Investigation into the cellular radiosensitivity of the LEC rat and analysis of candidate genes [Elektronische Ressource] / vorgelegt von Alesia Ivashkevich

Alesia Ivashkevich

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

INVESTIGATION INTO THE CELLULAR RADIOSENSITIVITY

OF THE LEC RAT

AND ANALYSIS OF CANDIDATE GENES
















Dissertation
zur Erlangung des Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München
Vorgelegt von
Alesia Ivashkevich aus Aspa, Russland
am 16 Juni 2008




2008

































1. Berichterstatter: Prof. Dr. F. Eckardt-Schupp

2. Berichterstatter: PD Dr. Anna A. Friedl

Betreuer: Dr. M. Rosemann

Tag der mündlichen Prüfung: 19 Dezember 2008








ii
Ehrenwörtliche Versicherung



Ich versichere, dass die Dissertation von mir selbständig und ohne unerlaubte Hilfsmittel
angefertigt wurde. Die Dissertation habe ich keiner anderen Fakultät oder Universität zur
Prüfung vorgelegt. Außerdem habe ich noch nicht versucht, eine Dissertation einzureichen
oder mich einer Doktorprüfung zu unterziehen.


Die Arbeit wurde von Dr. Michael Rosemann, Helmholtz Center Munich, Institute for Radiation
Biology, Neuherberg, betreut.

Melbourne, May 2008


Alesia Ivashkevich














iii


The most beautiful thing we can experience is the mysterious. It is the source of all true
art and science
Albert Einstein (1879-1955)


The important thing in science is not so much to obtain new facts as to discover new ways
of thinking about them
Sir William Bragg (1862 – 1942)





…dedicated to my family





















iv


ACKNOWLEDGEMENTS




I would like to thank everyone who was involved in the successful completion of this work.


The Institute of Pathology for providing me the possibility to conduct my PhD Thesis and
gain a valuable experience in scientific research during this time.

Special thanks to:

Prof. Dr. Friederike Eckardt-Schupp for scientific supervision, many fruitful discussions
and encouragement.

Prof. Dr. Michael J. Atkinson, Dr. Michael Rosemann and Prof. Dr. Summer for providing
an interesting topic for my project, which attracts great interest in the field of radiobiology
and their support during my time at the Institute.

Furthermore, I would like to thank Dr. Anna Friedl, Dr. Beisker, Dr. Maria Gomolka,
Dr. Tom Meindl, and Dr. Guido Drexler for many discussions and comments on my work.

Josef Lichtmanneger, Bahar Sanli-Bonazzi for their help with lots of RT-PCR and
sequencing and even more important, for cheering me up.

All other colleagues from the Institutes of Pathology, Toxicology and Radiobiology, at the
GSF for their collaboration and professional help.

Last but not least I would like to thank my family, and friends for their cordial
encouragement and emotional involvement in the process of my experimental work and
writing of my thesis.












v OUTLINE



OUTLINE ...................................................................................................................................................................VI
ABSTRACT............................................................................................................................................................. - 1 -
I. INTRODUCTION........................................................................................................................................ - 3 -
1.1 RADIOSENSITIVITY ............................................................................................................................- 4 -
1.1.1 Radiation effects in tissues and organs: stochastic versus deterministic and acute versus chronic
effects............................................................................................................................................... - 4 -
1.1.2 Effects of acute total body irradiation at the level of the organism.
Definition of the Lethal Dose in human and rodents ....................................................................... - 5 -
1.1.3 Physical and biological effects of ionizing radiation at the cellular level ........................................ - 6 -
1.1.4 Experimental end-points to measure cellular sensitivity in vitro ..................................................... - 8 -
1.1.5 Radiotherapy related aspects of radiation sensitivity ....................................................................... - 9 -
1.1.6 Molecular basis of radiosensitivity ................................................................................................ - 10 -
1.1.7 Experimental models of radiosensitivity........................................................................................ - 13 -
1.2 LEC RAT – A RODENT MODEL OF HUMAN DISEASES.........................................................................- 14 -
1.2.1 Presence of pathogenic mutations.................................................................................................. - 15 -
1.2.2 Radiosensitivity in LEC rat............................................................................................................ - 15 -
1.2.3 Sensitivity to radiation and chemical agents in strains related to LEC rat - LEA and LE rats....... - 22 -
1.3 THE AIMS OF THE STUDY..........................................................................................................- 23 -
II. MATERIALS AND METHODS............................................................................................................... - 24 -
2.1 MATERIALS......................................................................................................................................- 24 -
2.1.1 Equipment...................................................................................................................................... - 24 -
2.1.2 Consumable materials and chemicals ............................................................................................ - 24 -
2.1.3 Rat strains....................................................................................................................................... - 26 -
2.1.4 Kits and reagents............................................................................................................................ - 26 -
2.1.5 Length standards ............................................................................................................................ - 26 -
2.1.6 Enzymes......................................................................................................................................... - 26 -
2.1.7 Oligonucleotides ............................................................................................................................ - 27 -
2.1.8 Antibodies...................................................................................................................................... - 29 -
2.1.9 Buffers............................................................................................................................................ - 29 -
2.1.10 Software ......................................................................................................................................... - 31 -
2.1.11 Bioinformatic resources ................................................................................................................. - 31 -
2.2 METHODS ........................................................................................................................................- 32 -
2.2.1 Rat strains....................................................................................................................................... - 32 -
2.2.2 Establishment and cultivation of fibroblasts .................................................................................. - 32 -
2.2.3 The experimental systems.............................................................................................................. - 33 -
2.2.4 Growth curves................................................................................................................................ - 34 -
2.2.5 Clonogenic survival assay.............................................................................................................. - 35 -
2.2.6 Pulsed field gel electrophoresis...................................................................................................... - 38 -
2.2.7 Analysis of γH2AX foci..................................................................................................