Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Sequence-specific PCR markers were derived from one new and eight previously identified random amplified polymorphic DNA (RAPD) markers for the purpose of identifying grape (Vitis) rootstocks. The markers were developed because the RAPD assay was found to be inconsistent and the original RAPD markers unreliable. Southern hybridization analysis of the RAPD gels with cloned RAPD bands as probes revealed deficiencies of scoring RAPD bands based solely on ethidium bromide staining. In one case, bands of the same size generated by the same primer in different rootstocks -- normally scored as the same marker -- failed to cross-hybridize, implying a lack of homology between the bands. In another case, a band scored as absent based on ethidium bromide staining was detected by hybridization. One of nine RAPD bands was cloned in the present study. All nine were partially sequenced and sequence-specific pairs of primers were synthesized for each for use under stringent PCR conditions. Three of the primer pairs generated products only from the rootstocks from which the original RAPD bands had been cloned; three others produced products from additional rootstocks while still generating polymorphisms; two others generated apparent length variants from some accessions; and one primer pair resulted in a loss of polymorphism. Based on the eight polymorphic markers, five of nine rootstocks could be unambiguously distinguished. / Graduation date: 1996

For ras gene mutation analysis in the rainbow trout (Oncorhynchus mykiss) model system, a partial trout ras sequence was identified using the polymerase chain reaction (PCR). Two synthetic oligonucleotides based on rat K-ras gene sequence were used as primers for the PCR procedure. A 90 base pair (bp) sequence, referred to as the trout K-ras, was amplified from trout genomic DNA and cDNA. Cloned 90 by PCR products from several normal liver tissues were sequenced resulting in the same sequence. Large-sized PCR products, 111 and 237 bp, were also cloned and sequenced indicating that these fragments included the 90 by sequence information expressed in mRNA. This 5'-terminal partial trout K-ras nucleotide sequence was 88% homologous to that of the goldfish ras gene, and less homologous to those of mammalian ras genes. Based on the partial sequence information of two trout ras genes, K-ras and H-ras, DNA from trout tumors induced by chemical carcinogens, aflatoxin B1 (AFB1) and N-methyl-N'-nitro-N-nitrosoguanidene (MNNG), were analyzed for the presence of point mutations. Using the PCR and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated liver tumor DNA indicated evidence for ras point mutations. Of the 10 mutant ras genotypes, seven were probed as G to T transversions at the second position of codon 12, two were G to T transversions at the second position of codon 13, and one was a G to A transition at the first position of codon 12. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive mutation evidence in each case, which seemed to occur in only a fraction of the neoplastic cells. However, no mutations were detected in exon 1 of the trout K-ras gene, nor in DNA from trout normal livers. Results indicated that the hepatocarcinogen AFB1 induced similar ras gene mutations in trout as in rat liver tumors. By comparison, the mutation specificity of MNNG in trout liver tumors was for G to A transitions, but no ras mutations were detected in trout kidney tumors. This investigation was the initial study of experimentally induced ras gene point mutations in a lower vertebrate fish model. / Graduation date: 1991

Chlorinated aliphatic hydrocarbons (CAHs) include tetrachloroethene (PCE), trichloroethene (TCE), and others. The industrial solvent TCE is among the most ubiquitous chlorinated compounds found in groundwater pollution. TCE in environment can be removed by physical, chemical and biological procedures. Dehalorespiration is a biological pathway from which bacteria can derive energy from the reductive dechlorination of chlorinated ethenes using hydrogen or organic acids as electron donors and yielding chloride and ethene as degradation products. Dehalorespiration can be used to remediate chlorinated ethene contaminated aquifers if an appropriate aquifer ecosystem exists including populations of dechlorinating bacteria and companion organisms that contribute to the biogeochemical environment conducive to dehalorespiration activity. Enhanced in-situ aerobic or anaerobic bioremediation of chlorinated solvents is a cost-effective, expanding technology for the clean-up of chlorinated solvent-contaminated sites. The objective of this pilot-scale study was to apply an enhanced in situ bioremediation technology to remediate TCE-contaminated groundwater. Both aerobic and anaerobic remedial systems were evaluated at a TCE-spill site located in southern Taiwan. In the aerobic bioremediation zone, the effectiveness of air, nutrient, and sugarcane molasses injection to enhance the aerobic cometabolism on TCE degradation was evaluated. Results show that the decreases in TCE concentration were observed over 204 days operating period. Up to 73¢H-99¢H of TCE removal efficiency was obtained in this treatment system. In the anaerobic test zone, the effectiveness of nutrient and sugarcane molasses injection to enhance the anaerobic dechlorination on TCE degradation was also evaluated. Results show that the decreases in TCE concentration were observed over a 193-day operating period. Up to 53¢H-91¢H of TCE removal efficiency was obtained in this treatment system. Polymerase chain reaction was applied to analyze the gene variation in TCE-microbial degraders during the treatment process. Results from this study indicate that the aerobic TCE-degraders (type¢¹methanotrophs and type ¢º methanotrophs) and the gene of degradation enzymes (toluene monooxygenase, toluene dioxygenase, particulate methane monooxygenase) were detected after the treatment process in the aerobic test zone. In the anaerobic treatment zone, Dehalococcoides (anaerobic TCE-degrader) and the gene of degradation enzyme (vcrA and tceA) were detected and a significant drop of TCE concentration was also observed. Based on 16S rDNA sequence analysis, samples of groundwater from aerobic/anaerobic bioremediation zone are close related to the genera of Dehalococcoides sp. MB, Dehalococcoides ethenogenes 195, Dehalococcoides sp. VS, Acidovorax sp., Alicycliphilus sp., Burkholderiales, Caulobacter sp., Caulobacter tuntrae, Caulobacter vibrioides, Comamonadaceae, Hydrogenophaga sp., Iron-reducing bacterium, Mitsuaria chitosanitabida, Rhodocyclacea, Pseudomonas sp., Rhodoferax ferrireducens, Acinetobater sp., actinomycete, Pseudomonas aeruginosa and Variovorax sp. Results reveal that both the aerobic cometabolism and anaerobic dechlorination are feasible and applicable technologies to clean up TCE contaminated aquifers. Thus, the in situ bioremediation technology has the potential to be developed into an environmentally, economically and naturally acceptable remediation technology.

