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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Inducibility and overexpression studies of antiquitin in HEK293 and HepG2 cells. / Inducibility & overexpression studies of antiquitin in HEK293 and HepG2 cells

January 2005 (has links)
Wong Wei-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 221-242). / Abstracts in English and Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract in Chinese --- p.iv / Abstract in English --- p.vi / List of abbreviations --- p.viii / List of figures --- p.xi / List of tables --- p.xv / Content: --- p.xvi / General introduction --- p.1 / Aldehyde dehydrogenase superfamily --- p.3 / Background of antiquitin --- p.5 / Plant antiqutins (ALDH7B) --- p.5 / Animal antiquitins (ALDH7A) --- p.8 / Human antiquitin information on NCBI --- p.14 / Rationale of studying the inducibility of annquitin and overexpression of it in HEK293 and HepG2 cells --- p.16 / Flowchart 1 Procedure of antiquitin expression studies in the HEK293 and HepG2 cells under stress --- p.19 / Flowchart 2 Procedure to study antiquitin expression in the HEK293 and HepG2 cells after in silico promoter search --- p.20 / Flowchart 3 Procedure to study antiquitin overexpressed HEK293 and HepG2 cells --- p.21 / Chapter Chapter 1 --- Inducibility of antiquitin in the HEK293 and HepG2 cells under hyperosmotic stress / Chapter 1.1 --- Introduction --- p.22 / Chapter 1.1.1 --- Cellular response to hyperosmotic stress --- p.22 / Chapter 1.1.2 --- Methods to study the responses of cells under hyperosmotic stress --- p.24 / Chapter 1.2 --- Materials --- p.26 / Chapter 1.2.1 --- Cell culture media --- p.26 / Chapter 1.2.2 --- Buffers for RNA use --- p.26 / Chapter 1.2.3 --- Buffers for DNA use --- p.27 / Chapter 1.2.4 --- Other chemicals --- p.27 / Chapter 1.3 --- Methods --- p.28 / Chapter 1.3.1 --- Culture of HEK293 and HepG2 cells --- p.28 / Chapter 1.3.2 --- Hyperosmotic stress on HEK293 and HepG2 cells --- p.29 / Chapter 1.3.3 --- MTT assay --- p.29 / Chapter 1.3.4 --- Total RNA extraction --- p.30 / Chapter 1.3.5 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 1.3.6 --- Polymerase chain reaction (PCR) --- p.31 / Chapter 1.3.7 --- Quantification of PCR products --- p.31 / Chapter 1.3.8 --- Statistical analysis --- p.33 / Chapter 1.4 --- Results --- p.34 / Chapter 1.4.1 --- Viability of HEK293 and HepG2 cells under hyperosmotic stress --- p.34 / Chapter 1.4.2 --- Validation of RNA quality --- p.34 / Chapter 1.4.3 --- Validation and determination of PCR conditions --- p.40 / Chapter 1.4.4 --- Inducibility of antiquitin in HEK293 cells under hyperosmotic stress / Chapter 1.4.5 --- Inducibility of antiquitin in HepG2 cells under hyperosmotic stress --- p.43 / Chapter 1.4.6 --- Inducibility of aldose reductase under hyperosmotic stress --- p.43 / Chapter Chapter 2 --- "In silico studies of human antiquitin promoter, genomics sequences and open reading frame" --- p.54 / Chapter 2.1 --- Introduction --- p.54 / Chapter 2.1.1 --- Eukaryotic promoters --- p.55 / Chapter 2.1.2 --- Key events in transcriptional initiation --- p.55 / Chapter 2.1.3 --- Alternative splicing of mRNA --- p.57 / Chapter 2.1.4 --- Bipartite nuclear localization signal (NLS) --- p.57 / Chapter 2.2 --- Methods --- p.60 / Chapter 2.2.1 --- Putative promoter studies of human antiquitin --- p.60 / Chapter 2.2.2 --- Putative promoter studies of Arabidopsis thaliana antiquitin --- p.60 / Chapter 2.2.3 --- Analysis for the alternative splicing of human antiquitin mRNA --- p.60 / Chapter 2.2.4 --- Analysis for the nuclear localization signal (NLS) of human antiquitin amino acid sequence --- p.61 / Chapter 2.2.5 --- Nucleotide / amino acid sequence analyses --- p.61 / Chapter 2.3 --- Results --- p.62 / Chapter 2.3.1 --- Computer search for the putative cis-acting elements on human antiquitin promoter --- p.62 / Chapter 2.3.2 --- Comparison of cis-acting elements found on human antiquitin promoter with those on Arabidopsis thaliana antiquitin promoter --- p.62 / Chapter 2.3.3 --- Possibilities of alternative splicing isoforms of human antiquitin / Chapter 2.3.4 --- Possibilities of bipartite nuclear localization signals on human antiquitin protein --- p.83 / Chapter Chapter 3 --- Overexpression of antiquitin in HEK293 and HepG2 cells and their characterization / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- Cell cycle of a human somatic cell --- p.88 / Chapter 3.1.2 --- Detection of changes in the transcriptome --- p.90 / Chapter 3.1.3 --- Human genome U133 Plus 2.0 array --- p.95 / Chapter 3.1.4 --- Detection