Previous studies revealed that α-275-Cys provides an essential ligand to one of the Fe atoms on the FeMo-cofactor, and its substitutions resulted in inactive nitrogenase. In order to study the structural-functional relationship of the protein environment in this region with respect to the FeMo-cofactor, subtle changes were introduced through substitutions using a site-directed mutagenesis and gene-replacement method at α-274-His, α-276-Tyr and α-277-Arg in Azotobacter vinelandii nitrogenase. Characterization of mutants strains resulting from amino acid substitutions at residues, α-274-His, α-276-Tyr or α-277-Arg, using activity assays, resulted in mixed Nif phenotypes. Therefore, none of these residues is absolutely required for nitrogenase activity. However, the changed EPR spectra of the altered MoFe proteins from some strains with substitutions at either α-276-Tyr or α-277-Arg indicated that the FeMo-cofactor environment had been perturbed by these substitutions. Together with its changed EPR spectrum, substituting α-277-Arg with His showed some extraordinary catalytic features, such as its inability to reduce N₂ while retaining respectable C₂H₂- and H⁺-reduction activities. It was also found that this altered protein used a higher percentage of total electron flux for H₂ evolution under an C₂H₂/Ar atmosphere than did wild type. Further characterization of the purified α-277^his MoFe protein in parallel with its wild type counterpart revealed that the alteration in the α-277^his MoFe protein caused a lower affinity for C₂H₂ binding, whereas it did not affect the CO binding. Interestingly, CO-induced cooperativity during C₂H₂ reduction was observed in this altered MoFe protein clearly indicating two sites for C₂H₄ evolution, one of which might be in the vicinity of this residue. Furthermore, the α-277^his MoFe protein does not bind or reduce N₂ leading to the proposal of a nonexistent E₄ redox state in the MoFe protein catalytic cycle which was supported by stopped-flow spectrophotometric evidence. This altered α-277^his MoFe protein showed comparable physical stabilities to that of the wild-type protein, and its ATP hydrolysis rates remained constant under a number of substrates assayed. Therefore, the substitution has not affected the overall protein structure, rather, it has changed the local FeMo-cofactor environment. When we studied the purified α-276^his and α-274^gin/α-276^his MoFe proteins and compared the results with the data from the α-277^his and wild-type MoFe protein, we found that there is no direct correlation between the additional set of EPR signals observed in these altered MoFe proteins and their catalytic activities. The current understanding concerning the functionality of these residues is that they are involved in maintaining a proper environment for FeMoco to bind and in stabilizing the different redox states of the enzyme during catalysis. / Ph. D.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Prostate cancer is the most common form of non-skin cancer and the second leading cause of cancer deaths within the United States. The five year survival rate has increased from 69% to 99% over the last 25 years for the local and regional disease, but has remained fairly low (approximately 34%) for the advanced disease. Therefore, current research is aimed at finding complementary or alternative treatments that will specifically target components of the signal transduction, cell-cycle and apoptosis pathways to induce cell death, with little or no toxic side effects to the patient. In this study we investigated the effect of genistein on expression levels of genes involved in these pathways. Genistein is a (4 , 5 , 7-trihydroxyisoflavone) is a major isoflavone constituent of soy that has been shown to inhibit growth proliferation and induce apoptosis in cancer cells. The mechanism of genistein-induced cell death and potential molecular targets for genistein in LNCaP prostate cancer c ells was investigated using several techniques. The chemosensitivity of genistein towards the prostate cancer cells was investigated using the ATP and MTS assays and apoptosis induction was determined using apoptosis and caspase assays. Several molecular targets were also identified using cDNA microarray and RT-PCR analysis. Our results revealed that genistein induces cell death in a time and dose-dependent manner and regulates expression levels of several genes involved in carcinogenesis and immunogenicity. Several cell cycle genes were down-regulated, including the mitotic kinesins, cyclins and cyclin dependent kinases, indicating that genistein is able to halt cell cycle progression through the regulation of genes involved in this process. / Several members of the Bcl-2 family which are involved in apoptosis were also affected and a number of genes involved in immunogenicity were up-regulated including the DefB1 and HLA membrane receptors. The results of this study provide evidence of genistein's ability to inhibit growth proliferation and induce apoptosis and indicates its potential as an adjuvant in chemotherapy and immunotherapy. / by Kendra Merchant. