Table of Contents

Table of Contents

TABLE OF CONTENTS………………………………………………………................I

ABSTRACT……………………………………………………………………………….III

LIST OF TABLES….…………………………………………………………...............IV

LIST OF FIGURES…….……………………………………………………..................V

NOMENCLATURE…………………………………………………………..................VI

1.         INTRODUCTION………………………………………………………. ...1

1.1      CHARACTERISTICS OF THE PRION DISEASES……………1

1.1 .1        General Features of the Prion Diseases………………..1

1.1.2         Prion Diseases: Etiology, Transmission, Clinical                                Presentation and Treatment……………………………...2

                                  

1.2      MOLECULAR PROPERTIES OF THE PRION PROTEIN……5

1.2.1         Structure of the Prion Protein…………………………….5

1.2.2         Function of the Prion Protein…………………………. …7

1.2.3         Genetics of the Prion Protein…………………………….8

1.2.4         Prions in Yeast and Fungi………………………………..9

1.3      CELLULAR TRAFFICKING OF THE PRION PROTEIN ……10

1.4      THE PRINCIPLES AND APPLICATIONS OF THE ATOMIC 

            FORCE MICROSCOPY ………………………………………..13

      

1.4.1           Atomic Force Microscope……………………………….13

1.4.2           General Principles of AFM………………………………13

1.5      AMYLOID IN PRION DISEASES………………………………15

                 1.5.1          Formation of Amyloid……………………………………15

     1.5.2          Amyloid under AFM……………………………………..17

                      

           

 2.     MATERIALS AND METHODS……………………………………….....19

2.1      MATERIALS……………………………………………………...19

2.1.1         Chemicals, Buffers, Solutions, Reagents and Media...19

2.1.2            Disposable Materials…………………………………… 19

2.1.3            Antibodies…………………………………………………20

2.1.4            Cell Lines………………………………………………….20

2.1.5            Hardware………………………………………………….20

2.1.6            Software…………………………………………………..20

                                  

2.2      METHODS………………………………………………………..21  

2.2.1         Cell Fixation for AFM and Immunofluorescence……...21

2.2.2         Immunofluorescence Detection of PrP^c / PrP^Sc ………21 

2.2.3            Imaging of Immunofluorescence Labeled Samples….22

2.2.4            Atomic Force Microscopy Setup……………………….22

2.2.5            Image Processing………………………………………..22

3.       RESULTS…………………………………………………………………23

                        3.1      INDIRECT IMMUNOFLUORESCENCE OF PrP ON THE                                         SURFACE OF NORMAL AND INFECTED N₂A CELLS…….23

                        3.2      CELL SURFACE STRUCTURES IN SCRAPIE-INFECTED                                                AND NON-INFECTED CELL CULTURES……………………24

4.       DISCUSSION…………………………………………………………….35

REFERENCES…………………………………………………………………………..38

HONOUR STATEMENT……………………………………………………................43

Acknowledegements

ACKNOWLEDGEMENTS

I would like to express my profound thanks to Prof. Dr. Daniel Müller, who gave me the invaluable educational opportunity at the Cellular Machines Lab, BIOZ. Prof. Dr. Daniel Müller has encouraged me to open my mind to new challenges, and a competitive team work. Being in a competitive lab environment, with his other brilliant PhD students increased my enthusiasm to learn as much as possible. I am grateful to Prof. Dr. Müller for letting me to experience a great research environment in his distinguished lab.

I would like to express my sincere thanks to Susanne Wegmann for her invaluable instructions and suggestions. Susanne Wegmann´s high level of professionalism and commitment as a PhD student are her valuable assets. She has given me the idea of combining my medicine and molecular bioengineering knowledge to deal with AFM images. Her experience in the field and her kindness to teach have made valuable contributions to this research.

            I would like to thank Arch. Joanna Lorenc for her kind contributions to the processing of the AFM pictures.

            I would like to thank Prof. Dr. Sadik Ersöz and Prof. Dr. Nejat Akar from Ankara University School of Medicine for supporting me for my studies in Germany.

This thesis is dedicated to my family, my father Arch. Hüseyin Yüksel, my mother Prof. Dr. Öznur Yüksel and my sister PhD student Seniha Esen Yüksel, MSc. I can not thank them enough, but shall remain grateful for all they have done. 

First Supervisor:          Prof. Dr. Daniel J.  Müller

Second Supervisor:    Prof. Dr. Petra Schwille

Completed at:             Cellular Machines Group, BIOZ

                                        TU Dresden, Germany

Dresden, August 2006

Atomic Force Microscopy of Changes in Cell Morphology after Scrapie Infection

Thesis for the Degree of MSc

by

Mehmet Eren Yüksel

Submitted to

Faculty of Biology

Dresden Technical University

International Master Program in Molecular Bioengineering

Bioinnovations Zentrum

TU Dresden