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Publié par | Thesee |
Nombre de lectures | 12 |
Langue | English |
Poids de l'ouvrage | 1 Mo |
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UNIVERSITÉ D’ORLÉANS
ÉCOLE DOCTORALE ou SCIENCES ET TECHNOLOGIES
Centre de Biophysique Moléculaire/Académie des Sciences de Pologne
THÈSE EN COTUTELLE INTERNATIONALE présentée par :
Magdalena TOMCZYŃSKA
soutenue le : 28 septembre 2009
pour obtenir le grade de :
Docteur de l’université d’Orléans
et de l’Académie Polonaise des Sciences
Discipline : Aspects moléculaires et cellulaires de la biologie
Biologie de l’endothélium vasculaire isolé de
souris transgéniques YAC67 et YAC84-
modèles murins du syndrome de Down
THÈSE dirigée par :
Dr Claudine KIEDA Directrice de recherche – CNRS – Orléans – France
Dr Danura DUŚ Directrice de recherche – Académie des Sciences – Wrocław -
Pologne
RAPPORTEURS :
Pr Maciej KURPISZ Académie Polonaise des Sciences - Poznań - Pologne
Pr Irena FRYDECKA Académie Polonaise des Sciences - Wrocław - Pologne
___________________________________________________________________
JURY :
Pr Wojciech GORCZYCA Académie Polonaise des Sciences – Pologne - Président du jury
Dr Claudine KIEDA CNRS - Centre de biophysique moléculaire – Orléans-France
Dr Danuta DUŚ Académie Polonaise des Sciences – Pologne
Zofia BŁACH-OLSZEWSKA Académie Polonaise des Sciences – Pologne
Dr Janusz BORATYŃSKI Académie Polonaise des Sciences – Pologne
Pr Andrzej GAMIAN Académie Polonaise des Sciences – Pologne
Pr Leon STRZĄDAŁA Académie Polonaise des Sciences – Pologne
Pr Andrzej MAZUR Unité de Nutrition Humaine UMR 1019, INRA, France
Pr Maciej UGORSKI Académie Polonaise des Sciences – Pologne
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tel-00520763, version 1 - 24 Sep 2010
PhD student obtained fellowships from:
Jérôme Lejeune Fondation (2003 – 2007)
This work was supported by:
Grant of Jérôme Lejeune Fondation (2003 – 2007)
Grant of the Polish Ministry of Science and Higher Education
(N401 1335 33)
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tel-00520763, version 1 - 24 Sep 2010
I thank dr hab. Claudine Kieda and
doc. dr hab. Danuta Duś for supervision and
guidance; Univesity of Orléans, Centre
National de la Recherche Scientifique and
Polish Academy of Science for the
opportunity for realization of presented work.
I would like to thank also Aleksandra
Bielawska-Pohl, Agnieszka Krawczenko,
Maria Paprocka, Nathalie Lamerant-Fayel and
Michèle Mitterrand for continuous support.
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tel-00520763, version 1 - 24 Sep 2010TABLE OF CONTENS
LIST OF ABBREVIATIONS..................................................................................... 6
PREFACE................................................................................................................... 8
1 INTRODUCTION.......................................................................................... 10
1.1 Down syndrome: generalities .......................................................................... 11
1.2 Down syndrome features................................................................................ 12
1.2.1 Down syndrome disabilities................................................................ 12
1.2.2 Immunological status in Down syndrome........................................... 14
1.3 Genetics of Down syndrome............................................................................ 16
1.4 Murine models for Down syndrome............................................................... 17
1.5 Transgenic mice YAC67 and YAC84............................................................. 20
1.6 Endothelial cells.............................................................................................. 23
1.6.1 Generalities on the biology of endothelial cells..... 23
1.6.2 Angiogenesis................................................................................................... 25
1.6.3 The role of adhesion in lymphocyte homing and recirculation....................... 27
1.6.3.1 Cell Adhesion Molecules (CAMs).................................................... 27
1.6.3.2 Integrins............................................................................................ 28
1.6.3.3 Immunoglobulin-like cell adhesion molecules (Ig CAMs)............... 30
1.6.3.4 Mucin-like molecules......................................................................... 31
1.6.3.5 Chemokines......................................................................................... 32
1.6.3.6 Chemoattraction.................................................................................. 34
1.6.3.7 Rolling................................................................................................. 34
1.6.3.8 Activation............................................................................................ 34
1.6.3.9 Tight adhesion..................................................................................... 35
1.6.3.10 Transmigration.................................................................................. 35
AIM OF THE STUDY................................................................................................ 36
2 MATERIALS AND METHODS................................................................... 37
2.1 Animals........................................................................................................... 38
2.2 Cell lines.......................................................................................................... 38
2.3 Isolation and culture of microvascular endothelial cells ................................ 38
2.4 Immortalization and selection of endothelial cell lines................................... 39
2.5 Murine lymphocytes isolation......................................................................... 40
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tel-00520763, version 1 - 24 Sep 20102.6 Antibodies ...................................................................................................... 40
2.7 Flow cytometry analysis.................................................................................. 40
2.8 Intracellular von Willebrand factor (vWf) and Angiotensin-Converting 41
Enzyme (ACE) detection................................................................................
2.9 Adhesion assay ............................................................................................... 41
2.10 Pseudovessels formation assay....................................................................... 42
2.11 cDNA microarray analysis of gene expression profile................................... 43
3 RESULTS........................................................................................................ 46
3.1 Establishment of mouse endothelial cell lines................................................ 47
3.2 The presence of general markers of endothelial cell lines: vWf and
ACE................................................................................................................. 48
3.3 Characterization of the phenotype of transgenic mouse ECs lines from
YAC67 and YAC84 mice vs non-transgenic FVB cells................................. 49
3.4 Adhesive properties of organospecific ECs from transgenic animals toward
T and B lymphoma cells compared to FVB endothelial cells......................... 54
3.5 Adhesive properties of organospecific ECs from transgenic animals toward
lymphocytes isolated from FVB mice............................................................ 57
3.6 Comparison of angiogenic properties of ECs from transgenic mice with
additional copies of GIRK2 gene............................................................ 60
3.7 Gene expression assessment by cDNA microarrays for the whole murine
genome............................................................................................................ 64
4 DISCUSSION................................................................................................. 77
CONCLUSIONS......................................................................................................... 86
SUMMARY................................................................................................................ 88
BIBLIOGRAPHY....................................................................................................... 90
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tel-00520763, version 1 - 24 Sep 2010LIST OF ABBREVIATIONS
ACE Angiotensin converting enzyme
AD Alzheimer disease
AMKL Acute megakaryoblastic leukemia
AML Acute myeloid leukemia
Ang-1 Angiopoietin-1
aRNA Amplified RNA
ATCC American Type Culture Collection
BBB Blood-brain barrier
bFGF Basic fibroblast growth factor
BM Bone marrow
BSA Bovine serum albumin
CAMs Cell Adhesion Molecules
cDNA Complementary DNA
CNS Central nervous system
c-PBS Complete phosphate buffered saline
CRD Carbohydrate binding domain
Cy Cyanine
DMSO Dim