Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells [Elektronische Ressource] / Nina Weichert. Betreuer: Matthias Griese
94 pages
English

Découvre YouScribe en t'inscrivant gratuitement

Je m'inscris

Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells [Elektronische Ressource] / Nina Weichert. Betreuer: Matthias Griese

Découvre YouScribe en t'inscrivant gratuitement

Je m'inscris
Obtenez un accès à la bibliothèque pour le consulter en ligne
En savoir plus
94 pages
English
Obtenez un accès à la bibliothèque pour le consulter en ligne
En savoir plus

Description

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.

Sujets

Informations

Publié par
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...............................................................

  • Univers Univers
  • Ebooks Ebooks
  • Livres audio Livres audio
  • Presse Presse
  • Podcasts Podcasts
  • BD BD
  • Documents Documents