1 Introduction ................................................. 1
1.1 Integrative biology: what does it mean? ................. 1
1.2 RNA interference as a tool in molecular biology ......... 3
1.2.1 RNA interference ................................. 3
1.2.2 RNAi screens ..................................... 6
1.2.2.1 High-throughput RNAi screens (RNAi
HTSs) ................................... 7
1.2.2.2 RNAi reagents and off-target effects
(OTEs) .................................. 8
1.2.3 Integrative analysis of RNAi screens ............ 10
1.3 Endocytosis ............................................ 14
1.3.1 Vesicle formation, coating and fusion ........... 17
1.3.2 Molecular machineries and compartments in the
endocytic system ................................ 21
1.3.3 Endocytosis and signaling ....................... 24
2 Aims of the project ......................................... 29
2.1 The objectives of this thesis work ..................... 29
2.2 Achievements of this thesis work ....................... 30
3 Results ..................................................... 33
3.1 The idea: integrate different layers of information .... 34
3.2 Development of a suite of new integrative methods for
RNAi data .............................................. 35
3.3 Evaluation of the global performance of the
integrative approach ................................... 41
3.3.1 Improved detection of known endocytosis
related genes ................................... 41
3.3.2 Phenotypic space ................................ 45
3.4 Enrichment analysis .................................... 46
3.5 Endocytic parameters ................................... 53
3.5.1 Proof of principle and novel complexes
recovered with the integrative analysis ......... 53
3.5.1.1 EGFR-SOS1-GRB2 ......................... 54
3.5.1.2 ESCRT complexes ........................ 55
3.5.1.3 Adaptor complex 2 ...................... 56
3.5.1.4 V-ATPase-V0 domain ..................... 57
3.5.1.5 SNARE complex containing STX4 .......... 58
3.5.1.6 SNARE complex containing STX12 ......... 59
3.5.1.7 HOPS complex ........................... 59
3.5.2 KEGG human pathways with an enriched phenotype .. 61
3.5.3 Molecular modules detected with the
integrative analysis ............................ 66
3.5.3.1 EGFR-IGFR module ....................... 66
3.5.3.2 TGF β-Notch1 module .................... 67
3.5.3.3 Activins module ........................ 68
3.5.3.4 Integrins-cytoskeleton module .......... 69
3.5.4 RAB5 effectors .................................. 70
3.6 Integrative analysis of the cell morphological
parameters ............................................. 75
3.6.1 Exploring the morphological changes in the
endocytic screen ................................ 75
3.6.1.1 Immunoproteasome ....................... 75
3.6.1.2 Cytoplamic dynein complex .............. 76
3.6.1.3 Module containing ITGB5 ................ 76
3.6.1.4 Module containing GPCRs ................ 79
3.6.2 Exploring the morphological parameters in
combination with the endocytic ones ............. 79
3.6.2.1 Module containing FRAP1 ................ 79
3.7 Experimental validation of the crosstalk between EGFR
and IGFR: preliminary results .......................... 82
4 Discussion .................................................. 85
4.1 General remarks ........................................ 85
4.2 Development of a new approach for analyzing RNAi
screens ................................................ 87
4.3 Application to an endocytosis screen ................... 88
4.3.1 Computational validation ........................ 89
4.3.2 Detection of known endocytic genes and
machineries ..................................... 90
4.3.3 Endocytosis and signaling ....................... 91
4.3.4 Cross-talk of EGFR-IGFR and its impact on
endocytosis ..................................... 92
4.4 Conclusions and outlook ................................ 93
4.4.1 What has been done before and the present work .. 93
4.4.2 Methodological improvements and new
applications .................................... 94
5 Matherials and methods ...................................... 97
5.1 Gene-set based approach ................................ 97
5.1.1 General idea .................................... 97
5.1.2 Implementation .................................. 97
5.1.3 Statistical assessment .......................... 99
5.2 Network-based approach ................................. 99
5.2.1 Seed Selection ................................. 100
5.2.2 Module expansion ............................... 100
5.2.3 Module assessment: solution to deal with
frequent phenotypes ............................ 100
5.2.4 Module fusion .................................. 103
5.3 Evaluation performances via Receiver Operating
Characteristic curves ................................. 103
5.4 False Discovery Rate estimation ....................... 104
5.5 Execution pipeline .................................... 105
5.6 Distributed calculation ............................... 105
5.7 Management system for exploring the data results ...... 106
5.7.1 Database ....................................... 106
5.7.2 Matlab application ............................. 106
6 Appendix ................................................... 107
6.1 Lists of parameters ................................... 107
List of Figures ............................................... 111
List of Tables ................................................ 113
References .................................................... 115
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