Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 103-124).

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 136-144). Available also in a digital version from Dissertation Abstracts.

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 153-164).

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 169-186). Also available in print.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains vii, 124 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Prokaryotes are single-cell microorganisms. These creatures can be further classified to bacteria and archaea. Their DNA genetic meterials are spread around the cytoplasm rather than residing in the nucleus. Unlike eukaryotes, a high percentage of prokaryotic genome is composed of genes. The evolution of prokaryotes is different from that of the eukaryotes. Prokaryotes are found almost everywhere including the harshest environments on Earth. Understanding the whole pictures of their genomes will benefit us a lot in terms of new enzyme discovery, decoding drug resistance, biofuel development, etc. 　High-throughput sequencing technology is becoming increasingly popular in various sequencing projects. With different platforms, scientists are able to achieve millions ~ billions of sequences within days. In the last two years, there are a lot of prokaryotic genomes being sequenced under these platforms. However, there are only 1,542 complete chromosomes available in NCBI GenBank (September 2011) since the first complete genome of Bacillus subtilis was published in 1997. The most difficult step in finishing a complete genome is closing all gaps among different contigs. In this thesis, a series of comprehensive simulation studies based on 1,542 complete chromosomes have been performed in search of a cost-effective way to achieve complete prokaryotic genomes. Solutions to both draft and complete genome sequencing were provided by computer simulation. Moreover, classification studies have been performed to identify special prokaryotic phyla/orders (if any) dissatisfying our proposed strategies. 　Our results indicate that: 1) low coverage (6x-10x) pyrosequencing with long reads (400 bp) is sufficient to produce highly continuous and complete assemblies, presenting a tiny proportion of false gene duplication/loss. High quality draft genomes could be generated by this strategy; 2) Long repeats to some extent influence the assembly quality, especially for the genome coverage and contig number. The number of contigs and genome coverage rate are significantly correlated with the total size of repeat regions; 3) With a combination of one run of single-end reads (10x, 400bp read length) and one run of paired end reads (10x, 8kb library, 400bp read length), ~90% of chromosome assemblies are less than 10 scaffolds and ~95% of chromosome assemblies are less than 150 contigs. Most of the chromosomes can be assembled into high quality draft chromosomes (<50 contigs, ~4 scaffolds, >370kb contig N50 size, >99.99% single base accuracy and <0.5% false gene duplication/loss rate in average); 4) Similar patterns found in both simulated and real reads imply that our simulation analysis is not overestimated; 5) Greater attention is needed regarding the orders Thiotrichales, Enterobacteriales and Nostocales, when applying the above strategies for complete genome sequencing; 6) For prokaryotic species with multiple chromosomes, Pulse Field Gel Electrophoresis is needed to separate all their chromosomes which will be individually collected by electroelution prior to draft/complete genome sequencing. A comprehensive computer simulation study based on 1,542 chromosomes (all availabe prokaryotic complete chromosomes, September 2011) has been performed in this thesis. The sequencing strategies for both prokaryotic draft and complete genome proposed by the simulation study could facilitate the ongoing prokaryotic complete genome sequencing projects. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Tuberculosis (TB), a severe disease caused by Mycobacteria tuberculosis (MTb), remains a globally severe health problem. In modern days, emergence of drug resistant TB is a new threat to public health since it can lead to treatment failure, increased transmission of TB to other hosts and further development of drug resistant complications. The traditional diagnostic method for TB by Acid Fast Bacilli (AFB) smear and Löwenstein–Jensen medium (LJ) culture are poor in sensitivity and time consuming respectively. There is a need for rapid diagnosis and identification of MTb. Nucleic acid amplification like PCR and real-time PCR are options for rapid diagnosis. However, such techniques require sophisticated technique and complex equipment. The high cost would constitute a barrier for countries with a high demand but only limited resources. Loop-mediated isothermal amplification (LAMP) assay is a novel technique, proven by many studies as to its high sensitivity and highly specificity to MTb. Most importantly, LAMP assay is economical and affordable by developing countries. The first objective of this study was to evaluate the analytical sensitivity and specificity of LAMP assay. The specificity of LAMP assay was determined by performing LAMP assay on 19 clinical isolates, which had already been identified previously. The clinical isolates included 14 mycobacteria tuberculosis complex (MTb), and five mycobacteria other than tuberculosis (MOTT) strains that were positive for AFB smear and LJ culture but negative for IS6110 single-tube nested real time PCR. The specificity was 100%. The analytical sensitivity as well as the limit of detection (LOD) were determined by testing on a duplicate set of serial DNA dilution, where each duplicate consisted of dilution of 100,000, 10,000, 1000, 300, 100, 10 and 1 colony forming unit/milliliter (CFU/ml). The LOD of LAMP assay was about 3 CFU per reaction. The second objective of this study evaluated the diagnostic performance of LAMP assay against AFB culture and IS6110 single tube nested real time PCR for identification of MTb in 200 respiratory specimens from 123 patients. All the specimens have already been tested for IS6110 single tube nested real time PCR, and culture results and AFB smears results have been obtained for all the specimens. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of three diagnostic methods (AFB smear microscopy, LAMP assay amplification and IS6110 single-tube nested real time PCR) were calculated with 95% confidence interval using LJ culture as gold standard. The LAMP assay had a sensitivity of 80%, specificity of 92.6%, PPV of 60% and NPV of 97.1%. However, MTb might fail to grow on the LJ culture medium for various reasons, for instance, MTb might already be dead after antibiotic treatment of the patient, or there might be poor laboratory practices during the processing of the specimens. Since the LJ culture method could produce false negatives in the situations described above, an alternative to the LJ culture method, ‘Hybrid Method’ was used as the gold standard. Under this method, a specimen was regarded as positive if the LJ culture result was positive. On the other hand, if a specimen generated a negative result using the LJ culture method, the results from the LAMP assay and IS6110 nested real time PCR would be considered, i.e., if both the LAMP assay and IS6110 nested real time PCR gave positive results while the results of LJ culture were negative, the specimen was referred to be positive in this case. In other words, a specimen would be regarded as negative if and only if the LJ culture result was negative and at least one of the LAMP assay or IS6110 was negative at the same time. Along the same line, the sensitivity, specificity, PPV and NPV of the three diagnostic methods (AFB smear microscopy, LAMP assay amplification and IS6110 single-tube nested real time PCR) were calculated with 95% confidence interval against the Hybrid Method. After resolution, the LAMP assay had a sensitivity of 87.0%, specificity of 100%, PPV of 100% and NPV of 97.1%. Our results showed that the LAMP assay has a great potential to be a new TB diagnostic test, especially in developing countries, with its lot of advantages like ease of use, cheap and fast. The LAMP assay in the study showed a high specificity, however, the sensitivity has to be improved before application in clinical use. For comparison of clinical performance, IS6110 single tube nested real time PCR had a higher sensitivity than that of LAMP assay (100% vs 80% using culture as gold standard; 100% vs 87% using ‘Hybrid Method’ as gold standard). However, LAMP assay had a higher specificity than that of IS6110 single tube nested real time PCR (92.6% vs 90.7% using culture as gold standard; 100% vs 98% using ‘Hybrid Method’ as gold standard). LAMP had been proven to be a potential and powerful tool in clinical diagnosis of MTb. Further improvement on its sensitivity is required to enable its extensive use in the clinic in the future. / published_or_final_version / Medical Sciences / Master / Master of Medical Sciences