Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Heidelberg
2018
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| Beschreibung: | 92 Seiten Illustrationen, Diagramme 30 cm |
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| 245 | 1 | 0 | |a Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics |c presented by Master of Science Ya-Yun Cheng ; referees: Prof. Dr. Stefan Fröhling [und ein weiterer Gutachter] |
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
1.
ZUSAMMENFASSUNG..............................................................................................................1
2.
ABSTRACT................................................................................................................................3
3.
INTRODUCTION.........................................................................................................................
4
3.1 GENOME-WIDE RNA INTERFERENCE
SCREENS....................................................................
4
3.2
SARCOMAS.......................................................................................................................
7
3.2.1 BONE AND SOFT-TISSUE
SARCOMAS.............................................................................7
3.2.2
LIPOSARCOMAS.........................................................................................................
9
3.2.3 FUS-DDIT3 AND MYXOID LIPOSARCOMA
.................................................................
11
3.3 HIPPO
PATHWAY.............................................................................................................13
3.3.1 DISCOVERY OF THE HIPPO
PATHWAY..........................................................................13
3.3.2 REGULATION OF THE HIPPO
PATHWAY.........................................................................14
3.3.3 YAP1 AND TAZ AS TRANSCRIPTIONAL
CO-FACTORS.......................................................17
3.3.4 HIPPO PATHWAY AND
CANCER..................................................................................
18
3.3.5 THERAPEUTIC TARGETING OF THE HIPPO PATHWAY FOR CANCER TREATMENT
.....................
19
3.3.6 HIPPO PATHWAY AND SARCOMAS
............................................................................
21
3.4 AIMS OF THE
STUDY........................................................................................................
21
4. MATERIALS AND
METHODS...................................................................................................23
4.1 CELL CULTURE
....
..............................................................................................................23
4.2
CLONING.........................................................................................................................24
4.2.1 T
RANSFORMATION.....................................................................................................24
4.2.2 CLONING OF YAP1 INTO THE PLENTI6.2A/5-DEST
VECTOR.........................................24
4.2.3 CLONING SHRNA OLIGOS INTO THE PRSI12
VECTOR.................................................... 27
4.3 VIRUS PRODUCTION, TRANSDUCTION, AND CELL
SELECTION.......................................................29
4.3.1 VIRUS
PRODUCTION...................................................................................................29
4.3.2 VIRUS TRANSDUCTION AND CELL
SELECTION..................................................................
30
4.4 RNA
ANALYSIS...............................................................................................................
30
4.4.1 TOTAL RNA EXTRACTION AND CDNA SYNTHESIS
........................................................
30
4.4.2 REVERSE TRANSCRIPTION-PCR (RT-PCR) OF
FUS-DDIT3........................................31
4.4.3 REVERSE TRANSCRIPTION-QUANTITATIVE PCR
(RT-QPCR)..........................................31
4.5 PROTEIN
ANALYSIS...........................................................................................................
33
4.5.1 PROTEIN
EXTRACTION.................................................................................................33
4.5.2 BRADFORD
ASSAY.....................................................................................................33
4.5.3 WESTERN BLOTTING (W
B).........................................................................................
34
4.5.4 CELL
FRACTIONATION..................................................................................................35
4.5.5 CO-IMMUNOPRECIPITATION (CO-IP)
.........................................................................
36
4.6 DECIPHER SHRNA SCREENS AND DATA
ANALYSIS..........................................................37
4.6.1 DECIPHER
LIBRARY................................................................................................37
4.6.2 SHRNA
SCREENS....................................................................................................
37
4.6.3 DATA
ANALYSIS........................................................................................................
40
4.7 FUNCTIONAL
ASSAYS.......................................................................................................
41
4.7.1 MTS
ASSAY...........................................................................................................
