Novel fluorescent sensors for dynamic intracellular signalling [Elektronische Ressource] / presented by Andreas Schleifenbaum

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

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













presented by

Diplom-Chemiker Andreas Schleifenbaum
born in Munich, Germany

Oral-examination: ________________



Title:

Novel Fluorescent Sensors for Dynamic
Intracellular Signalling

















Referees:

Prof. Dr. Nils Metzler-Nolte, University of Heidelberg
PD Dr. Carsten Schultz, University of Dortmund/EMBL Heidelberg

Inaugural-Dissertation
zur
Erlangung der Doktorwürde
der Naturwissenschaftlich-Mathematischen Gesamtfakultät der
Ruprecht-Karls-Universität Heidelberg
















vorgelegt von

Diplom-Chemiker Andreas Schleifenbaum
aus München

Tag der mündlichen Prüfung: ________________



Titel der Arbeit:

Neue Fluoreszenzsonden für intrazelluläre
Signalwege


















Gutachter:

Prof. Dr. Nils Metzler-Nolte, Universität Heidelberg
PD Dr. Carsten Schultz, Universität Dortmund/EMBL Heidelberg
























Hiermit bestätige ich, dass ich die Arbeit selbständig verfasst und keine anderen als
die angegebenen Quellen und Hilfsmittel benutzt habe.
Heidelberg, im September 2005




______________________________________
Andreas Schleifenbaum




















Wenn man’s nur probiert so geht’s,
das heißt mitunter – doch nicht steht’s.
Wilhelm Busch









Table of contents Table of contents
Table of contents _____________________________________________ VII
Abstract _____________________________________________________ X
Zusammenfassung____________________________________________XIII
Abbreviations ________________________________________________XVI
1 Introduction_______________________________________________ 1
1.1 Phosphate esters regulate cells____________________________ 2
1.2 Protein kinase C _______________________________________ 2
1.3 Pleckstrin _____________________________________________ 8
1.4 Fluorescent proteins ____________________________________ 9
1.5 FRET _______________________________________________ 11
1.6 Fluorescent probes in live cell imaging _____________________ 13
2 Aim of the project _________________________________________ 18
3 Results _________________________________________________ 20
3.1 Design and cloning of KCP-1_____________________________ 21
3.2 In vitro Experiments____________________________________ 22
3.3 Characterization of KCP-1 in live cell experiments ____________ 25
3.4 Technical aspects _____________________________________ 29
3.5 Difficulties with live cell imaging___________________________ 35
3.6 Engineering the probe __________________________________ 35
3.7 Applications of KCP-1 52
4 Discussion ______________________________________________ 62
4.1 Introduction __________________________________________ 63
4.2 Design of the probe ____________________________________ 63
4.3 Fluorimeter experiments ________________________________ 63
4.4 Characterization of KCP-1 _______________________________ 64
4.5 Comparison of KCP-1 and CKAR _________________________ 65
4.6 KCP-1 mutants _______________________________________ 67
4.7 Drug screening possibilities ______________________________ 73
4.8 Multiparameter imaging _________________________________ 74
5 Outlook _________________________________________________ 78
6 Experimental part _________________________________________ 81
6.1 Material and assets ____________________________________ 82
6.2 Cloning and cell culture 87
6.3 DNA spotting 98
VIII Table of contents
6.4 In vitro experiments ____________________________________ 99
6.5 Microscopy__________________________________________ 100
6.6 Image processing 102
7 References _____________________________________________ 104
8 Appendix_______________________________________________ 114
8.1 Plugin and macro scripts _______________________________ 115
8.2 Publications _________________________________________ 128
Acknowledgements __________________________________________ 129



IX Abstract