Influence of papillomavirus early proteins on the expression of tumor-progression promoting genes [Elektronische Ressource] / presented by Sabrina Mühlen

Honeybee

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

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





























presented by

Sabrina Mühlen, MSc
born in Braunschweig, Germany
Oral Examination:







Influence of Papillomavirus Early Proteins
on the Expression of
Tumor-Progression Promoting Genes

































































































“Look to this day, for yesterday is already a dream and tomorrow is only a vision.
But today well lived makes every yesterday a dream of happiness, and every tomorrow a
vision of hope.”

























































PREFACE

Acknowledgements

There are a lot of people who have made this thesis possible, not only scientifically but
also personally. To these people I owe thanks.


Firstly and most importantly, I would like to thank my supervisor PD Dr. Christian Simon
offering this interesting project and work environment, and also for the great funding. It
is tempting to speculate just how he influenced this piece of work.

I would further like to thank Prof. L Gissmann for agreeing to act as my biological
supervisor and Professors P. Angel and R. Bartenschlager for being part of my scientific
committee. I am in debt to all three for the discussions at my progress reports and their
openness to arising problems.

An impaccably huge thank you goes to Dr. Andreas Behren who was the good soul of the
lab (and also the only other soul). His patience, openness to discussions, and knowledge
was irreplacable and unparalleled.

I am grateful to Prof. T. Iftner (Tübingen) for supplying plenty of material needed for the
research to this thesis.

Furthermore, the most sincere thank you to Marc and Nina for always always helping out
when help was needed and for their support throughout the past years. Thank you!

To Sabrina, Thalia, Desi, Mircea, Imad, Stefan, Anja, Ifey, Johannes, Martin, Andreas, all
the others I forgot, and most importantly to Steff, without you the past three years
would not have been what they were. Thanks for BBQs, Nightouts, Aquafitness,
Lunches, for listening, and fun.

A huge thank you to my parents for everything, yet again but never enough!

Andreas, just for being who he is, for his undivided support and understanding, and for
his trust in me.

And the last line is, as always through these years, reserved for my dearest friend Katie.
There is not much left to say this time. Same as usual, live your dreams!














I PREFACE




II INDEX

INDEX

I SUMMARY...................................................................................................... VI

II ZUSAMMENFASSUNG.................................................................................... VII

III ABBREVIATIONS .......................................................................................... XI

11 IINNTTRROODDUUCCTTIIOONN

1.1 HUMAN PAPILLOMAVIRUSES...........................................................................1
1.1.1 The regualtory early proteins 1 and 2........................................................2
1.1.2 The early protein E4 and E5.....................................................................3
1.1.3 The oncogenes E6 and E7........................................................................4
1.1.4 The structural proteins L1 and L2 .............................................................6
1.1.5 HPV Infection and Cancer .......................................................................7

1.2 THE COTTONTAIL RABBIT PAPILLOMAVIRUS (CRPV)..........................................9

1.3 TUMOR PROGRESSION.................................................................................10

1.4 PROTEASES................................................................................................11

1.5 THE MITOGENACTIVATED KINASE (MAPK) PATHWAYS....................................13

1.6 THE TRANSCRIPTION FACTOR AP1...............................................................16

1.7 AIM OF THIS STUDY ....................................................................................18

22 EEXXPPEERRIIMMEENNTTAALL PPRROOCCEEDDUURREESS

2.1 MATERIALS ................................................................................................19
2.1.1 Chemicals............................................................................................19
2.1.2 Oligonucleotides...................................................................................19
2.1.3 siRNAs ................................................................................................22
2.1.4 Plasmids..............................................................................................22
2.1.5 Cell Lines and Bacterial Strains...............................................................25
2.1.6 Antibodies ...........................................................................................26

2.2 MOLECULAR BIOLOGY..................................................................................28
2.2.1 Amplification of DNA .............................................................................28
2.2.2 Agarose Gel Electrophoresis...................................................................28
2.2.3 Construction of a DNAlibrary.................................................................28
2.2.4 DNAsequencing...................................................................................29
2.2.5 Cloning ...............................................................................................30
2.2.5.1 Restriction Reactions..................................................................30
2.2.5.2 AlkalinePhosphatase Treatment..................................................30
2.2.5.3 Ligation Reactions......................................................................30
2.2.6 Transformation of chemically competent bacteria .....................................30
2.2.6.1 Preparation of chemically competent E. coli...................................30
2.2.6.2 Transformation of cells ...............................................................31
2.2.7 Mutagenesis.........................................................................................32
2.2.8 Plasmid Purification...............................................................................32
2.2.9 Isolation of genomic DNAfrom cells and tissue samples .............................32
2.2.10 RNA Isolation .....................................................................................32
2.2.11 Quantification of DNA and RNA Samples ................................................32
2.2.12 ReverseTranscription (RT) PCR ..........................................................32
2.2.13 Quantitative EndPoint PCR..................................................................33

III INDEX

2.3 CELL BIOLOGY............................................................................................34
2.3.1 General Cell Culture..............................................................................34
2.3.2 Cell Culture Media and Supplements .......................................................34
2.3.3 Thawing and Freezing of Cells ................................................................34
2.3.4 Transfection.........................................................................................34
2.3.4.1 Transfection of DNA ...................................................................35
2.3.4.2 Transfection of siRNA .................................................................35
2.3.5 Nuclear staining ...................................................................................35
2.3.6 Preparation of whole cell lysates....................