41
4.7.2 SHRNA/RFP COMPETITION
ASSAY...........................................................................41
4.7.3 CELL CYCLE
ANALYSIS................................................................................................42
4.7.4 SENESCENCE
ASSAY...............................................................................................42
4.7.5 ANNEXIN V/7-AAD APOPTOSIS ASSAY
.....................................................................
43
4.7.6 IMMUNOFLUORESCENCE (IF
)....................................................................................
43
4.7.7 RNA
SEQUENCING..................................................................................................43
4.8 CHICKEN CHORIOALLANTOIC MEMBRANE (CAM) ASSAY
.....................................................
44
5.
RESULTS................................................................................................................................45
5.1 SCP-1 ISOGENEIC CELL LINES
CHARACTERIZATION...............................................................
45
5.2 DECIPHER SHRNA
SCREENS.......................................................................................
46
5.2.1 SCREENING
LAYOUT..................................................................................................46
5.2.2 SCREENING DATA
ANALYSIS......................................................................................
48
5.2.2.1 OVERVIEW OF THE SCREENING
DATA....................................................................
48
5.2.2.2 CANDIDATE GENE SELECTION AND VALIDATION: 1ST
METHOD..................................49
5.2.2 3 CANDIDATE GENE SELECTION AND VALIDATION: 2ND METHOD
................................
53
5.3 IMPORTANCE OF YAP1 IN MYXOID LIPOSARCOMA
CELLS......................................................55
5.3.1 EFFECTS OF YAP 1 KNOCKDOWN IN MYXOID LIPOSARCOMA
CELLS...................................55
5.3.2 EXPRESSION AND LOCALIZATION OF YAP 1 IN LIPOSARCOMA CELL LINES
...........................
61
5.3.3 INTERACTION BETWEEN YAP1 AND
FUS-DDIT3........................................................64
5.4 RNA SEQUENCING
ANALYSIS............................................................................................66
5.5
IN VIVO
VALIDATION OF
YAP1............................................................................................72
6.
DISCUSSION.........................................................................................................................
74
7.
REFERENCES.........................................................................................................................
80
8.
ABBREVIATIONS....................................................................................................................
90
|
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| author | Cheng, Ya-Yun |
| author_GND | (DE-588)1153924668 |
| author_facet | Cheng, Ya-Yun |
| author_role | aut |
| author_sort | Cheng, Ya-Yun |
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| bvnumber | BV045101705 |
| ctrlnum | (OCoLC)1136272851 (DE-599)DNB1155446941 |
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| physical | 92 Seiten Illustrationen, Diagramme 30 cm |
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| spelling | Cheng, Ya-Yun Verfasser (DE-588)1153924668 aut Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics presented by Master of Science Ya-Yun Cheng ; referees: Prof. Dr. Stefan Fröhling [und ein weiterer Gutachter] Heidelberg 2018 92 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto-Carola University of Heidelberg 2018 Mit einer Zusammenfassung in englischer und deutscher Sprache (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1155446941/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030492280&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Cheng, Ya-Yun Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics |
| subject_GND | (DE-588)4113937-9 |
| title | Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics |
| title_auth | Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics |
| title_exact_search | Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics |
| title_full | Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics presented by Master of Science Ya-Yun Cheng ; referees: Prof. Dr. Stefan Fröhling [und ein weiterer Gutachter] |
| title_fullStr | Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics presented by Master of Science Ya-Yun Cheng ; referees: Prof. Dr. Stefan Fröhling [und ein weiterer Gutachter] |
| title_full_unstemmed | Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics presented by Master of Science Ya-Yun Cheng ; referees: Prof. Dr. Stefan Fröhling [und ein weiterer Gutachter] |
| title_short | Identification of YAP1 as an essential gene in myxoid liposarcoma using functional genomics |
| title_sort | identification of yap1 as an essential gene in myxoid liposarcoma using functional genomics |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1155446941/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030492280&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT chengyayun identificationofyap1asanessentialgeneinmyxoidliposarcomausingfunctionalgenomics |
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