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Mak, Miu. / "April 2008." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 207-230). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Thesis (MSc (Hons))--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2006. / Bibliography: leaves 153-158. / Wheat is a major crop in Australia with around 25 million tonnes of grain harvested in an average year. Improved wheat grain cultivars and wheat grain milling can result in higher biological yields and flour quality. The introduction covers the general aspects of the wheat grain from bran development and structure through to millings and the importance of flour quality in flour-based products. It also highlights the problem with bran contamination in flour during milling and other factors that may have an effect on flour quality. Proteomics was used to identify proteins in three separate bran tissue fractions: the inner fraction (aleurone), intermediate fraction (nucellar tissue, testa, tube cells and cross cells) and the outer faction (hypodermis and epidermis). The aim of the project was to identify proteins in bran tissue fractions which may potentially be useful in improvements in wheat quality for farmers and consumers and flour yield for millers. The results show that more than 80% of the identified proteins in the outer and intermediate tissue factions are defence-and stress-related proteins (chitinase, xylanase, thaumatin-like protein, wheatwin 1, lipid-transfer protein, oxalatae oxidase (OXO), polyphenol oxidase (PPO), peroxidase (POX)). Almost 60% of the proteins identified in the inner tissue fraction are 7S Globulin storage proteins and around 15% are protein synthesis-and energy-related. Water-soluble proteins were also identified and it was found that endochitinase, OXO, PPO and POX all leach out from the grain durings imbibition. This study has added to the knowledge of bran tissue-specific proteins and has broad implications for improving crop yield and flour quality. / Mode of access: World Wide Web. / xiii, 158 leaves ill

[Truncated abstract] Introduction: Breast cancer is a heterogeneous disease, resulting in very different outcomes for women with apparently similar tumour characteristics. In order for patients to have optimal treatment, a better understanding of the molecular nature of their disease is required. Aims: The aims of this thesis were: 1) To determine whether methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes are associated with phenotypic features of breast cancer and the prognostic significance of methylation of these genes. 2) To investigate for possible associations between the frequency of methylation at RARβ2, CDH1, ER, BRCA1, CCND2, p16 and TWIST genes and the presence of germ-line variants in the TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes, as well as for possible correlations between these polymorphisms and clincopathological features of breast cancer including patient outcome. 3) To determine whether PIK3CA mutations determined clinical phenotype and the prognostic significance of PIK3CA mutations in a large and well characterized cohort of breast cancer patients. Methods: A large and well characterized series of primary breast tumours were selected for methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes using MSP, and for polymorphisms in TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes using PCR, PCR-RFLP and PCR-SSCP. Mutations to PIK3CA were detected using F-SSCP. Results and Conclusions: Methylation frequencies ranged from 11% for CCND2 to 84% for ER. More frequent hypermethylation was observed in tumours with poor histological differentiation compared to those with well/moderate differentiation, as well as trends for association with larger tumour size and mutant TP53. Tumours with ER and CDH1 methylation were associated with significantly lower hormone receptor levels, younger age at diagnosis and the presence of mutant p53. TWIST methylation is firstly reported to be associated with significantly older patient age at diagnosis and larger tumour size. Our data suggests that gene methylation may be linked to various pathological features of breast cancer. However, there appears to be little support for a distinctive CpG island methylator phenotype in breast cancer.

