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Publié par | ludwig-maximilians-universitat_munchen |
Publié le | 01 janvier 2011 |
Nombre de lectures | 39 |
Langue | English |
Poids de l'ouvrage | 8 Mo |
Extrait
Some ABCA3 mutations elevate ER stress and initiate
apoptosis of lung epithelial cells
Nina Weichert Aus der Kinderklinik und Kinderpoliklinik
im Dr. von Haunerschen Kinderspital
der Ludwig-Maximilians-Universität München
Direktor: Prof. Dr. med. Dr. sci. nat. Christoph Klein
Some ABCA3 mutations elevate ER stress and initiate
apoptosis of lung epithelial cells
Dissertation
zum Erwerb des Doktorgrades der Humanmedizin
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München
Vorgelegt von
Nina Weichert
aus Heidelberg
2011
Mit Genehmigung der Medizinischen Fakultät
der Universität München
1. Berichterstatter: Prof. Dr. Matthias Griese
2. Berichterstatter: Prof. Dr. Dennis Nowak
Mitberichterstatter: Priv. Doz. Dr. Angela Abicht
Prof. Dr. Michael Schleicher
Mitbetreuung durch den
promovierten Mitarbeiter: Dr. Suncana Kern
Dekan: Herr Prof. Dr. med. Dr. h. c. Maximilian Reiser,
FACR, FRCR
Tag der mündlichen Prüfung: 24.11.2011
Table of Contents
1.Abstract ................................................................................................................... 1
2.Zusammenfassung................................................................................................. 2
3.Intoduction.............. 3
3.1 Pediatric interstitial lung disease............................................... 3
3.1.1 Epidemiology of pILD............................................................................................... 3
3.1.2 Classification of pILD 4
3.1.3 Genetic surfactant dysfunction disorders................................................................. 5
3.1.4 Clinical diagnostics and therapy of pILD.................................................................. 6
3.1.5 Prognosis of pILD .................................................................................................... 6
3.2 ATP- binding cassette protein A3 (ABCA3) ............................................................... 7
3.2.1 ABC transporters ..................................................................................................... 7
3.2.2 General on the ABCA3 protein ................................................................................ 8
3.2.3 Function of ABCA3 .................................................................................................. 8
3.2.3.1 Localization and lamellar body biogenesis ........................................... 8
3.2.3.2 ABCA3 is a lipid transporter.................................................................. 9
3.2.3.3 Categorization of ABCA3 mutations ..................................................... 9
3.2.4 ABCA3 in lung disease .......................................................................................... 10
3.2.4.1 Early and late onset of lung disease ................................................... 10
3.2.4.2 Genotype-phenotype interplay............................................................ 11
3.2.4.3 Outer stressor ..................................................................................... 11
3.2.4.4 Histopathological pattern .................................................................... 11
3.2.4.5 Therapy............................................................................................... 12
3.2.5 ABCA3 mutations in this study............................................................................... 12
3.3 How misfolded proteins disturb cell homeostasis.................................................. 14
3.3.1 Function of the endoplasmic reticulum .................................................................. 14
3.3.2 Induction of the quality control system by unfolded proteins ................................. 15
3.3.3 Induction of apoptosis ............................................................................................ 17
3.4 ER stress and apoptosis contribute to disease pathogenesis .............................. 18
3.5. An objective ...............................................................................................................20
4. Materials ............................................................................................................... 21
4.1 Chemicals.................................................................................................................... 21
4.2 Equipment.21
4.3 Enzymes and kits ....................................................................................................... 22
4.4 Primers ........................................................................................................................ 23
I4.5 Vectors ........................................................................................................................ 24
4.6 Antibodies ................................................................................................................... 25
4.7 Bacterial strains and cell lines .................................................................................. 27
5. Methods................................................................................................................ 27
5.1 Molecular biological methods ................................................................................... 27
5.1.1 Cloning strategy: Generation of pUB6-ABCA3-HA vector ..................................... 27
5.1.2 Site-directed point mutagenesis............................................................................. 28
5.1.3 DNA- sequencing................................................................................................... 29
5.1.4 E.coli DH5 culture .................................................................................................. 29
5.1.5 Generation of competent E.coli DH5 .................................................................... 29
5.1.6 Transformation of E.coli DH5 ................................................................................ 30
5.1.7 Plasmid-DNA isolation ........................................................................................... 30
5.1.8 Restriction .............................................................................................................. 30
5.2 Mamalian cell culture ................................................................................................. 31
5.2.1 Media and growth conditions ................................................................................. 31
5.2.2 Mycoplasma testing ............................................................................................... 31
5.2.3 Transfection of A549 cells...................................................................................... 31
5.3 Biochemical methods ................................................................................................ 31
5.3.1 Whole cell lysates preparation ............................................................................... 31
5.3.2 Crude membrane preparation................................................................................ 32
5.3.3 Determination of protein concentration .................................................................. 32
5.3.4 Deglycosylation-assay ........................................................................................... 32
5.3.5 SDS-Polyacrylamide Gel Electrophoreses (SDS-PAGE) ...................................... 33
5.3.6 Western Blotting..................................................................................................... 33
5.3.7 Liposome preparation and NBD-lipid uptake ......................................................... 34
5.3.8 RNA isolation from A549 cells ............................................................................... 34
5.3.9 RT-PCR ................................................................................................................. 34
5.3.10 Densitrometric analysis of the intensity of protein and DNA bands ..................... 35
5.3.11 Immunofluorescence............................................................................................ 35
5.3.12 FACS analyses .................................................................................................... 36
5.3.13 Statistical analyses .............................................................................................. 36
6. Results.................................................................................................................. 37
6.1 Creating a model system to analyse the effect of ABCA3 mutations on alveolar
type II cells´homeostasis ................................................................................................. 37
6.1.1 Generation of pUB6-hABCA3 vector and site-directed point mutagenesis............ 37
6.1.2 Optimization of transfection procedure in A549 cell line ........................................ 39
6.2 General characterization of R43L, R280C and L101P ABCA3 mutations.............. 40
II6.2.1 Localization and trafficking...............................................................