Thesis (M.S.)--Georgia State University, 2008. / Title from file title page. Markus W. Germann, committee chair; W. David Wilson, Kathryn B. Grant, committee members. Electronic text (51 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed July 14, 2008. Includes bibliographical references (p. 50-51).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

No description available.

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, accounting for over 600,000 deaths annually. The mortality rate of CRC can be significantly reduced if it is detected early, suggesting the importance of cancer screening in CRC management. Currently, colonoscopy is the gold standard for CRC diagnosis as it is accurate and reliable. However, it is an invasive procedure that is associated with risks of complications, which contributes to the lack of patient compliance in colonoscopy screening. Other noninvasive detection methods suffer from poor sensitivity and specificity. Thus, there remains a great demand for the development of a noninvasive and accurate test for CRC diagnosis. Recently, studies using next-generation sequencing techniques have revealed compositional changes in the intestinal microbiome associated with CRC, implicating the possibility of using fecal microbiome as potential diagnostic markers. Specifically, the level of the gram-negative bacterium, Fusobacterium nucleatum, has been shown to be elevated in CRC patients compared to healthy controls. The work described in this thesis aims to develop unique RNA-cleaving DNAzymes that can distinguish between healthy and CRC stool microbiomes. RNA-cleaving DNAzymes are single stranded DNA molecules that are extensively used as analytical tools for metal ion sensing and bacterial detection. We conducted an in vitro selection experiment and isolated a F. nucleatum-responsive RNA-cleaving DNAzyme sensor, named RFD-FN1, that is activated by a heat stable protein marker by this bacterium. RFD-FN1 is highly specific for F. nucleatum and it has a limit of detection of 107 CFU/mL without culture and a single cell when cultured for 36 hours. The discovery of this novel molecular probe for F. nucleatum presents an important step forward towards the development of a novel DNAzyme-based detection method for colorectal cancer. / Thesis / Master of Science (MSc)

DNA provides the blueprint for cell function and growth, as well as ensuring continuity from one cell generation to the next. In order to compact, protect, and regulate this vital information, DNA is packaged by histone proteins into nucleosomes, which are the fundamental subunits of chromatin. Reactive oxygen species, generated by both endogenous and exogenous agents, can react with DNA, altering base chemistry and generating DNA strand breaks. Left unrepaired, these oxidation products can result in mutations and/or cell death. The Base Excision Repair (BER) pathway exists to deal with damaged bases and single-stranded DNA breaks. However, the packaging of DNA into chromatin provides roadblocks to repair. Damaged DNA bases may be buried within nucleosomes, where they are inaccessible to repair enzymes and other DNA binding proteins. Previous in vitro studies by our lab have demonstrated that BER enzymes can function within this challenging environment, albeit in a reduced capacity. Exposure to ionizing radiation often results in multiple, clustered oxidative lesions. Near-simultaneous BER of two lesions located on opposing strands within a single helical turn of DNA of one another creates multiple DNA single-strand break intermediates. This, in turn, may create a potentially lethal double-strand break (DSB) that can no longer be repaired by BER. To determine if chromatin offers protection from this phenomenon, we incubated DNA glycosylases with nucleosomes containing clustered damages in an attempt to generate DSBs. We discovered that nucleosomes offer substantial protection from inadvertent DSB formation. Steric hindrance by the histone core in the nucleosome was a major factor in restricting DSB formation. As well, lesions positioned very close to one another were refractory to processing, with one lesion blocking or disrupting access to the second site. The nucleosome itself appears to remain intact during DSB formation, and in some cases, no DNA is released from the histones. Taken together, these results suggest that in vivo, DSBs generated by BER occur primarily in regions of the genome associated with elevated rates of nucleosome turnover or remodeling, and in the short linker DNA segments that lie between adjacent nucleosomes. DNA ligase IIIα (LigIIIα) catalyzes the final step in BER. In order to facilitate repair, DNA ligase must completely encircle the DNA helix. Thus, DNA ligase must at least transiently disrupt histone-DNA contacts. To determine how LigIIIα functions in nucleosomes, given this restraint, we incubated the enzyme with nick-containing nucleosomes. We found that a nick located further within the nucleosome was ligated at a lower rate than one located closer to the edge. This indicated that LigIIIα must wait for DNA to spontaneously, transiently unwrap from the histone octamer to expose the nick for recognition. Remarkably, the disruption that must occur for ligation is both limited and transient: the nucleosome remains resistant to enzymatic digest before and during ligation, and reforms completely once LigIIIα dissociates.