Identification and characterization of novel cellular cholesterol regulators:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Zsfassung in engl. und dt. Sprache |
| Physical Description: | XVIII, 191 S. Ill., graph. Darst. |
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TABLE OF CONTENTS
SUMMARY I
ZUSAMMENFASSUNG III
ACKNOWLEDGMENTS V
PUBLICATIONS XVII
1 INTRODUCTION 1
1.1 UNDERSTANDING THE PATHOLOGICAL MECHANISMS OF GENETIC DISEASES 1
1.2 ASSESSING THE HERITABILITY OF GENETIC DISEASES 2
1.3 GENETIC MAPPING OF HERITABLE DISEASES 2
1.3.1 LINKAGE ANALYSIS FOR MENDELIAN DISEASES 3
1.3.2 GENOME-WIDE ASSOCIATION STUDIES FOR COMPLEX DISEASES 4
1.3.3 IDENTIFYING THE CAUSATIVE ELEMENTS 6
1.4 CARDIOVASCULAR DISEASE IS A COMMON COMPLEX DISEASE 6
1.5 ELEVATED BLOOD LIPID LEVELS ARE THE MAJOR RISK FACTOR FOR
CARDIOVASCULAR DISEASE 7
1.6 REGULATION OF CELLULAR CHOLESTEROL SYNTHESIS AND UPTAKE 9
1.6.1 CHOLESTEROL SYNTHESIS 10
1.6.2 SREBP-SCAP PATHWAY REGULATES CELLULAR CHOLESTEROL LEVELS 12
1.6.3 CHOLESTEROL UPTAKE BY LDL ENDOCYTOSIS 13
1.6.4 CHOLESTEROL EXPORT 14
1.7 GENETICS OF CHOLESTEROL REGULATION 15
1.7.1 HERITABILITY 15
1.7.2 MONOGENIC DISORDERS OF CHOLESTEROL REGULATION 15
1.7.3 COMPLEX LIPID TRAITS AND CARDIOVASCULAR DISEASE 16
1.8 HIGH CONTENT SCREENING USING RNA INTERFERENCE 17
1.8.1 RNAI SCREENING FOR REGULATORS OF CELLULAR CHOLESTEROL 18
HTTP://D-NB.INFO/1028020368
IMAGE 2
2 AIM OF STUDY 19
3 RESULTS 21
3.1 ASSAY CHARACTERIZATION AND DEVELOPMENT O F A NEW ANALYSIS PIPELINE
21
3.1.1 KNOCKDOWN O F KEY CHOLESTEROL REGULATORS 21
3.1.2 DEVELOPMENT O F AN AUTOMATED CELLPROFILER-R PIPELINE 23
3.2 DIFFERENT CANDIDATE SELECTION STRATEGIES 24
3.2.1 LUNA SCREEN 25
3.2.2 CHOLESTEROL MODULE SCREEN 25
3.2.3 GENOME-WIDE ASSOCIATION STUDY (GWAS) SCREEN 27
3.3 RNAI SCREENING RESULTS 31
3.3.1 LUNA SCREEN 31
3.3.2 CHOLESTEROL MODULE SCREEN 34
3.3.3 GENOME-WIDE ASSOCIATION STUDY (GWAS) SCREEN 36
3.5 SECONDARY SCREENS 42
3.5.1 CORRELATING FUNCTIONAL EFFECT WITH MRNA LEVEL 42
3.5.2 VALIDATION OF RESULTS USING AMPLEX RED ASSAY 47
3.5.3 VALIDATION O F RESULTS WITH LIVER-DERIVED CELL LINE HUH7 48
3.5.4 PREDICTION AND TESTING FOR SREBP-TARGET GENES 50
3.5.5 EFFECT O F KNOCKDOWN ON LDLR LEVELS 53
3.5.6 TESTING GENETIC INTERACTION WITH DOUBLE KNOCKDOWN 58
3.5.7 SUBCELLULAR LOCALIZATION OF FIVE SELECTED CANDIDATES 59
3.6 BIOINFORMATIC ANALYSIS OF RESULTS 62
3.6.1 OVERLAP WITH OTHER GENOME WIDE RNAI SCREENS 62
3.6.2 COMPARISON OF HIT RATE WITH DIFFERENT CANDIDATE SETS 63
3.7 CELL-BIOLOGICAL CHARACTERIZATION OF SELECTED HITS 65
3.7.1 SIK3 REGULATES LDLR LEVELS AT THE PLASMA MEMBRANE 65
3.7.2 COMPLEMENTATION WITH PAFAH1B1 ALLOWS TESTING OF INDIVIDUAL
MUTATIONS 66
4 DISCUSSION 71
4.1 ASSESSING PERFORMANCE OF FUNCTIONAL ASSAYS 72
4.1.1 HIGH SENSITIVITY ESTIMATED BASED ON DETECTION RATE O F KNOWN
CHOLESTEROL REGULATORS
73
4.1.2 HIGH SPECIFICITY IN DETECTING CHOLESTEROL REGULATORS 75
4.1.3 HELA KYOTO AS A SUITABLE CELL LINE TO STUDY CHOLESTEROL REGULATION
75
IMAGE 3
4.1.4 HIGH STRINGENCY OF THRESHOLDS APPLIED 76
4.2 COMPARING CANDIDATE SELECTION STRATEGIES 77
4.3 RNAI SCREENING RESULTS 78
4.3.1 NOVEL REGULATORS OF CELLULAR CHOLESTEROL REGULATION 78
4.3.2 SIMILARITY O F REGULATION O F CELLULAR CHOLESTEROL AND ER STRESS
82
4.3.3 COMPLEX INTERACTIONS WITHIN SINGLE ASSOCIATED LOCI 83
4.4 CHOLESTEROL REGULATION IS A NETWORK REGULATED BY FEEDBACK MECHANISMS
83
4.5 HYPOTHESES FOR MECHANISMS OF CHARACTERIZED GENES 85
4.5.1 SIK3 IS A PUTATIVE NOVEL REGULATOR O F SREBP 85
4.5.2 PAFAH1B1 COULD LINK LDL-CONTAINING ENDOSOMES TO MICROTUBULES 86
