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Engineering a synthetic p53-Mdm2 network in budding yeast [Elektronische Ressource] / presented by Barbara Di Ventura

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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 Engineering a synthetic p53-Mdm2 network in budding yeast presented by Diplom: Barbara Di Ventura Born in: Latina, Italia 2006 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 Diplom: Barbara Di Ventura Born in: Latina, Italia Engineering a synthetic p53-Mdm2 network in budding yeast Referees: Prof. Dr. Michael Knop Prof. Dr. Karsten Rippe A mamma, papa’ e Chicca "Experience is the name everyone gives to their mistake" Oscar Wilde Acknowledgments If I am here, writing the acknowledgements section in my phd thesis, I owe it first of all to my boss, Luis.

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Published 01 January 2007
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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








Engineering a synthetic p53-Mdm2
network in budding yeast






presented by

Diplom: Barbara Di Ventura
Born in: Latina, Italia





2006





















































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

Diplom: Barbara Di Ventura
Born in: Latina, Italia



















Engineering a synthetic p53-Mdm2
network in budding yeast









Referees: Prof. Dr. Michael Knop
Prof. Dr. Karsten Rippe

































A mamma, papa’ e Chicca





















































"Experience is the name everyone gives to their mistake"
Oscar Wilde
Acknowledgments

If I am here, writing the acknowledgements section in my phd thesis, I owe it first
of all to my boss, Luis. I am grateful that he wasn’t discouraged with the idea of getting
an engineer to work in his lab. On the opposite, he often told me that it was an advantage
to be naïve and ask the simplest questions. He offered me the opportunity to do my own
experiments, to make many mistakes, to develop my own way to do research. Most of all,
he has always comforted me in moments of discouragement, assuring me that lack of
results or experiments gone wrong are an inevitable feature of research. Luis, your
positive attitude, your extreme generosity, your humanity, make of you an example to
follow and give me hope that one can be a scientist and lead a happy life at the same
time!
Among the many people who have helped me with the basics of molecular
biology and that have patiently listened to me talking about the many doubts or troubles
related to my project, I first would like to thank Massimiliano. Despite his being totally
unrelated to my work, he has always found the time to explain me things, to give me
advice, without ever making me feel stupid or incompetent. I will not forget that night
when he sat with me for hours and together we went through my contradicting data and
finally came out with the idea that my protein could be sumoylated…Thanks Massi, you
do know what “sharing knowledge” means!
I am deeply grateful to Kostas, who accepted (despite the short notice!) to read
the entire thesis and gave me precious advices for improving it. Kostas, I thanked you at
least millions time during our email exchange of revisions of this thesis, but it’s never
going to be enough to express to you my gratitude!
These four years of endless nights in the lab would have not been the same
without my beloved friend, Caro. She is the most amazing person I have ever met.
Intelligent, genuine, stubborn and contradictory at times, but always, always willing to
help. With her I laughed and fought, cried and played. Caro, if we managed to stay
friends after our review-writing period, I am confident that we will be forever ☺
I thank all the people in the lab – past and present – for having created a
wonderful atmosphere to work in. Especially Mark and Kostas, with whom I shared the centrifuge, the fridge, the buffers, but also jokes, scientific discussions and music in the
absence of Christina. Remember guys, the time has come to push the button!!
JB, Paulo, Martin, Rob, Markus, Tom, Nga and The Fishpuddings: thanks for the
great time spent together playing and singing! Our rehearsals and concerts have been the
highlight of my life in Heidelberg indeed.
Fabiana, my dear, you have rescued me many times when I was sinking into the
library between introduction and results. Maybe we were drinking too many coffees
together, getting a bit distracted…but it was good to keep the mood high ☺
For the constant supply of materials, the yeast strains, the plasmids, the
antibodies, all the protocols, I am grateful to the entire Knop lab, especially Christof and
Michael. You have been extremely generous, helpful and patient, even when I was
coming ten times a day to ask questions…
I thank Charlotta Funaya and Claude Antony for their commitment in the electron
microscopy analysis. Thanks to Stefan and Arne who assisted me with the DeltaVision (I
still believe this microscope has something against me…), to Vladimir and Tomi who did
the microarrays analysis, to Andy for the FACS, to Sigfried Labeit for the human cDNA
libraries, to the Böettcher lab for the protease inhibitors.
I am also grateful to the members of my thesis advisory committee, Peer, Karsten,
Francois and Michael, who tried many times, for my own good, to persuade me to change
project. In the end, I am happy I have persisted ☺
I thank all the wonderful friends with whom I shared these years in EMBL. I
would like to mention you all, guys, but it would end up in almost as many pages as the
whole thesis!

Last but not least, I’d like to express my gratitude to those who indirectly helped
me getting this work done, bringing joy and happiness into my life: mamma, papa’,
Chicca, my fluffy love-addicted bunny Caffelatte, the most wonderful cat in the whole
world Chris, and my living-miracle Robert, who makes every day so worth living. Contents

Dedication
Acknowledgments
Contents
Abbreviations
Summary
Zusammenfessung
Aim of this study

Chapter One Introduction
Synthetic biology 2
Synthetic oscillators 9
The p53 natural oscillator 10
The p53-Mdm2 feedback loop 12
p53 regulation 13
Mmd2 15
Mono- versus polyubiquitylation 16
Redundancy in the p53 network 17
ARFOther players in p53 regulation: HAUSP, MdmX, p14 and
nucleolar proteins 20
p53 sumoylation 21
Regulation of protein function through post-translational modifications 22
Eukaryotic protein degradation and the ubiquitin pathway 23
Ubiquitin 28
E1, the ubiquitin-activating enzyme 30
E2, the ubiquitin-conjugating enzyme 32
E3, the ubiquitin ligase 34
Sumoylation 37
Mathematical Modelling of biological networks 42
Qualitative modelling 43 Quantitative modelling 44
Space in modelling 46

Chapter Two Results
Results Part One 47
Mathematical-formalism-independent errors 48 alism-dependent errors 50
Choice of formalism 50 Effect of localization of species on cellular processes 51
Continuous versus discrete concentrations 53
Results Part Two 55
Building the p53/Mdm2 synthetic oscillator in budding yeast
(Saccharomyces cerevisiae) 56
p53-ECFP is stable when expressed alone in yeast 56
p53-ECFP does not interfere with yeast gene expression
under normal growth conditions 57
p53-ECFP is diffuse throughout the yeast cell nucleus and
can be found at the septin ring 59
Mdm2-EYFP is degraded in yeast 61
Mechanisms underlying Mdm2-EYFP degradation in yeast 62
Mdm2-EYFP degradation is proteasome-dependent 64
Mdm2-EYFP localizes to the yeast nucleus and to one or
several nuclear speckles 64
Mdm2-EYFP localization is very dynamic 67
p53-ECFP is not degraded in the presence of Mdm2-EYFP 69
p53-ECFP and Mdm2-EYFP interact and co-localize to a dot
inside the yeast nucleus 70
On the road to degradation, first attempt: adding the
human ubiquitin chain elongation factor p300 to the network 75
On the road to degradation, second attempt: Removing
the fluorescent proteins from p53 and Mdm2 76

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