Wong, Tak Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 96-104). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgements --- p.vii / Abbreviation --- p.ix / Table of Content --- p.xi / List of figures --- p.xiv / List of tables --- p.xv / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Problem of soil salinization and sodification: reducing crop productivity --- p.1 / Chapter 1.2 --- Effects of high salinity on plant growth --- p.2 / Chapter 1.2.1 --- Ion toxicity --- p.2 / Chapter 1.2.2 --- Osmotic stress --- p.3 / Chapter 1.2.3 --- Oxidative stress --- p.3 / Chapter 1.3 --- Overview of salt tolerance mechanisms in plant --- p.4 / Chapter 1.3.1 --- Maintenance of ion homeostasis --- p.4 / Chapter 1.3.2 --- Maintaining osmotic homeostasis --- p.5 / Chapter 1.3.3 --- Detoxification of Reactive oxygen species --- p.5 / Chapter 1.4 --- The important role of CI- in plant salt stress tolerance research --- p.6 / Chapter 1.5 --- Introduction to chloride channel (CLC) family --- p.7 / Chapter 1.6 --- E. coli CLC-ecl: The first CLC member found to function as antiporter --- p.8 / Chapter 1.7 --- Yeast GEF1: eukaryotic model for early plant CLC complementation studies --- p.9 / Chapter 1.8 --- Mammalian CLC family: 4 channels and 5 antiporters --- p.10 / Chapter 1.8.1 --- CLC-4 and -5: First eukaryotic CLC member found to be function as antiporter --- p.13 / Chapter 1.8.2 --- CLC-7 function as antiporter and regulate lysosomal acidification --- p.13 / Chapter 1.8.3 --- "CLC-6 select nitrate over chloride, unlike other mammalian CLC members" --- p.14 / Chapter 1.9 --- Introduction to Plant CLC members --- p.14 / Chapter 1.10 --- Tobacco CLC-Ntl co-localized with mitochondrial markers in plant and may cause current on Xenopus oocytes membrane --- p.15 / Chapter 1.11 --- Rice CLCs may involved in salt tolerenace and growth regulation --- p.16 / Chapter 1.12 --- Arabidopsis CLC members are extensively studied --- p.18 / Chapter 1.12.1 --- AtCLCa regulates nitrate accumulation --- p.20 / Chapter 1.12.2 --- "AtCLCb, a nitrate/proton antiporter with unclear physiological role" --- p.22 / Chapter 1.12.3 --- "AtCLCc selective chloride over nitrate, involved in salt tolerance" --- p.23 / Chapter 1.12.4 --- AtCLCd and AtCLCf both localized on Golgi network --- p.25 / Chapter 1.12.5 --- AtCLCe may regulate ionic strength of chloroplast thylakoid membrane --- p.26 / Chapter 1.13 --- Previous work in Prof. Lam's laboratory --- p.26 / Chapter 1.14 --- "Reason, Hypothesis, Objective and long term significance" --- p.28 / Chapter 2. --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Bacterial strains, animals, plants and plasmid vectors" --- p.30 / Chapter 2.1.2 --- Chemicals and Enzymes --- p.33 / Chapter 2.1.3 --- Commercial kits --- p.33 / Chapter 2.1.4 --- Primers --- p.35 / Chapter 2.1.5 --- Equipments and facilities used --- p.36 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.36 / Chapter 2.1.7 --- Software --- p.36 / Chapter 2.2 --- Methods --- p.37 / Chapter 2.2.1 --- Growth and treatment of soybean seedling --- p.37 / Chapter 2.2.2 --- RNA extraction from root tissue --- p.37 / Chapter 2.2.3 --- RNA denaturing gel electrophoresis --- p.39 / Chapter 2.2.4 --- Generation and testing of single-stranded DIG-labeled PCR probes --- p.39 / Chapter 2.2.5 --- Northern blot analysis --- p.41 / Chapter 2.2.6 --- Transformation of V7/GmCLCl electro-competent Agrobacterium tumefaciens --- p.42 / Chapter 2.2.7 --- PCR screening of transformed Agrobacterium tumefaciens colonies --- p.43 / Chapter 2.2.8 --- DNA gel electrophoresis --- p.43 / Chapter 2.2.9 --- Agrobacterium-mediated transformation of tobacco BY-2 cells --- p.44 / Chapter 2.2.10 --- Verifying the expression of GmCLCl in transgenic tobacco BY-2 cells --- p.45 / Chapter 2.2.11 --- Salt treatment of tobacco BY-2 cells and cell viability assay --- p.46 / Chapter 2.2.12 --- Subcloning of GmCLCl cDNA into pgh21 vector --- p.47 / Chapter 2.2.13 --- In vitro synthesis of GmCLCl cRNA --- p.51 / Chapter 2.2.14 --- Obtaining oocyte from Xenopus laevis ovaries --- p.52 / Chapter 2.2.15 --- Microinjection of