5 IMPLICATIONS FOR FURTHER RESEARCH 89
6 MATERIALS 91
6.1 BACTERIA STRAINS 91
6.2 EUKARYOTIC CELL LINES 91
6.3 CULTURE MEDIA 91
6.3.1 MEDIA FOR BACTERIAL CELL CULTURE 91
6.3.2 MEDIA FOR MAMMALIAN CELL CULTURE 92
6.4 BUFFERS AND SOLUTIONS 92
6.5 REAGENTS 93
6.6 NUCLEIC ACIDS AND OLIGONUCLEOTIDES 94
6.6.1 PLASMIDS 94
6.6.2 PRIMER OLIGONUCLEOTIDES 94
6.6.3 SIRNA OLIGONUCLEOTIDES 95
6.7 KITS 96
6.8 ANTIBODIES 96
6.8.1 PRIMARY ANTIBODIES 96
6.8.2 SECONDARY ANTIBODIES 97
6.9 EQUIPMENT 97
6.9.1 MICROSCOPES 97
6.9.2 LABWARE 97
6.9.3 CENTRIFUGES 98
6.9.4 OTHER EQUIPMENT 98
6.10 SOFTWARE 98
IX
IMAGE 4
6.10.1 R PACKAGES 99
7 METHODS 101
7.1 MOLECULAR BIOLOGY 101
7.1.1 ELUTION OF SAMPLE CDNA FROM FILTER DISCS 101
7.1.2 HEAT SHOCK TRANSFORMATION 101
7.1.3 ISOLATION AND PURIFICATION O F PLASMIDS 101
7.1.4 EXTRACTION OF RNA AND REVERSE-TRANSCRIPTION INTO CDNA 102
7.1.5 QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) 103
7.1.6 SEPARATING DNA ON AGAROSE GEL 103
7.1.7 HANDLING OF SIRNAS 103
7.2 BIOCHEMISTRY 104
7.2.1 SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) .... 104
7.2.2 WESTERN BLOTTING 104
7.2.3 STRIPPING OF NITROCELLULOSE MEMBRANES 105
7.2.4 HIGH-THROUGHPUT WESTERN BLOTTING 105
7.2.5 CO-IMMUNOPRECIPITATION 105
7.3 CELL BIOLOGY 106
7.3.1 MAMMALIAN CELL CULTURE 106
7.3.2 TRANSFECTION OF MAMMALIAN CELLS 107
7.3.3 IMMUNOFLUORESCENCE AND FLUORESCENCE MICROSCOPY 109
7.3.4 ENDOCYTOSIS ASSAYS 110
7.3.5 LDL-BINDING ASSAY I L L
7.3.6 MEASURING CELLULAR CHOLESTEROL I L L
7.3.7 STEROL DEPLETION PROTOCOLS 112
7.4 COMPUTATIONAL BIOLOGY 112
7.4.1 IMAGE ANALYSIS 112
7.4.2 ANALYSIS OF CHIP DATA 115
7.4.3 ANALYSIS OF SCREENING RESULTS 115
7.4.4 DESIGN OF SIRNAS 121
8 ABBREVIATIONS 123
9 APPENDIX 125
9.1 SUPPLEMENTARY FIGURES 125
9.2 SUPPLEMENTARY TABLES 130
9.3 SCRIPTS AND MACROS 161
X
IMAGE 5
9.3.1 CELLPROFILER LDL-UPTAKE ASSAY ANALYSIS SCRIPT 161
9.3.2 CELLPROFILER FILIPIN ASSAY ANALYSIS SCRIPT 164
9.3.3 R FUNCTIONS FOR ANALYSIS SCRIPT 167
9.3.4 OTHER R FUNCTIONS 174
10 REFERENCES 181
XI
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| spelling | Blattmann, Peter Nils Verfasser (DE-588)1027206980 aut Identification and characterization of novel cellular cholesterol regulators [presented by Peter Nils Blattmann] 2012 XVIII, 191 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in engl. und dt. Sprache Heidelberg, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025363943&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Blattmann, Peter Nils Identification and characterization of novel cellular cholesterol regulators |
| subject_GND | (DE-588)4113937-9 |
| title | Identification and characterization of novel cellular cholesterol regulators |
| title_auth | Identification and characterization of novel cellular cholesterol regulators |
| title_exact_search | Identification and characterization of novel cellular cholesterol regulators |
| title_full | Identification and characterization of novel cellular cholesterol regulators [presented by Peter Nils Blattmann] |
| title_fullStr | Identification and characterization of novel cellular cholesterol regulators [presented by Peter Nils Blattmann] |
| title_full_unstemmed | Identification and characterization of novel cellular cholesterol regulators [presented by Peter Nils Blattmann] |
| title_short | Identification and characterization of novel cellular cholesterol regulators |
| title_sort | identification and characterization of novel cellular cholesterol regulators |
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