Low copy number anonymous nuclear loci were used to search for species markers in four species of Klamath Basin suckers. We sequenced 28 randomly chosen loci representing 10,421 bp; 21 loci were similar to sequences in GenBank. Eight fixed sequence differences were found among Klamath species. Locus 120 contained rare but diagnostic markers for Deltistes luxatus and for Catostomus rimiculus. Locus 4 also contained three rare but unique sites in Catostomus rimiculus. No sequence differences were found between Chasmistes brevirostris and Catostomus snyderi. Loci 4 and 120 exhibited allele frequency differences between Rogue River C. rimiculus and all Klamath Basin suckers. Genotype BB of locus 4 was a fixed diagnostic marker and genotype BB of locus 120 was a frequency dependent marker for Rogue C. rimiculus. Although Klamath suckers represent three genera, very limited variation was found among 10,431 base pairs. We examined phylogenetic patterns of five loci in eleven catostomid genera and 25 species to determine if the homogeneity in the Upper Klamath Basin was due to massive hybridization and introgression or to retention of ancestral sequences. Two loci with no similarity to GenBank sequences (non-coding loci) and three loci with substantial similarity to GenBank sequences (coding loci) gave similar results, providing support for various subfamilies and tribes, more support for eastern genera and little support for western genera. Each locus was a mosaic of species or population markers, sometimes providing discriminatory power for allopatric populations of a species, such as C. macrocheilus, while not discriminating other species. Upper Klamath Basin species were noteworthy in their lack of autapomorphies, but had similar numbers of derived informative sites as other catostomins. Upper Klamath Basin species consistently shared ancestral or equivocal informative sites either with moxostomatins or a variable group of western species and shared derived sites with other western species, especially C. occidentalis. The data suggest that Upper Klamath Basin species have retained a largely ancestral genome at these loci. Thus, the failure of this technique to uncover significant variation in Upper Klamath Basin species may be a reflection of their plesiomorphic genome at these loci and not necessarily hybridization. / Graduation date: 2004

Yip Pui Ying. / Thesis submitted in: November 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-184). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgment --- p.v / Abbreviations --- p.vii / Table of contents --- p.viii / List of Figures --- p.xii / List of Tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- The importance of characterization of Chinese medicinal materials and the development of Chinese medicine in Hong Kong --- p.1 / Chapter 1.2. --- Methods for characterization of Chinese medicinal materials --- p.5 / Chapter 1.3. --- Molecular characterization of Chinese medicinal materials --- p.8 / Chapter 1.3.1. --- DNA sequencing --- p.9 / Chapter 1.3.2. --- DNA fingerprinting --- p.14 / Chapter 1.3.3. --- Nucleic acid hybridization --- p.19 / Chapter 1.4. --- Objectives --- p.20 / Chapter Chapter 2 --- Characterization of Plant and Fungal Materials by rDNA ITS Sequence Analysis --- p.22 / Chapter 2.1. --- Introduction --- p.22 / Chapter 2.2. --- Materials and Methods --- p.22 / Chapter 2.2.1. --- Chinese medicinal materials used in this study --- p.22 / Chapter 2.2.1.1. --- Plants and fungi for interspecific ITS study --- p.22 / Chapter 2.2.1.2. --- Plant for intraspecific ITS study and locality study --- p.33 / Chapter 2.2.2. --- Extraction of total DNA --- p.35 / Chapter 2.2.3. