Lee Kai Woo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 90-104). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Thesis Committee --- p.iii / Abstracts --- p.v / 論文槪要 --- p.vii / List of figures --- p.viii / List of abbreviations --- p.ix / Chapter Chapter one --- Introduction --- p.1 / Chapter 1.1 --- Drug metabolism and UGTs --- p.1 / Chapter 1.2 --- Natural substrates of UGTs --- p.4 / Chapter 1.3 --- Functions of UGT isoforms: roles of UGT polymorphisms --- p.6 / Chapter 1.4 --- Evolution of the UGT1 gene locus in vertebrates --- p.8 / Chapter 1.5 --- Multiple Variable First Exons: A Mechanism for Cell- and Tissue-Specific Gene regulation --- p.13 / Chapter 1.6 --- Evolutionary Origin of the Variable and Constant Genomic Organization --- p.14 / Chapter 1.7 --- Variable and Constant Genomic Organizations Exist in Mammalian UGTs --- p.20 / Chapter 1.8 --- The history of recombinant UGT expression --- p.20 / Chapter 1.9 --- UGT1A8 --- p.21 / Chapter 1.10 --- Licorice and its active component --- p.24 / Chapter 1.11 --- Enzyme induction in the liver --- p.25 / Chapter 1 12 --- Objectives --- p.28 / Chapter Chapter two --- Methods and Materials --- p.29 / Chapter 2.1 --- UGT1A8 induction studies --- p.30 / Chapter 2.1.1 --- Drug preparation --- p.30 / Chapter 2.1.2 --- Cell viability study with Neutral Red Assay Rat treatment --- p.30 / Chapter 2.1.3 --- Cell treatment --- p.31 / Chapter 2.1.4 --- Rat treatment --- p.31 / Chapter 2.1.5 --- RNA extraction from rat liver and cell culture --- p.31 / Chapter 2.1.6 --- Quantization of RNA --- p.32 / Chapter 2.1.7 --- Denaturing gel electrophoresis for RNA --- p.33 / Chapter 2.1.8 --- Northern hybridization --- p.33 / Chapter 2.1.9 --- Probe for Northern Blotting --- p.34 / Chapter 2.1.10 --- Agarose Gel analysis and Northern Blot analysis --- p.34 / Chapter 2.2 --- Recombinant expression of UGT1A8 in E.coli JM109 --- p.35 / Chapter 2.2.1 --- cDNA synthesis --- p.35 / Chapter 2.2.2 --- Polymerase chain reaction --- p.35 / Chapter 2.2.3 --- Agarose gel electrophoresis for DNA --- p.35 / Chapter 2.2.4 --- "Amplification of target gene, UGT1A8" --- p.36 / Chapter 2.2.5 --- Restriction enzyme digestion of plasmid and insert --- p.36 / Chapter 2.2.6 --- Ligation of plasmid and insert DNA --- p.37 / Chapter 2.2.7 --- Amplification of target plasmid --- p.37 / Chapter 2.2.8 --- Screening of target plasmid --- p.37 / Chapter 2.2.9 --- DNA sequencing --- p.38 / Chapter 2.2.10 --- Transformation of protein expression host --- p.38 / Chapter 2.2.11 --- Confirmation of transformation of protein expression host --- p.38 / Chapter 2.2.12 --- Protein expression --- p.39 / Chapter 2.2.13 --- Protein purification --- p.39 / Chapter 2.2.14 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.40 / Chapter 2.2.15 --- Confirmation of the protein --- p.40 / Chapter 2.3 --- Characterization of recombinant UGT1A8 --- p.41 / Chapter 2.3.1 --- UGT assay --- p.41 / Chapter 2.4 --- Routine experiment methods --- p.41 / Chapter 2.4.1 --- Determination of protein --- p.41 / Chapter 2.4.2 --- Nucleic acid purification --- p.42 / Chapter 2.4.3 --- Preparation of chemically competent bacterial cells --- p.42 / Chapter 2.4.4 --- Colony PCR --- p.43 / Chapter 2.4.5 --- Plasmid rescue by alkaline lysis --- p.44 / Chapter 2.4.6 --- Charging of His-tagged column --- p.44 / Chapter 2.4.7 --- Washing of His-tagged column --- p.45 / Chapter Chapter three --- Results --- p.46 / Chapter 3.1 --- UGT1A8 Expression in clone9 and H4IIE after treatment with licorice and 18 β glycyrrhentinic acid --- p.46 / Chapter 3.2 --- UGT1A8 induction in wistar and j/j rats after treatment --- p.63 / Chapter 3.3 --- Construction of pRset-UGT 1A8 Vector --- p.70 / Chapter 3.4 --- Purification of recombinant UGT1A8 --- p.75 / Chapter 3.5 --- Screening of substrate of the purified enzyme --- p.77 / Chapter Chapter four --- Discussion --- p.78 / Chapter 4.1 --- Effects of licorice and 18βglycyrrhetinic acid in the induction of UGT1A8 in different cell lines --- p.78 / Chapter 4.2 --- Comparison of wistar and j/j rats in the induction of UGT1A8 --- p.79 / Chapter 4.3 --- Comparison of licorice and 18(3 glycyrrhetinic acid in the induction of UGT1A8 in rats --- p.81 / Chapter 4.4 --- Comparison of in vivo and in vitro of drug treatment --- p.81 / Chapter 4.5 --- Expression of UGT1A7 after drug treatment in vitro --- p.82 / Chapter 4.6 --- Protein expression and purification --- p.83 / Chapter 4.7 --- Substrates of UGT1A8 --- p.83 / Chapter Chapter Five --- Conclusions --- p.86 / References --- p.90 / Appendix --- p.105
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325805 |
| Date | January 2006 |
| Contributors | Lee, Kai Woo., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xii, 106 leaves : ill. ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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