevent the activation of the immune system. However, virus-induced cell lysis releases viral molecules that can stimulate immune responses in neighbouring uninfected cells. This thesis examines the mechanism by which cells respond to extracellular viral nucleic acids. We showed that a protein present at the cell surface called ‘class A scavenger receptor 1’ is sufficient to internalize extracellular viral nucleic acids, leading to immune responses. The response is impaired when a channel protein, SIDT2, is absent in the cells. Further work is necessary to understand how this knowledge can be harnessed to develop vaccines and antiviral therapeutics.

<p> An improved method for the synthesis of oligoribonucleotides of defined sequence was developed. The general phosphotriester synthesis of Neilson and co-workers was modified by the introduction of a new condensing agent, mesitylenesulfonyl-1,2,4-triazole, and by the replacement of the bis(cyclohexylammonium) salt of mono-2,2,2-trichloroethyl phosphate with its acid salt. These modifications provided significant increases in the yields for the condensation of protected nucleosides - especially in the case of purine residues. Finally, modification of the three-step procedure for the deprotection of protected oligoribonucleotides resulted in the isolation of oligomers of exceptional purity and biological activity.</p> <p> Oligomers corresponding to natural sequences in transfer RNA molecules were obtained by this improved method of synthesis. These oligomers were then used to study: 1. The formation of short double-stranded RNA helices and 2. The interactions of aminoacyl-tRNA ligases with tRNA fragments.</p> / Thesis / Doctor of Philosophy (PhD)

No description available.

Glioblastoma multiforme (GBM) is a deadly grade IV brain tumor characterized by a heterogeneous population of cells that are drug resistant, aggressive, and infiltrative. The current standard of care, which has not changed in over a decade, only provides GBM patients with 12-14 months survival post diagnosis. We asked if the addition of a novel endoplasmic reticulum (ER) stress inducing agent, JLK1486, to the standard chemotherapy, temozolomide (TMZ), which induces DNA double strand breaks (DSBs), would enhance TMZ’s efficacy. Because GBMs rely on the ER to mitigate their hypoxic environment and DNA repair to fix TMZ induced DSBs, we reasoned that DSBs occurring during heightened ER stress would be deleterious. Treatment of GBM cells with TMZ+JLK1486 decreased cell viability and increased cell death due to apoptosis. We found that TMZ+JLK1486 prolonged ER stress induction, as indicated by elevated ER stress marker BiP, ATF4, and CHOP, while sustaining activation of the DNA damage response pathway. This combination produced unresolved DNA DSBs due to RAD51 reduction, a key DNA repair factor. The combination of TMZ+JLK1486 is a potential novel therapeutic combination and suggests an inverse relationship between ER stress and DNA repair pathways.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

Homologous Recombination (HR) can promote genome stability through its capacity to faithfully repair DNA gouble 2trand !;!reak2 (DSBs) and preventing the demise of stalled replication forks in part by catalysing template switching to enable DNA polymerase to bypass lesions. Despite these beneficial roles, inappropriate or untimely HR events can have deleterious consequences. HR can cause genome instability by recombining "inappropriate" homologous sequences, especially if the recombination intermediates are resolved to form crossovers. Over the past few years, study of the rare inherited chromosome instability disorder, Eanconi Anaemia (FA), has uncovered a novel DNA damage response pathway. Although the FA pathway is required primarily for interstrand DNA cross link repair, its precise role in DNA repair reactions is still unclear. FA.Qomplementation group M (FANCM) is the sole component within the FA core complex which possesses a DNA helicase/ATPase domain and an endonuclease domain (albeit non-functional), suggesting that FANCM could translocate along DNA and target the FA core complex to blocked replication forks. To further elucidate the role of FANCM in HR, I have purified Fm11, the FANCM orthologue in the fission yeast Schizosaccharomyces pombe and tested its activity on a range of synthetic replication and recombination intermediates in vitro. Fml1 binds both replication forks and Holliday Junctions (HJs) which are key intermediates of HR.

Plants have developed intricate mechanisms to control growth in response to a variety of environmental cues, to compensate its immobility and to survive in both normal and adverse conditions. The TCP proteins are a family of plant-specific, basic helix-loop-helix (bHLH) transcription factors that involve in different aspects in plant growth and developmental control. The Arabidopsis TCP20 has been shown to involve in coordinating cell growth and proliferation, and in growth arrest in response to DNA double-stranded breaks (DSB). In this thesis, the main interest is to examine the function of Arabidopsis TCP6, which shares the highest homology with TCP20, and like TCP20, contains a putative ATM phosphorylation motif that suggests potential involvement in the ATM/ATR-mediated DSB responses. Expressional analysis including transcript measurement and reporter gene tagging demonstrated that TCP6 is expressed in flowers, in particular in the first mitotic event of pollen and ovule/embryo sac development, indicating that TCP6 potentially involves in regulating the mitotic cell cycle during gametophyte development. Yet no gametophytic or fertility-affecting mutant phenotype was observed in the tcp6 single and tcp6/tcp20 double mutants, which may be due to high functional redundancy. The tcp6/tcp20 double mutant seedlings exhibited significantly higher growth performances in true leaf growth compared to wild type when treated with gamma radiation, implying that both functional TCP6 and TCP20 are involved in response to gamma radiation-generated DSBs. The work of this thesis provides the first expressional and functional characterizations of TCP6, with the results suggesting that TCP6 and other class I TCPs play a role in regulating growth under both normal and stress conditions.