Aspects of poriferan (Suberites domuncula) apoptosis and innate immune system and evolutionary implications [Elektronische Ressource] / Bérengère Luthringer

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2009
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Aspects of Poriferan
(Suberites domuncula)
Apoptosis and Innate Immune System
and Evolutionary Implications








Dissertation zur Erlangung des Grades
Doktor der Naturwissenschaften

Am Fachbereich Biologie
der Johannes Gutenberg-Universität in Mainz

Bérengère Luthringer

Geb. am 30 März 1979 in Plœmeur (Frankreich)

Mainz, 2009



























Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung: 23/04/2009
II
III— Table of Contents —

TABLE OF CONTENTS

I. ABSTRACT................................................................................................................... 1
II. INTRODUCTION ..................................................................................................... 3
A. Innate immunity ....................................................................................................... 3
B. Apoptosis .................................................................................................................. 7
1. Origin and history of apoptosis.............................................................................. 7
2. Triggering of apoptosis .......................................................................................... 8
3. Core component of apoptosis ................................................................................ 9
a. The Bcl-2 family ................................................................................................ 9
b. The tumor necrosis factors (TNF) super family and their receptors ................. 10
c. The soluble inter-membrane mitochondria protein (SIMP) family................... 11
d. The caspase family ........................................................................................... 12
e. The inhibitor of apoptosis protein (IAP) family ............................................... 14
C. Survivin .................................................................................................................. 15
D. Porifera................................................................................................................... 18
E. Porifera: a model to study apoptosis and immune pathways .................................... 23
III. AIM OF THE STUDY ............................................................................................. 26
IV. MATERIALS AND METHODS.............................................................................. 27
A. Materials ................................................................................................................. 27
1. Chemicals and ready-to-use solutions .................................................................. 27
2. Antibiotics ........................................................................................................... 29
3. Media .................................................................................................................. 29
4. Enzymes .............................................................................................................. 29
5. Antibodies 30
6. Markers ............................................................................................................... 30
7. Kits...................................................................................................................... 30
8. Bacteria strains and cell-lines................................................................................ 31
9. Vectors (cloning and expression) and libraries (cDNA and genomic)................... 31
10. Laboratory equipment and supplies ................................................................... 32
11. Experimental animals......................................................................................... 33
12. Computer and online softwares ......................................................................... 34
13. Primers .............................................................................................................. 35
B. Methods.................................................................................................................. 38
1. RNA extraction.................................................................................................... 39
IV— Table of Contents —

2. Spectrophotometric measurement of concentration and purity of nucleic acids ... 39
3. Reverse transcription............................................................................................ 40
4. GeneRacer™......................................................................................................... 41
5. Polymerase chain reaction (PCR)......................................................................... 42
a. Generation of primers ...................................................................................... 42
b. Enzymes .......................................................................................................... 44
c. Cycle profile for standard PCR 45
d. Standard PCR.................................................................................................. 46
e. Checking PCR ................................................................................................. 47
f. Sequencing PCR............................................................................................... 48
g. DIG labelling PCR .......................................................................................... 49
6. Agarose gel electrophoresis................................................................................... 50
7. Isolation of DNA from agarose gel....................................................................... 51
8. Cloning ............................................................................................................... 52
9. Transformation of competent cells 52
a. Preparation of “ultra-competent” E. coli: the Inoue method (adapted from Inoue
H et al. 1990) ...................................................................................................... 53
i. Monitoring of the bacterial growth via optic density (OD)........................... 54
10. Selection of recombinant vector of interest ........................................................ 54
a. Antibiotic resistance and white/blue selection................................................... 54
b. Checking PCR................................................................................................. 55
c. Overnight cultures ........................................................................................... 55
d. Plasmid digestion............................................................................................. 56
11. Isolation of plasmid DNA.................................................................................. 56
a. Isolation of plasmid DNA (mini-, midi-, and maxi-scale)................................. 56
b. Boiling method for mini-scale samples............................................................. 57
12. DNA sequencing ............................................................................................... 57
13. Sequence analysis (in silico) ................................................................................ 58
14. Cryopreservation................................................................................................ 59
15. Analysis on transcriptome .................................................................................. 60
a. Primmorphs preparation (adapted from Custodio MR et al. 1998) .................. 60
b. Apoptosis and innate immunity induction for transcriptome analysis .............. 61
c. Northern blotting............................................................................................. 62
d. In situ hybridization......................................................................................... 64
16. Analysis on proteome 66
a. Protein quantification ...................................................................................... 66
i. Assay according to bicinchoninic acid (BCA; adapted from Smith PK et al.
1987)............................................................................................................... 66
ii. Preparation of diluted albumin (BSA) standards .......................................... 67
V— Table of Contents —

iii. Experion™ Pro260 ...................................................................................... 67
b. Polyacrylamide gel electrophoresis (PAGE)...................................................... 68
i. Denaturing PAGE (SDS-PAGE) .................................................................. 68
ii. Native/semi-native PAGE - shift assay ......................................................... 69
iii. Coomassie staining of polyacrylamide gel ................................................... 70
c. Western blotting .............................................................................................. 71
d. General considerations on expression.........................................