3.1 Antibodies. . . 22
3.2 Secondary antibodies and isotype controls. . . 23
3.3 Dyes. . . 23
3.4 Primers. . . 24
3.5 Chemicals. . . 25
3.6 Kits. . . 28
3.7 Buffers and stock solutions. . . 28
3.8 Buffers and solutions for microarray. . . 31
3.9 Cell lines. . . 32
3.10 Media. . . 34
3.11 Laboratory animals. . . 34
3.12 Used laboratory equipment. . . 35
3.13 Disposable plastic ware and other disposables. . . 37
3.14 Providers. . . 38
4.1 Master mix dsDNA for microarray. . . 57
4.2 Master mix for generating labelled cRNA for microarray. . . 57
4.3 Fragmentation mix for one 4× 44K microarray. . . 60
4.4 Master mix reverse transcription. . . 61
4.5 Standard PCR reaction. . . 62
4.6 Standard PCR programme. . . 62
4.7 Master mix quantitative real-time PCR. . . 63
4.8 Quantitative real-time PCR programme. . . 64
5.1 Analysis of microarray data. . . 70
5.2 Selected genes for validation of microarray data by quantitative real-time PCR. . . 70
B.1 Quantification of labelled amplified cRNA for microarray. . . 171
B.2 All genes found to be regulated in micoarray analysis. . . 171
Acknowledgements
I would like to thank Prof. Dr. Detlef Doenecke and Prof. Dieter Heineke for being official reviewers of my thesis. I am furthermore thankful to PD Dr. Lutz Walter, PD Dr. Michael Hoppert, Prof. Dr. Sigrid Hoyer-Fender, and Prof. Dr. Peter Kappeler, who completed the committee for the evaluation of my thesis.
I would like to thank my supervisor PD. Dr. Ralf Dressel for giving me the opportunity to work on the topic of HSP70 and sulphatases 1 and 2 and their role in apoptosis.
Although, he should actually never had employed me, as he explained to me a few weeks ago with a friendly grin on his face referring to (Djerassi 1993), I am very thankful to complete my doctoral thesis in his lab. He is always open for discussions, has great ideas, and is always very accurate and reliable.
Leslie Elsner is the best technical assistant I have ever known. Not only for her practical skills and insider tricks using our laboratory equipment, but also for her personality, for showing me that there is a life outside science, and for the impressive demonstration that once one does enough sports, one can eat as much chocolate as one wants ;) Thanks.
My 3 years of laboratory work, reading papers, writing abstracts, proof-reading ab-stracts, designing posters and primers, and finally writing the thesis, and many other things, would not have been as efficient, well structured, funny, informative, and excited if there would not have been my nice colleagues Dr. Vijayakumar "VJ" Muppala, Dr. Peter Novota, and my special Kinderzimmerkollegin Miriam "Wie konnte das denn passieren"
Ensslen. Thank you for always being there for me.
Furthermore, I want to thank the GRK1034 for letting me participate in their edu-cational programme. I learned a lot. I am very thankful to all other doctoral students from the GRK1034 but especially Dr. Ines Ecke and Lars Böckmann for sharing and dis-cussing scientific problems but also feelings with me, one can just understand while doing a scientific doctoral thesis.
To avoid that water bombs are thrown at me when I will kiss the Gänseliesl, I also need to thank Dr. Gabriela Salinas-Riester and Lennart Opitz for their extraordinary help while performing the microarray analysis and the quantitative real-time PCR. I kept my promise!
I additionally want to thank Prof. Dr. Thomas Dierks for providing us with the Sulf MEFs, Prof. Dr. Christopher Froelich for giving us the labelled GrB, Dr. Tim Seidler for
Acknowledgements
allowing us to use his AdV and to work in the S2-laboratory, Anne Schmöle for organising papers I could not get, and Dr. Fridtjof Nußbeck for the statistical analysis using ANOVA.
Apart from that, I am deeply grateful to my best friends Franziska Jurk and Stephan Janz. Without your mental and technical support, lending me your ear for all my problems and giving me a shoulder to cry on, encouraging me to go on but also distracting me if neccessary, and making me happy, this thesis might not have been finished, at least not with having in mind that I am not alone. In short, thanks for your friendship.
I am of course also very grateful to my parents Bedri and Marion Demiroglu, who tried to support me always as good as they could, which is particularly not as easy due to the distance between Cologne and Göttingen, but I know for sure, that your thoughts were always with me.
Last but not least I want to thank my boyfriend Gunnar Nußbeck. I know that doing a doctoral thesis was not an easy thing for me, but it must have been even harder for you.
All my emotions, excitement, frustration, anger, stress, and happiness strongly correlated with the results of my experiments, but nevertheless you were always there for me. You gave me the energy and strength to complete this thesis, you teached me to be a “tank-rhino”, which was not always that easy, and you always believed in me. Thanks, I love you.
This work was financed by the DFG grant DR 394/2-3 and the DFG graduate college 1034: Genetic polymorphisms in Oncology.
Appendix A
Quantitative real-time PCR
A.1 Dissociation curves
Figure A.1: Dissociation curve for theHsp70primerDepicted is a screenshot of the dissociation curves of the cDNA of Ge-tet-2 triplicate amplified with theHsp70primer. The x-axis shows the temperature in◦C and the y-axis the derivative of the raw data of the SYBR green fluorescence. The dissociation curve is a quality control for the qRT-PCR, as it ensures an equal amplification of the products with one primer, if all curves overlap, as it is the case here.