Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts [Elektronische Ressource] / vorgelegt von Lars Hofmann

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Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Role and regulation of the p53-homolog p73
in the transformation of normal human
fibroblasts




Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg


vorgelegt von
Lars Hofmann

aus
Aschaffenburg







Würzburg 2007
























Eingereicht am

Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr. Dr. Martin J. Müller
Gutachter: Prof. Dr. Michael P. Schön
Gutachter : Prof. Dr. Georg Krohne

Tag des Promotionskolloquiums:

Doktorurkunde ausgehändigt am



































Erklärung

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen
als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in
gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher,
außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen
Grade erworben und zu erwerben gesucht.


Würzburg,




Lars Hofmann

Content
SUMMARY ................................................................................................................ IV
ZUSAMMENFASSUNG ............................................................................................. V
1. INTRODUCTION ................................................................................................. 1
1.1. Molecular basics of cancer .......................................................................................... 1
1.2. Early research on tumorigenesis ................................................................................. 3
1.3. Developing cell culture models of tumorigenesis ....................................................... 4
1.4. Key molecules in human cell transformation ............................................................ 6
1.4.1. hTERT .................................................................................................................. 6
1.4.2. H-RasV12 ............................................................................................................. 7
1.4.3. SV40 small t ......................................................................................................... 8
1.4.4.VLarge T ....................................................................................................... 8
1.5. Principles of tumor suppression .................................................................................. 9
1.6. p73, a transcription factor of the p53 family ........................................................... 10
1.6.1. Gene and protein organization of p73 ................................................................. 10
1.6.2. p53 family proteins have diverse biological functions ........................................ 11
1.6.3. p73 in cancer development ................................................................................. 12
1.6.4. Regulation of p73 ............................................................................................... 12
1.6.5. Regulation by p73 ............................................................................................... 13
1.7. Scope of the project .................................................................................................... 14
2. MATERIALS AND METHODS .......................................................................... 15
2.1. Materials and Equipment .......................................................................................... 15
2.1.1. Instruments and other technical equipment ......................................................... 15
2.1.2. Glass and plastic ware, consumables .................................................................. 16
2.1.3. Enzymes, PCR reagents and size standards ........................................................ 17
2.1.4. Antibodies........................................................................................................... 17
2.1.5. Oligonucleotides ................................................................................................. 18
2.1.6. Bacterial strains .................................................................................................. 18
2.1.7. Plasm ids .............................................................................................................. 19
2.1.8. Eukaryotic cell lines ........................................................................................... 19
2.2. Buffers, media and solutions ..................................................................................... 20
2.2.1. Protein detection ................................................................................................. 20
2.2.2. Flow cytometry ................................................................................................... 21
2.2.3. Cell culture ......................................................................................................... 21
2.2.4. Plasmid isolation from bacteria ........................................................................... 22
2.2.5. Other self-prepared buffers, media and solutions ................................................ 22
I
2.3. Cell culture .................................................................................................................. 23
2.3.1. Cell maintenance ................................................................................................ 23
2.3.2. Tumorigenicity assay .......................................................................................... 23
2.3.3. Growth curves ..................................................................................................... 23
2.3.4. Viability staining ................................................................................................ 24
2.3.5. Transfection ........................................................................................................ 24
2.3.6. Electroporation ................................................................................................... 24
2.3.7. Retroviral transduction ....................................................................................... 24
2.3.8. Adenoviral trtion ...................................................................................... 25
2.3.9. Cell stock freezing .............................................................................................. 25
2.4. DNA work ................................................................................................................... 25
2.4.1. Cloning ............................................................................................................... 25
2.4.2. Plasmid DNA isolation- Miniprep ...................................................................... 26
2.4.3. Plasmiation- Midiprep ...................................................................... 27
2.4.4. Plasmiation- Maxiprep
2.4.5. DNA purification- spin columns ......................................................................... 27
2.4.6. DNA purification- phenol-chloroform extraction ............................................... 27
2.4.7.quantification ............................................................................................ 28
2.4.8. DNA sequencing ................................................................................................. 28
2.4.9. Mutagenesis ........................................................................................................ 28
2.5. RNA work ................................................................................................................... 28
2.5.1. RNA isolation ..................................................................................................... 28
2.5.2.NA quantification ............................................................................................. 29
2.5.3. cDNA preparation ............................................................................................... 29
2.5.4. PCR and semi-quantitative RT-PCR ................................................................... 29
2.5.5. PCR primers and amplicon sizes ......................................................................... 30
2.5.6. Real Time PCR ................................................................................................... 30
2.5.7. DNA microarray ................................................................................................. 31
2.6. Protein work ............................................................................................................... 32
2.6.1. Western Blot .....................................................................................................