Graduate

Thesis (M.S.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Christa Merzdorf. Includes bibliographical references (leaves 51-55).

Thesis (Ph. D.)--University of Rhode Island, 2004. / Typescript. Includes bibliographical references (leaves 220-233).

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

This study provides information about the effects of polyamines on the restriction enzymatic cleavage of bacteriophage lambda DNA. The polyamines studied were spermine, spermidine, Nl-acetylspermidine and N8- acetylspermidine. The restriction enzymes studied were Xhoi, BamHI, EcoRI, and Hindiii. The electrophoretic pattern of lambda DNA digests by these enzymes were recorded photographically. These results were further analyzed by spectrographic digitization and replotting. Polyamines affect the electrophoretic pattern of restriction fragments in two ways: by causing DNA streaking and by decreasing ethidium bromide binding to DNA, which in turn affects DNA staining properties. The polyamines studied have effects which are increasingly dependent on the charge of the polyamine. The concentration necssary to alter the electrophoretic pattern decreases with increased positive charge of the polyamines. Spermine, the most highly charged polyamine studied, resulted in alterations at a lower concentration than any other polyamines studied. Following spermine was spermidine, and then the two acetylated polyamines, Nl-acetylspermidine, and N8- acetylspermidine.

A DNA probe was developed for the identification of the periodontal pathogen, Actinobacillus actinomycetemcomitans. Chromosomal DNA was extracted from A. actinomycetemcomitans, digested with a restriction enzyme, Sau3A, ligated to plasmid DNA (pUC18) and transformed into JM109 cells to give a partial A. actinomycetemcomitans library. The library was screened using Southern blot analysis. Out of the nine inserts tested, one was found to be species specific as it did not cross-hybridise to Haemophilus aphrophilus, a closely related organism which occurs in the normal oral microflora, nor did it cross-hybridise with 7 species of Bacteroides tested. A level of detection of 104 cells or 50ng of A. actinomycetemcomitans was obtained. The probe has a length of 779bp and out of 30 restriction enzymes tested, only SspI was found to have a restriction site in the insert. The probe was tested on clinical specimens obtained from five different periodontitis patient groups and was shown to correlate with culture results in eighteen out of twenty-two cases in detecting A. actinomycetemcomitans.

by Ka-chun Yiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1989. / Bibliography: leaves 162-192.

Graduation date: 1994

Giardia is the most common human parasite infection in the United States causing a lengthy diarrhea. Transmission of Giardia is by the fecal-oral route and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia in drinking water through the "Surface Water Treatment Rule." Current methods for detection of Giardia in water rely primarily on microscopic observation of water concentrates by immunofluorescent techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 base pairs long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of M13 vector with insert was isolated from lysed host E. coli XL1- Blue and used for production of the cDNA probe by nick translation with ³²P-labeled nucleotides. Seven different protocols were tested for extracting nucleic acids from the cysts. Using the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates by dot-blot hybridization assays. Environmental concentrates from secondary and tertiary treated sewage or surface waters were screened for Giardia cysts by immunofluorescent and the genespecific probe. Positive signals were observed in sewage and surface water samples without floatation at ten fold greater dilutions than after floatation. It appeared that gene probe detection was slightly more sensitive than microscopic detection of Giardia cysts for wastewater samples. In six surface water samples and two sewage sample no positive results were found either by the cDNA probe or immunofluorescent. Usually, DNA probes are radiolabeled and the most commonly used is ³²P. ³²P is expensive, hazardous and has an extremely short half-life of 14.3 days, necessitating frequent preparation of the nucleic acid probes. Three non-radioactive labeling methods, chemiluminescence, enzyme-linked immunoassay and enhanced chemiluminescence were evaluated. The cDNA probe was labeled by nick translation for chemiluminescence method. Biotinylated deoxyuridine triphosphate was used in place of deoxythymidine triphosphate to produce biotinylated DNA strands. The result of hybridization was visualized by chemiluminescenct detection of DNA. The sensitivity of the chemiluminescent method and the 32P labeled probe was 0.1 pg of DNA in a slot-blot hybridization assay.

Thesis (Ph. D. - Microbiology and Immunology)--University of Arizona, 1989. / Includes bibliographical references (leaves 165-170).