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Table of contents
Abstract
Chapter 1 Introduction to amyloid proteins and disease
1.1 Protein folding and misfolding
1.2 Protein folding
1.3 Amyloid Proteins
1.4 Amyloid fibril structure and amyloid formation
1.5 Factors of protein aggregation
1.6 Diabetes mellitus
1.7 The insulin secretory granule
1.8 IAPP expression
1.9 IAPP Post-translational modification
1.10 Physiological functions of IAPP
1.11 IAPP amyloidogenesis and structure
1.12 β-cell failure in type 2 diabetes
1.13 Apoptosis
1.14 hIAPP toxicity
1.15 Primlintide, an IAPP analogue
1.16 IAPP research
1.2 Objectives of the thesis
Chapter 2 Materials and methods
Biophysical methods
Monitoring the kinetics of fibril formation using Thioflavin T fluorescence
2.1.1 Principle
2.1.2 Advantages, drawbacks and requirements for ThT T assay
2.1.3. Experimental protocol for ThT T assay
Identifying the change in the structure of a protein with circular dichroism (CD)
2.2.1 Principle
2.2.3. Experimental protocol for CD
Observation of amyloid fibers with Transmission Electron Microscopy
2.3.1 Principle
2.3.2 Advantages, drawbacks and requirements for TEM technique
2.3.3 Experimental protocol for TEM technique
Studying the interaction of the membrane with the amyloid peptide with model membrane assays, the calcein fluorescent probe
2.4.1. Principle
2.4.2 Advantages, drawbacks and requirements for preparation of Large unilamellar vesicles by extrusion technique (LUV) and Calcein assay
2.4.3. Experimental protocol
Cell biological techniques
Confirming cytotoxicity with a cell viability test, The MTT Reduction Assay
2.5.1 Principle
2.5.2. Advantages, drawbacks and requirements for MTT assay
2.5.3 Experimental protocol for MTT assay
Chapter 3 The flanking peptides issues from the maturation of the human islet amyloid polypeptide (hIAPP) do not modify hIAPP-fibril formation nor hIAPP-induced cell death
3.1 Introduction
3.2 Materials and Methods
3.2.1 Materials
3.2.2 Peptide synthesis and preparation
3.2.3 Determination of peptide aggregation by thioflavin-T assay
3.2.4 Transmission Electron Microscopy (TEM)
3.2.5 Circular dichroism
3.2.6 Membranes preparation
3.2.7 Vesicle Dye Leakage Assay
3.2 8 Cell culture
3.2 9Human islet culture
3.2.10 Fibril formation in presence of cells or islets
3.2.11 MTT Cell Toxicity Assay
3.2.12 Statistics
3.3 Results and discussions
3.3.1 The flanking peptide are not amyloidogenic in solution
3.3.2 Do the flanking peptides influence mature hIAPP fibrillation in membrane models, in cells and in human islets?
3.4 Discussions
Chapter 4 β-pancreatic amyloid deposit, a protein conformational disease involved into type 2 diabetes: deleterious role of plasma membrane lipids
4.1 Kinetics of hIAPP fibrillation in presence of different cell lines
4.2 Determination of hIAPP toxicity in presence of different cell lines
4.3 Implication of IAPP receptor
4.4 Role of lipids membrane on hIAPP fibrillation
4.5 Effect of a diabetic environment on hIAPP fibrillation
4.6 hIAPP fibrillation ion human islets
V. Discussion of results
VIII. FRENCH ABSTRACT