Clostridium difficile is the most common infectious cause of nosocomial diarrhea, affecting thousands of patients annually and exacting enormous costs on the U.S. health care system. Early diagnosis is critical to prevent transmission and reduce morbidity and mortality, yet sensitive and specific diagnostic tests with a quick turnaround time are lacking. The objective of this study was to determine if a new commercially available real time polymerase chain reaction (PCR) test would prove more rapid, sensitive and specific than standard methods for the diagnosis of C. difficile infection (CDI). BD GeneOhm Cdiff assay, a real-time PCR assay for detection of C. difficile toxin B (tcdB) gene, was compared with Tox A/B II ELISA and a two-step algorithm which includes C. Diff Chek-60 Glutamate Dehydrogenase (GDH)-antigen assay followed by cytotoxin neutralization. Four-hundred liquid or semisolid stools submitted for diagnostic C. difficile testing were selected: 200 GDH antigen-positive and 200 GDH antigen-negative. All samples were tested by the C. Diff Chek-60 GDH antigen, cytotoxin neutralization, Toxin A/B II ELISA, and BD GeneOhm Cdiff assay. Discrepant specimens were tested by toxigenic culture as an independent gold standard. Chart review was performed on patients with discrepant specimens. As BD GeneOhm Cdiff assay was not FDA-cleared at the time of study, PCR results were not clinically reported. Of 200 GDH-positive samples, 71 were positive by Tox A/B II, 88 were positive by the two-step method, 93 were positive by PCR, and 96 were positive by GDH-antigen only. Of 200 GDH-negative samples, 3 were positive by PCR only. Toxigenic culture was performed on 41 samples with discrepant results and 39 were culture-positive. After culture resolution of discrepants, Tox A/B II detected 70 (66.7%), the two-step method detected 87 (82.9%), and PCR detected 96 (91.4%) of 105 true positives. The BD Gene-Ohm Cdiff assay was more sensitive in detecting toxigenic C. difficile than Tox A/B II (p <0.0001); however, the difference between PCR and the two-step method was not significant (p=0.1237). The BD GeneOhm Cdiff assay took a similar amount of time to perform as the Tox A/B II and was more rapid than the two-step method. Chart review revealed that 18 patients with cytotoxin-negative, PCR-positive discrepant samples were given 1-2 days of therapy (n=8), or no treatment at all (n=10). Yet symptoms resolved and no further C. difficile testing was requested for 13 of 18 patients for 6-8 months after hospital discharge. Only one patient had a subsequent cytotoxin positive stool submitted 22 days after the study sample was tested. Enhanced sensitivity and rapid turnaround time make the BD GeneOhm Cdiff assay an important advance in the diagnosis of toxigenic C. difficile infection. The BD GeneOhm Cdiff assay is significantly more sensitive than a commonly-used ELISA toxin assay and has a sensitivity and specificity comparable to the two-step method. Its turnaround time is similar to ELISA toxin assays and more rapid than the two-step method. Disadvantages to implementation of BD GeneOhm Cdiff assay include increased cost and potential treatment of asymptomatic carriers and mild, self-resolving disease.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "December 2000." Typescript. Vita. Includes bibliographical references (leaves 95-112). Also available on the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 120-144).

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 41-49).

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 183-207). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR39001

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 170-205).