Contents
INTRODUCTION
Abstract...1
1. Protein aggregation and amyloidoses...4
1.1. Amyloid diseases...4
1.2. Structure of amyloid fibrils...6
1.3. Amyloid formation...8
1.4. Cytotocity of amyloid aggregates...10
1.5. Structural basis and molecular features of amyloid cytotoxicity...11
1.6. Protein-membrane interaction and cytotoxicity...12
1.7. Phospholipid membranes as catalysts for protein aggregation...16
2. Diabetes mellitus and human amylin...17
2.1. The two kinds of diabetes mellitus...17
2.2. β-cell mass...17
2.3. Insulin secretion function...18
2.4. Physiological functions of human amylin...20
2.5. Factors of β-cell damage in NIDDM: genetic factors, glucotoxicity, lipotoxicity and islet amyloid deposits of human amylin...22
2.6. Molecular characterization, structural properties and fibril formation of human amylin...24
2.7. Possible causes of hIAPP aggregation and islet amyloid formation...30
2.8. Mechanisms of hIAPP cytotoxicity: from the amyloid hypothesis to the oligomer hypothesis and the interaction with the membrane...35
2.9. hIAPP-induced β-cell apoptosis...40
2.10. Appendix...42
3. Oleuropein aglycon and hIAPP amyloid aggregation...43
3.1. Structure and beneficial effects of oleuropein aglycon on human health...43
3.2. Polyphenols as antiamyloidogenic agents: oleuropein aglycon vs hIAPP...47
5. MATERIALS and METHODS
5. Materials and methods...50
5.1. Materials and cell culture...50
5.2. Oleuropein deglycosilation...50
5.3. Cytotoxicity assay...51
5.4. Caspase-3 activity assay...52
5.5. Lactate dehydrogenase (LDH) release assay...52
5.6. Immunofluorescence analysis...53
5.7. Preparation of phospholipid unilamellar vesicles...54
5.8. Calcein release assay...55
5.9. ThT assay...56
5.10. Circular dichroism...57
5.11. Electron microscopy...58
RESULTS
6. Results...60
6.1. The oleuropein aglycon reduces hIAPP cytotoxicity...60
6.1.1. MTT assay...60
6.1.2. Caspase-3 activity assay and LDH relaese assay...63
6.2. hIAPP aggregates grown with oleuropein aglycon do not interact with the cell...64
6.2.1. Immunofluorescence analysis...64
6.2.2. Permeabilization of synthetic phospholipid vesicles by hIAPP aggregates...65
6.3. In the presence of oleuropein, hIAPP aggregates through a different pathway...67
6.3.1. ThT assay...67
6.3.2. CD analysis...69
6.3.3. Electron microscopy analysis...71
DISCUSSION
7. Discussion...74
7.2. Structural effects of oleuropein aglycon on hIAPP aggregates...75
7.3. The nature of the cytoprotective action of oleuropein aglycon...75
7.4. Conclusions...77