GmCLCl cRNA into Xenopus oocyte and oocyte incubation --- p.53 / Chapter 2.2.16 --- Two electrode voltage clamp of Xenopus oocytes --- p.54 / Chapter 3. --- Results --- p.56 / Chapter 3.1 --- Phylogenetic analysis of GmCLCl --- p.56 / Chapter 3.2 --- Expression of GmCLCl in root was induced by NaCl and alkaline condition --- p.60 / Chapter 3.3 --- Construction of GmCLCl transgenic tobacco BY-2 cell line --- p.62 / Chapter 3.4 --- GmCLCl improve NaCl stress tolerance of transgenic tobacco BY-2 cells in a pH dependent manner --- p.67 / Chapter 3.5 --- Subcloning of GmCLCl into pgh21 --- p.70 / Chapter 3.6 --- GmCLCl cRNA synthesis by in vitro transcription --- p.72 / Chapter 3.7 --- Two electrode voltage clamp (TEVC) of GmCLCl cRNA injected Xenopus oocytes --- p.75 / Chapter 4. --- Discussion --- p.81 / Chapter 4.1 --- Implications from phylogenetic and sequence analysis on the function of GmCLCl --- p.81 / Chapter 4.2 --- Electrophysiological characterization of GmCLC 1 by Xenopus oocytes --- p.82 / Chapter 4.3 --- Some plant CLCs contributed in salt tolerance response --- p.84 / Chapter 4.4 --- Relationship between pH and physiological function of plant CLCs --- p.85 / Chapter 5. --- Conclusion and Perspectives --- p.88 / Chapter 6. --- Appendices --- p.90 / Chapter Appendix I: --- Major Chemicals and reagents used in this research --- p.90 / Chapter Appendix II: --- Enzymes used in this research --- p.92 / Chapter Appendix III: --- Major equipment and facilities used in this research --- p.93 / Chapter Appendix IV: --- "Buffer, solution, gel and medium formulation" --- p.94 / Chapter 7. --- References --- p.96

Indiana University-Purdue University Indianapolis (IUPUI) / Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4µg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4µg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability.

Indiana University-Purdue University Indianapolis (IUPUI) / Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 h exposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, singleand multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatics analysis of proteins identified by LFQMS. Interestingly, after exposure to a high concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT, respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins common to both kinds of nanotubes are associated with the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The decrease in expression of the majority proteins suggests a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure. To examine the effect of nanotubes on the plasma membrane, we investigated the interaction of short purified MWCNT with model lipid membranes using a planar bilayer workstation. Bilayer lipid membranes were synthesized using neutral 1, 2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) in 1 M KCl. The ion channel model protein, Gramicidin A (gA), was incorporated into the bilayers and used to measure the effect of MWCNT on ion transport. The opening and closing of ion channels, amplitude of current, and open probability and lifetime of ion channels were measured and analyzed by Clampfit. The presence of an intermediate concentration of MWCNT (2 μg/ml) could be related to a statistically significant decrease of the open probability and lifetime of gA channels. The proteomic studies revealed changes in response to CNT exposure. An analysis of the changes using multiple databases revealed alterations in pathways, which were consistent with the physiological changes that were observed in cultured cells exposed to very low concentrations of CNT. The physiological changes included the break down of the barrier function and the inhibition of the mucocillary clearance, both of which could increase the risk of CNT’s toxicity to human health. The biophysical studies indicate MWCNTs have an effect on single channel kinetics of Gramicidin A model cation channel. These changes are consistent with the inhibitory effect of nanoparticles on hormone stimulated transepithelial ion flux, but additional experiments will be necessary to substantiate this correlation.