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.35 / Chapter 2.2.4. --- Purification of PCR products --- p.38 / Chapter 2.2.5. --- Cloning using pCR-Script´ёØ Amp SK(+) Cloning Kit --- p.38 / Chapter 2.2.5.1. --- Polishing --- p.38 / Chapter 2.2.5.2. --- Ligation of inserts into pCR-Script´ёØ Amp SK(+) cloning vector --- p.38 / Chapter 2.2.5.3. --- Transformation --- p.40 / Chapter 2.2.5.4. --- PCR screening of white colonies --- p.40 / Chapter 2.2.5.5. --- Purification of PCR screening products --- p.41 / Chapter 2.2.6. --- Sequencing of ITS regions --- p.41 / Chapter 2.2.6.1. --- Cycle sequencing reaction --- p.41 / Chapter 2.2.6.2. --- Purification of sequencing extension products --- p.41 / Chapter 2.2.6.3. --- Electrophoresis by genetic analyzer --- p.42 / Chapter 2.2.7. --- Sequence analysis and alignment --- p.42 / Chapter 2.3. --- Results --- p.42 / Chapter 2.3.1. --- Extraction of total DNA --- p.42 / Chapter 2.3.2. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.44 / Chapter 2.3.2.1. --- Interspecific ITS study --- p.44 / Chapter 2.3.2.2. --- Intraspecific ITS study --- p.46 / Chapter 2.3.3. --- Sequence analysis and alignment --- p.47 / Chapter 2.3.3.1. --- Interspecific ITS study --- p.47 / Chapter 2.3.3.2. --- Intraspecific ITS study --- p.56 / Chapter 2.4. --- Discussions --- p.60 / Chapter 2.4.1. --- rDNA regions used for studying Chinese medicinal materials --- p.60 / Chapter 2.4.2. --- The results agreed with previously published works --- p.60 / Chapter 2.4.3. --- Explanation of interspecific results within the Ganoderma genus --- p.60 / Chapter 2.4.4. --- Implications from interspecific comparisons --- p.60 / Chapter 2.4.5. --- Implications from intraspecific comparisons --- p.61 / Chapter Chapter 3 --- .Characterization of Astragalus membranaceus by DNA Fingerprinting / Chapter 3.1 --- Introduction --- p.62 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Extraction of total DNA --- p.62 / Chapter 3.2.2 --- Generation and detection of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.2.3 --- Analysis of DNA fingerprints --- p.63 / Chapter 3.3 --- Results --- p.63 / Chapter 3.3.1 --- Generation of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.3.2 --- Fingerprint analysis --- p.69 / Chapter 3.4 --- Discussion --- p.85 / Chapter 3.4.1 --- RP-PCR has been used on Chinese medicinal materials --- p.85 / Chapter 3.4.2 --- AP-PCR used instead of RAPD --- p.85 / Chapter 3.4.3 --- Reproducibility and amount of bands --- p.86 / Chapter 3.4.4 --- Alternatives of electrophoresis process --- p.88 / Chapter 3.4.5 --- Explanation of results --- p.88 / Chapter 3.4.6 --- Distinguishing Neimengu and Shanxi samples --- p.89 / Chapter 3.4.7 --- Further studies --- p.90 / Chapter Chapter 4 --- Characterization of Plant and Fungal Materials by DNA-DNA Hybridization on Microarrays --- p.91 / Chapter 4.1 --- Introduction --- p.91 / Chapter 4.2 --- Materials and Methods --- p.92 / Chapter 4.2.1 --- Samples for microarray study --- p.92 / Chapter 4.2.2 --- Extraction of total DNA --- p.95 / Chapter 4.2.3 --- Amplification and sequencing of ITS 1 region of rRNA gene --- p.95 / Chapter 4.2.4 --- Preparation of labeled probe --- p.95 / Chapter 4.2.5 --- Amplification of ITS1 fragments --- p.97 / Chapter 4.2.6 --- Preparation of slides --- p.103 / Chapter 4.2.7 --- Hybridization and washing --- p.104 / Chapter 4.2.8 --- Scanning and data analysis --- p.105 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- DNA extraction --- p.105 / Chapter 4.3.2 --- Amplification and sequencing of ITS1 region of rRNA gene --- p.107 / Chapter 4.3.3 --- Preparation of labeled probe and amplification of ITS1 fragments… --- p.112 / Chapter 4.3.4 --- Preparation of slides --- p.112 / Chapter 4.3.5 --- Scanning and data analysis --- p.116 / Chapter 4.4 --- Discussion --- p.134 / Chapter 4.4.1 --- Implications --- p.134 / Chapter 4.4.2 --- Applying the findings --- p.134 / Chapter 4.4.3 --- Ways to maximize specificity --- p.137 / Chapter 4.4.4 --- Optimisation --- p.138 / Chapter 4.4.5 --- Microarray may be more advantageous over sequencing --- p.138 / Chapter