Role of impaired lysosomal trafficking in the development of lung fibrosis in a murine model of Hermansky-Pudlak syndrome [Elektronische Ressource] / vorgelegt von Poornima Mahavadi

Sujets

• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 58
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Role of impaired lysosomal trafficking in the
development of lung fibrosis in a murine model of
Hermansky-Pudlak syndrome





Inaugural - Dissertation
zur Erlangung
des
Doktorgrades der
Naturwissenschaften
-Dr. rer. nat.-



dem
Fachbereich Biologie
der
Justus-Liebig-Universität Gießen
vorlegt von

M.Sc. Poornima Mahavadi
aus
Manthani, Indien




Gießen, February 2009

D-26
































Dekan

Prof. Dr. Peter R. Schreiner


Gutachter

Prof. Dr. Andreas Guenther

Prof. Dr. Michael U. Martin






































Dedicated to my parents…











Erklärung

Hermit erkläre ich, dass ich die vorliegende Arbeit selbstaendig verfasst habe
und dabei keine anderen als die angegebenen Quellen und Hilfsmittel verwendet
habe. Zitate sind als solche gekenzeichnet.
Giessen, den 16.02.2009.

Poornima Mahavadi.
























































Index I

INDEX

Index ..................................................................................................................... I
Abbreviations .....................................................................................................IV
1. Introduction ..................................................................................................... 1
1.1. The Pulmonary Surfactant ............................................................................. 1
1.1.1. Synthesis, composition and secretion of pulmonary surfactant ...................... …….1
1.1.2. Surfactant lipids ..................................................................................................... 3
1.1.3. Surfactant proteins ................................................................................................ 4
1.1.3.1. The hydrophyllic surfactant proteins, SP-A & SP-D ............................................ 4
1.1.3.2. The hydrophobic surfactant proteins, SP-B & SP-C ............................................ 5
1.2. Disorders of the pulmonary surfactant system .................................................... 7
1.3. Idiopathic pulmonary fibrosis .................................................................................. 8
1.4. Genetically defined disorders that may end up in progressive lung fibrosis . 10
1.5. Hermansky-Pudlak Syndrome .............................................................................. 11
1.5.1. The BLOC complex ......................................................................................................... 12
1.5.2. The AP-3 complex ........................................................................................................... 13
1.5.3. Hermansky-Pudlak syndrome associated interstitial pneumonia ............................. 15
1.5.4. Types of HPS and corresponding mouse models ...................................................... 17
2. Aim of the study ....................................................................................................... 24
3. Materials ..................................................................................................................... 25
3.1. General materials ................................................................................................... 25
3.2. Materials for animal work ...................................................................................... 26
3.3. Materials for histology ............................................................................................ 26
3.4. Kits ............................................................................................................................ 27
3.5. List of primers .......................................................................................................... 27
3.6. List of antibodies ..................................................................................................... 28
3.7. Equipment & software ............................................................................................ 29
4. Methods ...................................................................................................................... 30
4.1. Animals..................................................................................................................... 30
4.2. Histology .................................................................................................................. 31
4.2.1. Hematoxylin & Eosin staining ........................................................................................ 31
4.2.2. Trichrome staining ........................................................................................................... 32
Index II

4.3. Western blot analysis ............................................................................................. 33
4.4. Phospholipid analysis ............................................................................................ 35
4.5. Lipidomics ................................................................................................................ 35
4.6. Isolation of RNA from mice lungs ........................................................................ 37
4.7. Preparation of cDNA from RNA probes .............................................................. 38
4.8. Semi-quantitative RT-PCR .................................................................................... 39
4.9. Agarose gel electrophoresis ................................................................................. 39
4.10. Immunohistochemistry ........................................................................................ 40
4.11. In-situ apoptosis assay ........................................................................................ 41
4.12. Microscopy ............................................................................................................ 42
4.13. Isolation of alveolar epithelial cells .................................................................... 42
4.14. Densitometry analysis ......................................................................................... 44
4.15. Statistics................................................................................................................. 44
5. Results ........................................................................................................................ 45
5.1. General appearance and phenotype of HPS mice ........................................... 45
5.2. Lung histology of HPS mice .................................................................................. 45
5.3. Surfactant alterations in HPS mice ...................................................................... 48
5.3.1. Altered processing and transport of the hydrophobic surfactant proteins .............. 48
5.3.1.1. Reduction in mature SP-B & mature SP-C in BALF occurs almost exclusively in
HPS1/2 mice ................................................................................................................................ 48
5.3.1.2. Extensive surfactant protein accumulation in HPS1/2 double mutant mice ...... 49
5.3.1.3. Surfactant protein alterations in HPS1/6 double mutant mice ........................... 51
5.3.1.4. Surfactant protein alterations in HPS mono mutant mice ...................................... 52
5.3.1.5. Comparitive analysis of surfactant protein alterations in HPS mice ..................... 53
5.3.2. Phospholipidosis in HPS mice ............................................................................. 55
5.3.3. Lipidomic profiling of HPS lung tissues ........................................................................ 57
5.3.3.1. Accumulation of PC and surfactant specific DPPC in HPS1/2 mice .................... 59
5.3.3.2. Glucosylceramides in HPS mice ................................................................................ 60
5.4. AECII undergo early and extensive apoptosis in HPS1/2 mice ...................... 61
5.5. Early lysosomal stress underlies AECII apoptosis in HPS1/2 mice ............... 64
5.5.1. Lysosomal stress is specific in HPS1/2 mice .............................................................. 65
5.5.2. Cathepsin D mediated apoptosis in HPS1/2 mice ...................................................... 66
5.6. Induction of ER stress