of changes in the proteome --- p.96 / Chapter 3.1.5 --- MALDI-TOF MS --- p.97 / Chapter 3.2 --- Materials --- p.99 / Chapter 3.2.1 --- Solutions for cell culture use --- p.99 / Chapter 3.2.2 --- Solutions for cloning --- p.99 / Chapter 3.2.3 --- Buffers for cell cycle analysis --- p.99 / Chapter 3.2.4 --- Buffers for two-dimensional (2D) electrophoresis --- p.100 / Chapter 3.2.5 --- Solutions for silver staining --- p.101 / Chapter 3.2.6 --- Solutions for Coomassie blue protein staining --- p.102 / Chapter 3.2.7 --- Solutions for Western blotting --- p.102 / Chapter 3.2.8 --- Solutions for mass spectrometry --- p.103 / Chapter 3.3 --- Methods --- p.104 / Chapter 3.3.1 --- Hypoosmotic stress --- p.104 / Chapter 3.3.2 --- Heat shock --- p.104 / Chapter 3.3.3 --- Oxidative stress treatment / Chapter 3.3.4 --- Chemical hypoxia --- p.104 / Chapter 3.3.5 --- Treatment of forskolin --- p.106 / Chapter 3.3.6 --- Culture of SHSY5Y cells and its differentiation --- p.106 / Chapter 3.3.7 --- Cloning of pBUDCE4.1/ATQ --- p.106 / Chapter 3.3.8 --- PCR product purification --- p.107 / Chapter 3.3.9 --- Preparation of pEGFP.N1 vector for co-transfection --- p.109 / Chapter 3.3.10 --- Transfection of HEK293 and HepG2 cells --- p.109 / Chapter 3.3.11 --- Assays to characterize transient transfected HEK293 and HepG2 cells --- p.110 / Chapter 3.3.11.1 --- Transfection efficiency monitoring --- p.110 / Chapter 3.3.11.2 --- Cell cycle analysis --- p.112 / Chapter 3.3.11.3 --- Cell doubling time measurement --- p.112 / Chapter 3.3.11.4 --- Stress responsiveness --- p.113 / Chapter 3.3.11.5 --- Oligonucleotide array analysis --- p.113 / Chapter 3.3.11.5.1 --- Total RNA extraction --- p.113 / Chapter 3.3.11.5.2 --- Oligonucleotide array preparations --- p.113 / Chapter 3.3.11.5.3 --- Data analysis --- p.114 / Chapter 3.3.11.6 --- Two-dimensional (2D) electrophoresis --- p.115 / Chapter 3.3.11.6.1 --- Total protein extraction --- p.115 / Chapter 3.3.11.6.2 --- Protein quantification --- p.115 / Chapter 3.3.11.6.3 --- First dimension electrophoresis: isoelectric focusing (IEF) --- p.115 / Chapter 3.3.11.6.4 --- Second dimension electrophoresis: SDS- --- p.116 / Chapter 3.3.11.6.5 --- Silver staining --- p.116 / Chapter 3.3.11.6.6 --- Spots detection --- p.117 / Chapter 3.3.11.7 --- Preparations of samples for MALDI-TOF MS --- p.117 / Chapter 3.3.11.7.1 --- Silver de-staining --- p.117 / Chapter 3.3.11.7.2 --- In-gel tryptic digestion --- p.118 / Chapter 3.3.11.7.3 --- Peptide extraction --- p.118 / Chapter 3.3.11.7.4 --- ZipTip® samples desalting and concentrating --- p.119 / Chapter 3.3.11.7.5 --- MALDI-TOF MS --- p.119 / Chapter 3.3.11.8 --- Western blotting --- p.119 / Chapter 3.3.11.8.1 --- Antibodies probing --- p.120 / Chapter 3.3.11.8.2 --- Enhanced chemiluminescence's (ECL) assay --- p.121 / Chapter 3.4 --- Results --- p.122 / Chapter 3.4.1 --- Inducibility of antiquitin in HEK293 cells under xenobiotic stimulus --- p.122 / Chapter 3.4.2 --- Inducibility of antiquitin in HEK293 and HepG2 cells under chemical hypoxia --- p.122 / Chapter 3.4.3 --- Inducibility of antiquitin in HEK293 and HepG2 cells under hypoosmotic stress --- p.122 / Chapter 3.4.4 --- Inducibility of antiquitin in HEK293 and HepG2 cells under heat shock --- p.122 / Chapter 3.4.5 --- Inducibility of antiquitin in HEK293 and HepG2 cells under forskolin challenge --- p.128 / Chapter 3.4.6 --- Expression of antiquitin in differentiating SHSY5Y cells by retinoic acid and N2 supplement --- p.128 / Chapter 3.4.7 --- Overexpression of antiquitin in HEK293 and HepG2 cells --- p.128 / Chapter 3.4.8 --- Viability of transfected HEK293 and HepG2 cells under hyperosmotic stress --- p.136 / Chapter 3.4.9 --- Cell doubling times of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.10 --- Cell cycle analysis of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.11 --- "Western blot analysis of cyclin D, cyclin A and cyclin B of transfected HEK293 and HepG2 cells" --- p.148 / Chapter 3.4.12 --- RNA quality control tests for oligonucleotide array analysis --- p.148 / Chapter 3.4.13 --- Oligonucleotide array analysis on transfected HEK293 and HepG2 cells --- p.155 / Chapter 3.4.14 --- Two-dimensional electrophoresis of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.15 --- MALDI-TOF MS of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.16 --- Genes and proteins upregulnted in the antiquitin transfected HEK293 and HepG2 cells --- p.190 / Discussion --- p.197 / Reference --- p.221 / Appendix Materials used in the project --- p.243

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