The proteomic approach to investigate Parkinson's desease: pathogenetic mechanisms and biomarkers discovery.

摘要: 4 1.INTRODUCTION 6 1.1 The proteomic approach to investigate pathogenethic mechanisms and find biomarkers for Parkinson‟s disease 7 1.2 8 1.2.1. Epidemiology Genetics 11 1.3 Alteration of dopamine homeostasis oxidative stress 13 1.4 α-Synuclein 14 1.5 DJ-1 19 1.6 Cellular models α-synuclein toxicity 20 1.7 as a mitochondrial 22 1.8 Biomarkers 27 1.8.1 Rationale peripheral PD 29 1.8.2 Potential source PD: Peripheral blood lymphocytes 30 2.AIM OF THE PROJECT 34 3.MATERIALS AND METHODS 36 3.1 A SH-SY5Y MODEL TO INVESTIGATE PATHOGENETIC MECHANISMS ......... 37 3.1.1 Cell cultures transfection 3.1.2 viability 3.1.3 2-DE electrophoresis statistical analysis 38 3.1.4 LC-MS-MS protein identification 39 3.1.5 Bioinformatics enrichment network clustering 40 3.1.6 Apoptosis 3.1.7 Transient luciferase gene reporter assay 41 3.1.8 Western blotting α-synuclein, HSP70 14-3-3 3.1.9 cycle 42 3.1.10 Voltage Dependent Anion Channels (VDACs) 43 MONODIMENSIONAL WESTERN BLOTTING (TIME COURSE) VDAC2 TWO-DIMENSIONAL 44 3.1.11 Tetramethylrhodamine methyl ester (TMRM) staining 3.1.12 Measurement Ca2+ flux mitochondria using Aequorin 45 CELL PREPARATION, CALCIUM-PHOSPHATE TRANSFECTION PROCEDURE RECONSTITUTION FUNCTIONAL AEQUORIN