List of Figures ............................................... vii
List of Tables ................................................. xi
Glossary ..................................................... xiii
1 Introduction ................................................. 1
1.1 Preface: Fats - a 'big' problem ......................... 1
1.2 Lipid droplets .......................................... 2
1.2.1 Cellular role of lipid droplets .................. 5
1.2.2 Diseases related to lipid droplets ............... 6
1.3 Lipid droplets: How to study? ........................... 7
1.3.1 High content screening ........................... 7
1.3.1.1 High content screening to study
lipid droplets .......................... 8
1.3.2 Data analysis .................................... 9
1.3.2.1 Quality control parameters .............. 9
1.3.2.2 Statistical methods to facilitate
hit identification ..................... 11
1.3.3 Screening approaches to study lipid droplet
metabolism ...................................... 17
1.3.3.1 Genome-wide RNAi screen in
Drosophila S2-cells .................... 17
1.3.3.2 Genome-wide RNAi screen in
C.elegans .............................. 18
1.3.3.3 Genome-wide analysis in
S.cerevisiae using non-essential
deletion strains ....................... 18
2 Aims of the thesis .......................................... 21
3 Results ..................................................... 23
3.1 Assay development ...................................... 23
3.1.1 Cellular system ................................. 24
3.1.1.1 Controls in the high content
screen ................................. 28
3.1.2 Readout and image analysis ...................... 32
3.1.3 Data analysis in the high content screen ........ 37
3.1.4 Test of the final protocol ...................... 39
3.2 The screen ............................................. 48
3.2.1 The primary high content screen ................. 48
3.2.1.1 Data analysis of the well-based
data set ............................... 50
3.2.1.2 Data analysis of the cell-based
data set ............................... 53
3.2.2 Verification of candidates ...................... 57
3.2.2.1 Verification by siRNA treatment ........ 57
3.2.2.2 Verification by esiRNA treatment ....... 62
3.2.3 The final candidate genes ....................... 65
3.2.3.1 Comparison with previous screens ....... 68
3.3 Validation of candidate genes .......................... 69
3.3.1 Validation of KIAA0551 .......................... 70
3.3.2 Validation of CSNK2B ............................ 76
3.4 The role of FLJ21820 ................................... 86
4 Discussion ................................................. 101
4.1 The HC screen ......................................... 101
4.1.1 Properties of the assay ........................ 101
4.1.2 Outcome of the HC screen ....................... 103
4.1.2.1 Advantages and disadvantages of
the assay ............................. 105
4.1.3 Conclusion and outlook of the HC screen ........ 108
4.2 The candidates ........................................ 109
4.2.1 KIAA0551 ....................................... 109
4.2.2 CSNK2B ......................................... 111
4.2.2.1 FLJ21820 .............................. 113
4.2.3 Conclusions and outlook ........................ 115
5 Contributions .............................................. 117
6 Material and Methods ...................................... 119
6.1 Materials ............................................. 119
6.2 Methods: cell cultivation of mammals .................. 122
6.2.1 Cell culture ................................... 122
6.2.1.1 Cell culture: human A431 cells ........ 122
6.2.1.2 Cell culture: human HEK293 cells ...... 122
6.2.1.3 Cell culture: mouse 3T3-L1 cells ...... 123
6.2.2 DNA transfection ............................... 123
6.2.2.1 DNA transfection in human A431
cells ................................. 123
6.2.2.2 DNA transfection in HEK293 cells ...... 123
6.2.2.3 DNA transfection in differentiated
3T3-L1 adipocytes ..................... 123
6.2.3 RNAi treatment ................................. 124
6.2.3.1 siRNA transfection in 24-well
plate formate ......................... 124
6.2.3.2 siRNA transfection in 96-well
plate formate ......................... 124
6.2.3.3 esiRNA transfection in 96-well
plate formate ......................... 124
6.2.3.4 siRNA transfection in 384-well
plate formate ......................... 124
6.2.4 Fluorescent light microscopy ................... 124
6.2.4.1 Modified Staining protocol of the
HC assay for manual image
acquisition ........................... 125
6.2.4.2 Staining protocol of the HC assay
for automated image acquistion ........ 125
6.2.5 Image acquisition and analysis ................. 125
6.2.5.1 Manual image acquisition and
analysis .............................. 125
6.2.5.2 Automated image acquisition and
analysis .............................. 125
6.2.6 Data analysis .................................. 126
6.2.7 Functional annotation .......................... 126
6.2.8 Hierarchical clustering ........................ 126
6.2.9 Homology modeling .............................. 127
6.2.10 Prediction of phosphorylation sites ............ 127
6.2.11 Isolation of LDs ............................... 127
6.2.12 Lipid analysis ................................. 128
6.2.12.1 Analysis of total lipid
composition ........................... 128
6.2.12.2 Lipid uptake experiments .............. 128
6.2.12.3 Activity assays ....................... 128
6.2.12.4 One-phase lipid extraction ............ 129
6.2.12.5 TLC analysis and quantification ....... 129
6.2.13 Treatment with CSNK2 inhibitor ................. 130
6.2.14 RNA extraction and quantitative RT-PCR ......... 130
6.2.15 SDS-PAGE and western blotting .................. 131
6.3 Antibody production ................................... 131
6.4 Methods: cell cultivation of S.cerevisiae ............. 131
6.4.1 Cell culture: S.cerevisiae ..................... 131
6.4.2 Transformation ................................. 132
6.4.3 Fluorescent light microscopy ................... 132
7 PhD publication record ..................................... 133
References .................................................... 135
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