The WNT/β-catenin [WNT-beta-catenin] pathway [Elektronische Ressource] : profibrotic signaling in fibroblasts in idiopathic pulmonary fibrosis / vorgelegt von Monika Kramer

Kramer C., Monika

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• Gesundheit

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2010
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

The WNT/β-catenin pathway -
Profibrotic signaling in fibroblasts in
Idiopathic Pulmonary Fibrosis
MONIKA KRAMER
édition scientifique
VVB LAUFERSWEILER VERLAG
INAUGURALDISSERTATION zur Erlangung des Grades eines Doktors der Medizin
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15 ISBN: 978-3-8359-5627-8 des Fachbereichs Medizin der Justus-Liebig-Universität GießenD-35396 GIESSEN
Tel: [phone] Fax: -5599890
[e-mail]
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 6 2 7 8
édition scientifique
VVB LAUFERSWEILER VERLAGVVB
MONIKA KRAMER WNT/β- CATENIN PATHWAY IN IPFDas Werk ist in allen seinen Teilen urheberrechtlich geschützt.
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1. Auflage 2010
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© 2010 by VVB LAUFERSWEILER VERLAG, Giessen
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édition scientifique
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: [phone] Fax: [phone]
email: [e-mail]
www.doktorverlag.de
The WNT/ β-catenin pathway
Profibrotic signaling in fibroblasts
in Idiopathic Pulmonary Fibrosis








INAUGURAL-DISSERTATION
zur Erlangung des Grades eines
Doktors der Medizin
des Fachbereichs Medizin der
Justus-Liebig-Universität Gießen









vorgelegt von




Monika Kramer
aus Balve




Gießen 2009
̶Aus dem Zentrum für Innere Medizin
Medizinische Klinik II,
Direktor: Prof. Dr. med. W. Seeger
Universitätsklinikum Gießen und Marburg GmbH Standort Gießen








































Gutachter: Prof. Dr. O. Eickelberg

Gutachter: Prof. Dr. K. Preissner

Tag der Disputation: 31.08.2010





























Meiner Familie















Teile der vorliegenden Arbeit wurden in folgenden Artikeln publiziert:

Königshoff M., Balsara N., Pfaff E. M., Kramer M., Chrobak I., Seeger W., Eickelberg O.
Functional Wnt signaling is increased in idiopathic pulmonary fibrosis. PLOS ONE, e2 142
(2008)

Königshoff M., Kramer M., Balsara N., Wilhelm J., Amarie O. V., Jahn A., Rose F., Fink L.,
Seeger W., Schaefer L., Gunther A., Eickelberg O. WNT1inducible signaling protein1
mediates pulmonary fibrosis in mice and is upregulated humans with idiopathic pulmonary
fibrosis. J ClinInvest 119, 772787 (2009)


Vorträge und Poster:

Posterpräsentation, PneumoUpdate, Innsbruck. 2007. Kramer M., Königshoff M.,
Eickelberg O. WNT/WISP1 signaling in lung fibroblasts: Novel profibrotic mediators in IPF.

Vortrag, Sektion Zellbiologie der Deutschen Gesellschaft für Pneumologie und
Beatmungsmedizin e.V. 2008. Kramer M., Königshoff M., Eickelberg O. WNT/WISP1
Signaltransduktion in Lungenfibroblasten: Neue profibrotische Mediatoren in der
idiopathischen pulmonalen Fibrose.
I Content I
I Content
I Content......................................................................................................................................I
II Figures ..................................................................................................................................IV
III Tables ..................................................................................................................................IV
IV Abbreviations ....................................................................................................................... V
V Summary X
VI Zusammenfassung...............................................................................................................XI
1. Introduction ............................................................................................................................ 1
1.1 Interstitial lung diseases ................................................................................................... 1
1.2 Idiopathic pulmonary fibrosis .......................................................................................... 2
1.2.1 Epidemiology ............................................................................................................ 2
1.2.2 Clinical and histopathological features ..................................................................... 2
1.2.3 Pathomechanisms of idiopathic pulmonary fibrosis ................................................. 5
1.3 WNT signaling ................................................................................................................. 8
1.3.1 WNT family of signaling molecules – classification and physiology ...................... 8
1.3.2 The WNT/βcatenin signaling pathway .................................................................... 8
1.3.2.1 WNT/βcatenin signaling in diseases............................................................... 10
1.3.2.2 WNT/βcatenin signaling in the lung and in IPF ............................................. 11
1.3.3 WNT1inducible signaling protein 1 in IPF............................................................ 12
2. Hypothesis and aim of the study .......................................................................................... 14
3. Materials and Methods ......................................................................................................... 16
3.1 Materials......................................................................................................................... 16
3.1.1 Cells / Cell line........................................................................................................ 16
3.1.2 Machines / Software................................................................................................ 16
3.1.3 Reagents .................................................................................................................. 16
3.1.3.1 Chemicals and biochemicals ............................................................................ 16
3.1.3.2 Ligands, recombinant proteins .............. 17
3.1.3.3 qRTPCR reagents............................................................................................ 18
3.1.3.4 Antibodies ........................................................................................................ 18
3.1.3.5 Buffer .............................................................................................................. 18
3.1.3.6 Gels................................................................................................................... 20
3.1.4 Kits .......................................................................................................................... 21
3.2 Methods 21 I Content II
3.2.1 Cell culture .............................................................................................................. 21
3
3.2.2 [ H]thymidine proliferation assay.......................................................................... 21
3.2.3 Protein extraction and quantification ...................................................................... 22
3.2.4 SDS polyacrylamide gel electrophoresis................................................................. 22
3.2.5 Immunoblotting....................................................................................................... 23
3.2.6. Densitometry .......................................................................................................... 23
3.2.7 Sircol Collagen Assay ............................................................................................. 24
3.2.8 RNA isolation and measurement............................................................................. 24
3.2.9 cDNA synthesis....................................................................................................... 25
3.2.10 Quantitative reverse transcription polymerase chain reaction (qRTPCR)........... 25
3.2.10.1 Measurement of fluorescence with SYBR Green I........................................ 26
3.2.10.2 Primerdesign and efficiency test .................................................................... 27
3.2.10.3 Melting curve analysis ................................................................................... 28
3.2.10.4 Quantification and