Chapter Five --- General Discussion and Summary --- p.140 / Chapter 5.1. --- Objectives of this study --- p.140 / Chapter 5.2. --- rDNA ITS sequencing --- p.140 / Chapter 5.2.1. --- Description of the approach and summary of the results --- p.140 / Chapter 5.2.2. --- Implications from the results --- p.140 / Chapter 5.2.3. --- Advantages and limitations of DNA sequencing --- p.141 / Chapter 5.3. --- AP-PCR fingerprinting --- p.141 / Chapter 5.3.1. --- Description of the approach and summary of the results --- p.141 / Chapter 5.3.2. --- Advantages and limitations of DNA fingerprinting --- p.142 / Chapter 5.4. --- DNA-DNA hybridization on microarrays --- p.143 / Chapter 5.4.1. --- Description of the approach and summary of the results --- p.143 / Chapter 5.4.2. --- Implications from the results --- p.143 / Chapter 5.4.3. --- Advantages and limitations of DNA hybridization on microarrays. --- p.144 / Chapter 5.5. --- Overall summary --- p.144 / Chapter 5.6. --- Future studies --- p.146 / References --- p.147 / Appendix --- p.185

Cheung, Chun Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 111-128). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iv / 摘要 --- p.vii / Table of content --- p.ix / List of Figures --- p.xvii / List of Tables --- p.xix / Abbreviations --- p.xxi / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Complementary and Alternative Medicine (CAM) and Traditional Chinese Medicine (TCM) --- p.1 / Chapter 1.2 --- The development of Traditional Chinese Medicine --- p.2 / Chapter 1.3 --- Quality control of Traditional Chinese Medicine --- p.3 / Chapter 1.4 --- Problems of adulteration --- p.5 / Chapter 1.4.1 --- Confusion by common names --- p.5 / Chapter 1.4.2 --- Erroneous and intentional adulteration --- p.6 / Chapter 1.5 --- Authentication of Traditional Chinese Medicine using DNA techniques --- p.7 / Chapter 1.6 --- Crocodile meat --- p.10 / Chapter 1.6.1 --- Crocodile meat as Traditional Chinese Medicine --- p.10 / Chapter 1.6.2 --- Crocodile meat as exotic meat --- p.10 / Chapter 1.6.3 --- Effects of crocodile meat on mice --- p.12 / Chapter 1.6.4 --- Adulteration of crocodile meat in Hong Kong --- p.13 / Chapter 1.6.5 --- Authentication of crocodile meat --- p.14 / Chapter 1.6.5.1 --- SCAR analysis --- p.14 / Chapter 1.6.5.2 --- 12S and 16S ribosomal DNA --- p.14 / Chapter 1.7 --- Fish air-bladder --- p.15 / Chapter 1.7.1 --- Fish air-bladder as Traditional Chinese Medicine --- p.15 / Chapter 1.7.2 --- A case study --- p.16 / Chapter 1.7.3 --- Authentication of fish air-bladder --- p.17 / Chapter 1.8 --- Radix Stellariae --- p.18 / Chapter 1.8.1 --- Stellaria dichotoma L. var. lanceolata Bge --- p.18 / Chapter 1.8.2 --- Radix Stellariae as Traditional Chinese Medicine --- p.19 / Chapter 1.8.3 --- Chemicals in Radix Stellariae and their uses --- p.19 / Chapter 1.8.4 --- Adulteration of Radix Stellariae --- p.20 / Chapter 1.8.5 --- Authentication of Radix Stellariae --- p.21 / Chapter 1.8.5.1 --- Internal Transcribed Spacers (ITS) --- p.21 / Chapter 1.8.5.2 --- trnH-psbA intergenic spacer --- p.23 / Chapter 1.9 --- Objectives --- p.25 / Chapter Chapter 2. --- Materials and Methods --- p.26 / Chapter 2.1 --- Samples used in the study --- p.26 / Chapter 2.1.1 --- Crocodile and monitor lizard samples --- p.26 / Chapter 2.1.2 --- Sequence from NCBI database --- p.26 / Chapter 2.1.3 --- Fish air-bladder samples --- p.30 / Chapter 2.1.4 --- Radix Stellariae samples and samples of related species --- p.33 / Chapter 2.1.5 --- Sequences from NCBI database --- p.33 / Chapter 2.2 --- Reagents and equipments --- p.36 / Chapter 2.2.1 --- Sample preparation and DNA extraction --- p.36 / Chapter 2.2.2 --- Polymerase Chain Reaction --- p.38 / Chapter 2.2.3 --- Agarose gel electrophoresis and Gene Clean --- p.39 / Chapter 2.2.4 --- Cloning --- p.40 / Chapter 2.2.5 --- Cycle sequencing --- p.41 / Chapter 2.3 --- Experimental procedures --- p.42 / Chapter 2.3.1 --- Sample preparation --- p.42 / Chapter 2.3.2 --- DNA extraction --- p.42 / Chapter 2.3.3 --- Polymerase Chain Reaction --- p.44 / Chapter 2.3.4 --- Agarose gel electrophoresis --- p.47 / Chapter 2.3.5 --- Gene Clean --- p.47 / Chapter 2.3.6 --- Cloning --- p.48 / Chapter 2.3.7 --- Cycle sequencing and sequence analyses --- p.51 / Chapter Chapter 3. --- Crocodile meat - Results and Discussion --- p.54 / Chapter 3.1 --- Results --- p.54 / Chapter 3.1.1 --- SCAR analysis --- p.54 / Chapter 3.1.2 --- Sequence analyses --- p.55 / Chapter 3.1.3 --- The dendrograms --- p.56 / Chapter 3.2 --- Discussion --- p.60 / Chapter 3.2.1 --- SCAR as a quick and inexpensive method for the authentication of crocodile meat --- p.60 / Chapter 3.2.2 --- DNA sequencing - A useful tool to identify the source species of the crocodile meat --- p.61 / Chapter 3.2.3 --- Adulteration of crocodile meat in Hong Kong --- p.63 / Chapter 3.2.4 --- Source species of the genuine crocodile meats and the adulterants --- p.63 / Chapter 3.2.5 --- Regulation of labeling of food in Hong Kong --- p.69 / Chapter 3.2.6 --- Source species of the lizard head and tail from AFCD --- p.69 / Chapter 3.3 --- Summary --- p.70 / Chapter Chapter 4. --- Fish air-bladders - Results and Discussion --- p.72 / Chapter 4.1 --- Results --- p.72 / Chapter 4.1.1 --- Identities of sample BH and F1 --- p.73 / Chapter 4.1.2 --- Identity of sample BS --- p.74 / Chapter 4.1.3 --- Identities of samples GD and ZG --- p.74 / Chapter 4.1.4 --- Identity of sample GG --- p.74 / Chapter 4.1.5 --- "Identities of samples HB, HT and SH" --- p.75 / Chapter 4.1.6 --- Identity of sample JL --- p.75 / Chapter 4.1.7 --- Identity of sample MS --- p.76 / Chapter 4.1.8 --- Identity of sample RE --- p.76 / Chapter 4.2 --- Discussion --- p.77 / Chapter 4.2.1 --- Sample RE was confirmed to have originated from rabbit ears --- p.77 / Chapter 4.2.2 --- Identities of the dry fish air-bladders sold in Hong Kong --- p.79 / Chapter 4.2.3 --- Identities of the fresh fish air-bladders sold in Hong Kong --- p.82 / Chapter 4.2.4 --- Limitations of the use of DNA sequences for source species identification --- p.83 / Chapter 4.2.5 --- Variation in prices of fish air-bladders --- p.87 / Chapter 4.3 --- Summary --- p.88 / Chapter Chapter 5. --- Radix Stellariae - Results and Discussion --- p.89 / Chapter 5.1 --- Results --- p.89 / Chapter 5.1.1 --- Sequence analyses --- p.90 / Chapter 5.1.2 --- The dendrograms --- p.90 / Chapter 5.2 --- Discussion --- p.97 / Chapter 5.2.1 --- Identities of the samples obtained from the market --- p.97 / Chapter 5.2.2 --- Identity of sample Sdl4 --- p.97 / Chapter 5.2.3 --- Identities of samples Sd02R and Sd04 --- p.100 / Chapter 5.2.4 --- Myosoton aquaticum in the Stellaria-Myosoton clade --- p.104 / Chapter 5.2.5 --- Medicinal uses of the substitutes of Radix Stellariae --- p.105 / Chapter 5.3 --- Summary --- p.106 / Chapter Chapter 6. --- Conclusion --- p.107 / Reference --- p.111 / Appendix 1. 12S rDNA sequences of crocodilian and Varanus species obtained from NCBI database for sequence analyses --- p.129 / Appendix 2. 16S rDNA sequences of crocodilian and Varanus species obtained from NCBI database for sequence analyses --- p.130 / "Appendix 3. ITS sequences of the species in the genera Arenaria, Myosoton, Silene, and Stellaria obtained from NCBI database for sequence analyses" --- p.131 / Appendix 4. 