Functional analysis of RACK1 as a novel interaction partner of BMPRII in pulmonary arterial hypertension [Elektronische Ressource] / vorgelegt von Anna Zakrzewicz

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2007
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 3 Mo

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FUNCTIONAL ANALYSIS OF RACK1 AS A NOVEL
INTERACTION PARTNER OF BMPRII IN
PULMONARY ARTERIAL HYPERTENSION
ANNA ZAKRZEWICZ
INAUGURAL-DISSERTATION
zur Erlangung des Grades eines
édition scientifique Doktors der Humanbiologie
VVB LAUFERSWEILER VERLAG des Fachbereichs Medizin der
VVB LAUFERSWEILER VERLAG Justus-Liebig-Universität GießenISBN 3-8359-5186-6
STAUFENBERGRING 15
D-35396 GIESSEN
Tel: [phone] Fax: -5599890
[e-mail]
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 1 8 6 0
édition scientifique
VVB VVB LAUFERSWEILER VERLAG
ANNA ZAKRZEWICZ RACK1 IN PAH. Das Werk ist in allen seinen Teilen urheberrechtlich geschützt.
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1. Auflage 2007
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1 Edition 2007
© 2007 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
VVB LAUFERSWEILER VERLAG
édition scientifique
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: [phone] Fax: [phone]
email: [e-mail]
www.doktorverlag.de
Functional analysis of RACK1 as a
novel interaction partner of BMPRII
in pulmonary arterial hypertension



Inaugural-Dissertation
zur Erlangung des Grades eines
Doktors der Humanbiologie
des Fachbereichs Medizin der
Justus-Liebig-Universität Gießen




vorgelegt von


Anna Zakrzewicz
aus Skarzysko-Kamienna, Polen



Gießen, 2006
Aus dem Zentrum für Innere Medizin
des Klinikums der Justus-Liebig-Universität Gießen
Director: Prof. Dr. W. Seeger



















Gutachter: Prof. Dr. W. Seeger / Dr. O. Eickelberg

Gutachter: PD Dr. S. Kanse




Tag der Disputation: 4. Juni 2007

Table of contents I


I Table of contents
I TABLE OF CONTENTS .................................................................................................I
II LIST OF FIGURES.................................................................................................... VII
III LIST OF TABLES ..................................................................................................... IX
IV ABBREVIATIONS ..................................................................................................... X
III SUMMARY.............................................................................................................. XIII
IV ZUSAMMENFASSUNG ..........................................................................................XIV
1 INTRODUCTION......................................................................................................1
1.1 Pulmonary arterial hypertension .......................................................................1
1.1.1 Characteristics of pulmonary arterial hypertension.........................................1
1.1.2 Histopathology of pulmonary arterial hypertension.........................................2
1.1.3 Genetic basis of pulmonary arterial hypertension...........................................3
1.1.4 Animal models of pulmonary arterial hypertension.........................................4
1.1.4.1 The monocrotaline rat model of pulmonary arterial hypertension................4
1.1.4.2 The hypoxia-induced model of pulmonary arterial hypertension .................5
1.1.4.3 Transgenic animals ....................................................................................5
1.2 BMP signalling ....................................................................................................6
1.2.1 Bone morphogenetic proteins ........................................................................6
1.2.2 BMP receptors...............................................................................................7
1.2.3 The BMP signalling pathways ........................................................................9
1.2.3.1 Smad-dependent pathways........................................................................9
1.2.3.2 BMP-MAPK dependent pathway ..............................................................11
1.3 BMP signalling in lung development and homeostasis .................................12
1.4 BMPRII and pulmonary arterial hypertension.................................................14
1.4.1 Genomic structure and function of BMPRII ..................................................14 Table of contents II


1.4.2 BMPR2 mutations in pulmonary arterial hypertension patients.....................15
1.4.3 Functional consequences of BMPR2 mutations...........................................16
1.4.3.1 Loss of transcriptional activitiy..................................................................16
1.4.3.2 Decreased ligand binding ability...............................................................17
1.4.3.3 Failure of BMPRII trafficking to the plasma membrane.............................17
1.4.3.4 Activation of Smad-independent BMP signalling pathways.......................17
1.4.3.5 Increased of BMP signalling .....................................................................18
1.4.3.6 Down-regulation of BMPRII expression....................................................18
1.4.3.7 Failure of antiproliferative effects on vascular cells...................................18
1.5 Experimental design and aim of the project ...................................................19
2 MATERIALS AND METHODS ...............................................................................20
2.1 Materials ............................................................................................................20
2.1.1 Equipment....................................................................................................20
2.1.2 Reagents .....................................................................................................21
2.1.3 Cell Lines.....................................................................................................23
2.1.3.1 Mammalian cell lines ................................................................................23
2.1.3.2 Yeast cells................................................................................................23
2.1.3.3 Prokaryotic cells .......................................................................................23
2.1.4 Animals........................................................................................................23
2.1.4.1 A monocrotaline rat model of pulmonary arterial hypertension .................23
2.1.4.2 Hypoxia mouse model of pulmonary arterial hypertension........................24
2.2 Methods.............................................................................................................24
2.2.1 RNA isolation ...............................................................................................24
2.2.2 Reverse Transcription..................................................................................24
2.2.2.1 RT - Mix....................................................................................................25
2.2.3 The Polymerase Chain Reaction (PCR).......................................................25
2.2.3.1 PCR - Mix.................................................................................................25
2.2.3.2 PCR program ...........................................................................................26
2.2.4 Site-directed mutagenesis............................................................................26
TM2.2.4.1 The QuikChange PCR-Mix....................................................................27
2.2.5 Gel electrophoresis......................................................................................27
2.2.5.1 Agarose gel electrophoresis .....................................................................28
2.2.5.2 SDS polyacrylamide gel electrophoresis (SDS-PAGE).............................28 Table of contents III


2.2.6 Recombinant DNA technology .....................................................................29
2.2.6.1 PCR product purification...........................................................................30
2.2.6.2 Ligation of PCR products into the pGEM-T Easy Vector...........................30
2.2.6.3 Ligation-Mix..............................................................................................30
2.2.7 Subcloning into expression vectors..............................................................30
2.2.7.1 DNA digestion using restriction endonucleases........................................31
2.2.8 Immunological methods ..........................