Molecular mediators of alveolarization [Elektronische Ressource] / by Jens-Christian Wolff

Wolff Rajzman, Jens-Christian

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2010
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 2 Mo

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MOLECULAR MEDIATORS OF ALVEOLARIZATION
JENS-CHRISTIAN WOLFF
INAUGURAL DISSERTATION
submitted to the Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree of the Facultiesédition scientifique
VVB LAUFERSWEILER VERLAG of Veterinary Medicine and Medicine
VVB LAUFERSWEILER VERLAG of the Justus Liebig University Giessen
STAUFENBERGRING 15 ISBN: 978-3-8359-5611-7
D-35396 GIESSEN
Tel: [phone] Fax: -5599890
[e-mail]
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 6 1 1 7
édition scientifique
VVB LAUFERSWEILER VERLAG
VVB
JENS-CHRISTIAN WOLFF MOLECULAR MEDIATORS OF ALVEOLARIZATIONDas Werk ist in allen seinen Teilen urheberrechtlich geschützt.
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1. Auflage 2010
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édition scientifique
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Molecular Mediators of Alveolarization







INAUGURAL DISSERTATION
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen





by

Jens-Christian Wolff
of Erlangen, Germany





Giessen, 2010



From the Department of Internal Medicine, Medical Clinic II
(Director: Prof. Dr. med. Werner Seeger)
of the Faculty of Medicine of the Justus Liebig University Giessen





















First Supervisor and Committee Member: Prof. Dr. Werner Seeger
Second Supervisor and Committee Member: Prof. Dr. Johannes C. Schittny (Bern, CH)
Committee Members: Prof. Dr. Wolfgang Kummer
Prof. Dr. Christiane Herden


rdDate of Doctoral Defense: July 23 , 2010

I Index
I Index.........................................................................................................................1
II List of tables and figures .......................................................................................... 4
III Abbreviations...........................................................................................................5
1 Introduction..............................................................................................................7
1.1 Lung functions and structure ............................................................................ 7
1.2 Regular lung development................................................................................ 9
1.3 Modifyers of lung development ..................................................................... 11
1.3.1 Varying concentrations of endogenous molecules ................................. 11
1.3.2 Extrapulmonary and environmental influences...................................... 13
1.4 Experimental modifications of lung growth................................................... 15
1.4.1 Glucocorticoid treatment........................................................................16
1.4.2 Calorie restriction and refeeding ............................................................ 16
1.4.3 Tracheal occlusion..................................................................................18
1.4.4 Compensatory lung growth .................................................................... 19
1.5 The present study: Intentions and technical approaches ................................ 21
1.6 Aims of the study............................................................................................ 23
2 Material and Methods............................................................................................. 24
2.1 Animal surgery...............................................................................................24
2.1.1 Pneumonectomy.....................................................................................24
2.1.2 Removal of (residual) lungs ................................................................... 25
2.2 Generation of array data ................................................................................. 25
2.2.1 Experimental design...............................................................................26
2.2.2 RNA extraction.......................................................................................26
2.2.3 Labelling.................................................................................................26
2.2.4 Hybridization, scanning and image analysis .......................................... 27
2.2.5 Statistical analysis..................................................................................28
2.3 Real-time PCR................................................................................................28
2.4 Western blot....................................................................................................29
2.5 Cloning...........................................................................................................30
2.6 In-situ hybridization.......................................................................................31
2.6.1 Generation of probes..............................................................................31
2.6.2 Sampling.................................................................................................32
1
2.6.3 Hybridization..........................................................................................32
2.7 Cell culture.....................................................................................................33
2.7.1 Culture conditions..................................................................................33
2.7.2 Isolation of murine AECs II ................................................................... 34
2.7.3 Transfection of cultured cells ................................................................. 34
2.8 Functional studies...........................................................................................35
2.8.1 Proliferation............................................................................................35
2.8.2 Adhesion assay.......................................................................................35
2.8.3 Migration assay......................................................................................36
2.8.4 Detection of apoptosis ............................................................................ 36
2.9 Immunofluorescence staining.........................................................................36
3 Results....................................................................................................................38
3.1 Array analysis.................................................................................................38
3.2 Top-regulated genes of each model................................................................ 39
3.3 Intersection: newborn and pneumonectomy mice.......................................... 41
3.4 Validation of array data .................................................................................. 43
3.4.1 Real-time PCR........................................................................................43
3.4.2 Western blot............................................................................................44
3.5 Localization of mRNA: in-situ hybridizations ............................................... 45
3.6 First candidate gene: Egr1 .............................................................................. 46
3.6.1 Proliferation46
3.6.2 Secreted mediators..................................................................................47
3.6.3 Apoptosis................................................................................................48
3.6.4 Migration49
3.6.5 Adhesion.................................................................................................49
3.6.6 Localization............................................................................................50
3.7 Second candidate gene: Stefin A1.................................................................. 52
3.7.1 Quantification.........................................................................................52
3.7.2 Localization53
3.7.3 Functional studies...................................................................................54
4 Discussion.......................