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ELECTROCHEMICAL MEASUREMENT OF PLASMA MEMBRANE CHOLESTEROL IN LIVE CELLS AND MOUSE TISSUES

Fang, Danjun
http://rave.ohiolink.edu/etdc/view?acc_num=case1251945019

Abstract Details

2010, Doctor of Philosophy, Case Western Reserve University, Chemistry.
In this study, cholesterol oxidase-modified platinum micro-cavity electrodes are used to measure cholesterol efflux from the plasma membrane surface of a single neuron in the buccal ganglion of Aplysia at room temperature. Background subtraction analog chronocoulometry is used to measure hydrogen peroxide accumulation resulting from cellular cholesterol efflux and enzymatic oxidation in the micro-cavity volume. The data are consistent with the aqueous diffusion model for cellular cholesterol efflux where plasma membrane cholesterol undergoes exchange with solution phase cholesterol. The notion that the cellular cholesterol set-point can be modeled as a thermodynamic state (i.e., activity; chemical potential) exhibited at the cell surface is explored. A titration assay is developed, using the electrode measurements as an indicator, where the solution cholesterol concentration required to drive net cholesterol influx into the cell is determined. When cholesterol influx to the cell occurs, plasma membrane cholesterol increases and larger electrode responses are observed. Because the experiments are equilibrium based, data are discussed assuming that the end-point estimates obtained reflect a thermodynamic cholesterol state that tracks the concentration of cholesterol in the membrane. Previous observations demonstrate that CFTR (cystic fibrosis transmembrane conductance regulator) -null cells and tissues exhibit an increase in plasma membrane cholesterol content compared to wild type (WT) controls. The hypothesis of this study is that these alterations in cholesterol processing will correlate with Cftr genotype. Membrane cholesterol contents are elevated in both R117H and Φrdquo;F508 mouse nasal epithelium compared to age-matched sibling WT controls demonstrating a genotype correlation to membrane cholesterol content. With the advent of new CFTR correctors and potentiators, determining membrane cholesterol content could prove to be an important biomarker of CFTR function and the cellular responses to various levels of CFTR function.
Daniel Scherson (Committee Chair)
Alfred Anderson (Committee Member)
Carlos Crespo (Committee Member)
Thomas Kelley (Committee Member)
James Burgess (Advisor)
123 p.
Analytical Chemistry
cholesterol oxidase; platinum micro-cavity electrode; titration assay; plasma membrane cholesterol; CFTR

Recommended Citations

Citations

  • Fang, D. (2010). ELECTROCHEMICAL MEASUREMENT OF PLASMA MEMBRANE CHOLESTEROL IN LIVE CELLS AND MOUSE TISSUES [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1251945019

    APA Style (7th edition)

  • Fang, Danjun. ELECTROCHEMICAL MEASUREMENT OF PLASMA MEMBRANE CHOLESTEROL IN LIVE CELLS AND MOUSE TISSUES. 2010. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1251945019.

    MLA Style (8th edition)

  • Fang, Danjun. "ELECTROCHEMICAL MEASUREMENT OF PLASMA MEMBRANE CHOLESTEROL IN LIVE CELLS AND MOUSE TISSUES." Doctoral dissertation, Case Western Reserve University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1251945019

    Chicago Manual of Style (17th edition)

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© 2009, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.