Fluorescence Platform Development for Detection of Cystic Fibrosis Transmembrane Conductance Regulator Trafficking
نویسنده
چکیده
........................................................................................................................................................ 3 List of Figures ................................................................................................................................................ 4 List of Tables ................................................................................................................................................. 4 Abbreviations ................................................................................................................................................ 5 Chapter 1 : Cystic Fibrosis and CFTR ............................................................................................................ 8 Ch.1 – Introduction ................................................................................................................................... 8 Cystic Fibrosis ........................................................................................................................................ 8 CFTR .................................................................................................................................................... 12 CFTR Quality Control at the Endoplasmic Reticulum .......................................................................... 14 Stability of CFTR in the Plasma Membrane ......................................................................................... 16 Methods Available to Study CFTR ....................................................................................................... 18 Fluorogen Activating Proteins ............................................................................................................. 21 Ch. 1 – Results ......................................................................................................................................... 24 Tagging the N-terminus of CFTR ......................................................................................................... 24 Fourth Extracellular Loop Tagging Strategy ........................................................................................ 30 Functional Validation of Fluorescent CFTR Reporters ........................................................................ 32 Biochemical Analysis of Fluorescent CFTR Reporters ......................................................................... 36 Ch. 1 – Discussion .................................................................................................................................... 38 Ch. 1 – Materials and Methods ............................................................................................................... 43 Chapter 2 : Detection of CFTR ΔF508 .......................................................................................................... 49 Ch. 2 Introduction ................................................................................................................................. 49 CFTR ΔF508 ......................................................................................................................................... 49 Temperature Sensitivity and Gating Defect of the ΔF508 mutation ................................................. 51 Chaperone Quality Control Checkpoints............................................................................................. 51 Repairing the Gating Defect ................................................................................................................ 53 Correctors ........................................................................................................................................... 55 High Content Screening ...................................................................................................................... 59 Corrector Additivity and Synergy ........................................................................................................ 60 Ch. 2 – Results ......................................................................................................................................... 62 Visualization of CFTR ΔF508 Rescue ................................................................................................... 63
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