Development and application of a high throughput cell based assay to identify apoptosis inducing proteins, and functional characterization of the candidate vacuole membrane protein 1 (Vmp1) [Elektronische Ressource] / presented by Mamatha Sauermann

Sabbela

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences

















presented by
Mamatha Sauermann, M.Sc. in Biotechnology
Born in Srikakulam, India




thDate of oral-examination: 27 September, 2006










Development and application of a high throughput cell based assay to
identify apoptosis inducing proteins, and functional characterization of the
candidate Vacuole Membrane Protein 1 (Vmp1)


















Referees: PD. Dr. Stefan Wiemann
PD. Dr. Frank Breitling

































To my husband and parents

Contents

SUMMARY ..................................................................................................................... 1
ZUSAMMENFASSUNG ................................................................................................ 2
1 INTRODUCTION ........................... 3
1.1 Types of cell death........................................................................................................ 4
1.1.1 Necrosis.................................................................................................................................. 5
1.1.2 Apoptosis ............................................................................................................................... 5
1.2 Organelle specific initiation of apoptosis pathways .................................................. 6
1.2.1 Death Receptor pathway from the plasma membrane (Extrinsic pathway)........................... 7
1.2.2 Mitochondrial pathway of apoptosis (Intrinsic pathway) ...................................................... 8
1.2.3 The nuclear death pathway .................................................................................................... 9
1.2.4 Endoplasmic reticulum (ER) mediated apoptosis................................................................ 10
1.2.4.1 Unfolded protein response and apoptosis ................................................................................... 10
2+1.2.4.2 Role of Ca in ER stress-induced apoptosis.............................................................................. 11
1.2.5 Golgi apparatus and apoptosis ............................................................................................. 12
1.2.6 Lysosome-mediated apoptosis ............................................................................................. 12
1.2.7 Role of cytoskeleton and cell adhesion in apoptosis............................................................ 13
1.2.8 Integration of different apoptosis pathways......................................................................... 14
1.3 Apoptosis dysregulation and its clinical implications ............................................. 14
1.3.1 Apoptosis and cancer ........................................................................................................... 15
1.3.2 Apoptosis and autoimmunity ............................................................................................... 15
1.3.3 Apoptosis and AIDS ............................................................................................................ 16
1.3.4 Apoptosis and Neurodegeneration....................................................................................... 16
1.4 Detection of apoptosis ................................................................................................ 16
1.4.1 Analysis of cell morphology................................................................................................ 17
1.4.2 Plasma membrane changes .................................................................................................. 17
1.4.3 Mitochondrial changes......................................................................................................... 18
1.4.4 DNA and nuclear changes.................................................................................................... 18
1.4.5 Biochemical changes ........................................................................................................... 20
1.5 cDNA resources for identification of novel apoptosis activators ........................... 21
1.5.1 Selection of novel full-length cDNAs for screening............................................................ 21
1.6 Aim of the project....................................................................................................... 22
2 MATERIALS AND METHODS.......................................................................... 23
2.1 Materials ............................................................................................ 23
2.1.1 Equipment............................................................................................................................ 23
2.1.2 Chemicals............................................................................................................................. 24
2.1.3 Kits....................................................................................................................................... 26
2.1.4 Plastic and glassware ...........................................................................................................26
2.1.5 Oligonucleotides .................................................................................................................. 27
2.1.6 siRNAs................................................................................................................................. 28
2.1.7 Peptides................................................................................................................................ 29
2.1.8 Antibiotics............................................................................................................................ 29
2.1.9 Restriction enzymes and buffers.......................................................................................... 29
Contents

2.1.10 Bacterial Strains...............................................................................................................30
2.1.11 Cell Lines......................................................................................................................... 30
2.1.12 Antibodies........................................................................................................................ 31
2.1.13 Plasmids........................................................................................................................... 32
2.2 Methods....................................................................................................................... 35
2.2.1 Molecular Biology methods................................................................................................. 35
2.2.1.1 Polymerase Chain Reaction (PCR)............................................................................................. 35
2.2.1.2 Purification of PCR products ...................................................................................................... 38
2.2.1.3 Ligation........................................................................................................................................ 39
2.2.1.4 Gateway Reactions...................................................................................................................... 40
2.2.1.5 Transformation of bacteria.......................................................................................................... 41
2.2.1.6 Small – scale preparation of plasmid DNA (mini prep)............................................................. 42
2.2.1.7 Large – scale preparation of plasmid DNA (maxi prep) ............................................................ 43
2.2.1.8 Measurement of DNA concentration.......................................................................................... 44
2.2.1.9 Restriction digest......................................................................................................................... 44
2.2.1.10 Gel electrophoresis...................................................................................................................... 45
2.2.1.11 Extraction of total RNA from cells............................................................................................. 45
2.2.1.12 Quantification of the RNA using Ribogreen .............................................................................. 46
2.2.1.13 Reverse transcription................................................................................................................... 46
2.2.1.14 Quantitative Real - time PCR (TaqMan) .................................................................................... 47
2.2.2 Cell biology methods ...........................................................................