7rnH-psbA intergenic spacer sequences of Silene species obtained from NCBI database for sequence analyses --- p.132 / Appendix 5. Sequence alignment of 12S rRNA gene sequences of crocodile and monitor lizard samples --- p.133 / Appendix 6. Sequence alignment of 16S rRNA gene sequences of crocodile and lizard samples --- p.141 / Appendix 7. Sequence alignment of coxl sequences of fish air-bladder samples --- p.149 / Appendix 8. Sequence alignment of 12S rRNA gene sequences of fish air-bladder samples --- p.151 / Appendix 9. Sequence alignment of 16S rRNA gene sequences of fish air-bladder samples --- p.153 / Appendix 10. Sequence alignment of coxl region of Vibrio parahaemolyticus and the coxl primers --- p.155 / Appendix 11. Sequence alignment of ITS sequences of Radix Stellariae and related samples --- p.156 / Appendix 12. Sequence alignment of trnH-psbA of Radix Stellariae and related samples --- p.163 / Appendix 13. Search results of coxl sequences of the fish air-bladder samples in BOLD-IDS --- p.167 / Appendix 14. Search results of coxl sequences of the fish air-bladder samples in NCBI nucleotide BLAST --- p.168 / Appendix 15. Search results of 12S rDNA sequences of the fish air-bladder samples in NCBI nucleotide BLAST --- p.169 / Appendix 16. Search results of 16S rDNA sequences of the fish air-bladder samples in NCBI nucleotide BLAST --- p.170 / Appendix 17. Sequence similarities (%) of coxl sequences of the fish air-bladder samples --- p.171 / Appendix 18. Sequence similarities (%) of 12S rDNA sequences of the fish air-bladder samples --- p.172 / Appendix 19. Sequence similarities (%) of 16S rDNA sequences of the fish air-bladder samples --- p.173

Li Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 146-161). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.III / TABLE OF CONTENTS --- p.VII / LIST OF FIGURES AND TABLES --- p.XIII / LIST OF ABBREVIATIONS --- p.XX / Chapter CHAPTER ONE --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Authentication of Chinese medicines --- p.1 / Chapter 1.1.1 --- The need for authentication of Chinese medicines --- p.1 / Chapter 1.1.2 --- Traditional methods for authentication --- p.2 / Chapter 1.1.3 --- Molecular methods for authentication --- p.4 / Chapter 1.1.3.1 --- DNA fingerprinting --- p.5 / Chapter 1.1.3.2 --- DNA sequencing --- p.6 / Chapter 1.1.3.2.1 --- Choosing a suitable region for DNA sequencing --- p.7 / Chapter 1.1.3.2.2 --- Chloroplast irnL-trnF region --- p.9 / Chapter 1.1.3.2.3 --- Complete sequence of ITS rDNA region --- p.10 / Chapter 1.1.3.2.4 --- 5S rDNA intergenic spacer --- p.11 / Chapter 1.1.3.2.5 --- Calculation of similarities among sequences --- p.12 / Chapter 1.1.3.2.6 --- Construction methods of phylograms --- p.12 / Chapter 1.2 --- The need for molecular authentication of three medicinal herbs --- p.14 / Chapter 1.2.1 --- The herb Baiying --- p.14 / Chapter 1.2.1.1 --- The poisoning case reported in Hong Kong --- p.14 / Chapter 1.2.1.2 --- The identity of genuine Baiying --- p.15 / Chapter 1.2.1.3 --- Morphological characters of the herb Baiying --- p.15 / Chapter 1.2.1.4 --- Medicinal values of Baiying --- p.17 / Chapter 1.2.1.5 --- Xungufeng as the adulterant of Baiying --- p.17 / Chapter 1.2.1.5.1 --- The toxic chemicals aristolochic acids --- p.18 / Chapter 1.2.1.6 --- The need for molecular authentication of Baiying --- p.19 / Chapter 1.2.2 --- The herb Baihuasheshecao --- p.19 / Chapter 1.2.2.1 --- The identity of Baihuasheshecao --- p.19 / Chapter 1.2.2.2 --- Morphological characters of the herb Baihuasheshecao --- p.20 / Chapter 1.2.2.3 --- Medicinal uses --- p.23 / Chapter 1.2.2.4 --- Chemical profile --- p.24 / Chapter 1.2.2.5 --- Adulterants of Baihuasheshecao --- p.24 / Chapter 1.2.2.6 --- Chemical studies of H. diffusa and H. corymbosa --- p.25 / Chapter 1.2.2.7 --- Existing methods for authentication --- p.26 / Chapter 1.2.2.8 --- The need for molecular authentication of Baihuasheshecao --- p.28 / Chapter 1.2.3 --- The herb Chuanlianzi --- p.28 / Chapter 1.2.3.1 --- The identity of Chuanlianzi --- p.28 / Chapter 1.2.3.2 --- Medicinal values --- p.29 / Chapter 1.2.3.3. --- The bioactive chemical --- p.31 / Chapter 1.2.3.4 --- Kulianzi as the substitute of Chuanlianzi --- p.31 / Chapter 1.2.3.5 --- Poisoning cases reported due to ingestion of Kulianzi --- p.32 / Chapter 1.2.3.6 --- Comparative studies of Chuanlianzi and Kulianzi --- p.32 / Chapter 1.2.3.7 --- The need for molecular authentication of Chuanlianzi --- p.33 / Chapter CHAPTER TWO --- OBJECTIVE --- p.35 / Chapter CHAPTER THREE --- MATERIALS AND METHODS --- p.36 / Chapter 3.1 --- Plant and herb samples --- p.36 / Chapter 3.2 --- Total DNA extraction --- p.48 / Chapter 3.2.1 --- Cetyltriethylammonium bromide extraction --- p.48 / Chapter 3.2.2 --- Commercial kit extraction --- p.49 / Chapter 3.3 --- DNA amplification --- p.50 / Chapter 3.4 --- DNA fingerprinting --- p.51 / Chapter 3.4.1 --- DNA concentration determination --- p.51 / Chapter 3.4.2 --- ISSR fingerprinting --- p.52 / Chapter 3.5 --- Agarose gel electrophoresis --- p.53 / Chapter 3.6 --- Purification of PCR product --- p.53 / Chapter 3.7 --- Cloning of PCR product --- p.54 / Chapter 3.7.1 --- Ligation --- p.54 / Chapter 3.7.2 --- Transformation --- p.55 / Chapter 3.7.3 --- Cell cultivation --- p.55 / Chapter 3.7.4 --- Plasmid extraction --- p.55 / Chapter 3.7.5 --- Insert confirmation --- p.56 / Chapter 3.8 --- Determination of DNA concentration --- p.56 / Chapter 3.9 --- DNA sequencing --- p.57 / Chapter 3.9.1 --- Cycle sequencing --- p.57 / Chapter 3.9.2 --- Purification of cycle sequencing products --- p.57 / Chapter 3.9.3 --- DNA analysis --- p.58 / Chapter 3.10 --- Sequence analysis --- p.58 / Chapter CHAPTER FOUR --- MOLECULAR AUTHENTICATION OF BAIYING --- p.59 / Chapter 4.1 --- Results --- p.59 / Chapter 4.1.1 --- Sequence alignment --- p.59 / Chapter 4.1.2 --- Percentage similarity analysis --- p.68 / Chapter 4.1.3 --- Phylogram study --- p.71 / Chapter 4.2 --- Discussion --- p.79 / Chapter 4.2.1 --- Evaluation of chloroplast trnL-trnF region in differentiation of Baiying and Xungufeng --- p.79 / Chapter 4.2.2 --- Molecular authentication of Baiying --- p.80 / Chapter 4.3 --- Conclusion --- p.81 / Chapter CHAPTER FIVE --- MOLECULAR AUTHENTICATION OF BAIHUASHESHECAO --- p.82 / Chapter 5.1 --- Results --- p.82 / Chapter 5.1.1 --- ITS region used for DNA sequencing --- p.82 / Chapter 5.1.2 --- Sequence alignment --- p.82 / Chapter 5.1.3 --- Percentage similarity analysis --- p.88 / Chapter 5.1.4 --- Phylogram study --- p.90 / Chapter 5.2 --- Discussion --- p.98 / Chapter 5.2.1 --- Evaluation of complete sequence of ITS region in differentiation of Hedyotis species --- p.98 / Chapter 5.2.2 --- Molecular authentication of retailed Baihuasheshecao --- p.99 / Chapter 5.2.3 --- Analysis of conflicting data between this study and published results --- p.99 / Chapter 5.2.3.1 --- Comparison of ITS-1 region --- p.101 / Chapter 5.2.3.2 --- Comparison of ITS-2 region --- p.104 / Chapter 5.2.3.3 --- Proposed reasons for the conflicting data --- p.108 / Chapter 5.3 --- Conclusion --- p.109 / Chapter CHAPTER SIX --- MOLECULAR AUTHENTICATION OF CHUANLIANZI --- p.110 / Chapter 6.1 --- Results --- p.110 / Chapter 6.1.1 --- DNA sequencing --- p.110 / Chapter 6.1.1.1 --- Complete sequence of ITS region used for DNA sequencing --- p.110 / Chapter 6.1.1.1.1 --- Sequence alignment --- p.111 / Chapter 6.1.1.1.2 --- Percentage similarity analysis --- p.113 / Chapter 6.1.1.2 --- 5S rDNA intergenic spacer used for DNA sequencing --- p.113 / Chapter 6.1.1.2.1 --- Sequencing alignment --- p.114 / Chapter 6.1.1.2.2 --- Percentage similarity analysis --- p.122 / Chapter 6.1.1.2.3 --- Phylogram study --- p.128 / Chapter 6.1.2 --- ISSR fingerprinting --- p.136 / Chapter 6.2 --- Discussion --- p.138 / Chapter 6.2.1 --- DNA sequencing results --- p.138 / Chapter 6.2.2. --- ISSR fingerprinting results --- p.139 / Chapter 6.2.3 --- Investigation of the identity of retailed Chuanlianzi --- p.140 / Chapter 6.2.4 --- Taxonomic interpretation for Melia species --- p.141 / Chapter 6.2.5 --- Kulianzi involved in this study --- p.141 / Chapter 6.3 --- Conclusion --- p.141 / Chapter CHAPTER SEVEN --- CONCLUSION --- p.143 / BILBIOGRAPHY --- p.146 / APPDENDIX - MATERIAL PREPARATION --- p.162