Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL:
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| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Aachen
[2018]
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| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | 84 Seiten Illustrationen 21 cm |
Internformat
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| 245 | 1 | 0 | |a Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL |c vorgelegt von Johanna Köhler |
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
1. INTRODUCTION 1
1.1. HEMATOPOIESIS 1
1.2. MYELOPROLIFERATIVE NEOPLASMS 3
1.2.1. THE FOUR CLASSIC MPNS 3
1.2.2. PATHOGENESIS AND MOLECULAR FEATURES OF PHILADELPHIA CHROMOSOME
NEGATIVE MPNS 4
1.3. CALRETICULIN MUTATIONS IN MPNS 6
1.3.1. SOMATIC CALRETICULIN MUTATIONS 6
1.3.2. CALRETICULIN 7
1.3.3. THE NOVEL CALRETICULIN PROTEIN 7
1.3.4. CLINICAL IMPACT AND THERAPY 8
1.4. ADDITIONAL CALRETICULIN MUTATIONS 10
1.4.1. A 9 BP DELETION IN CALR EXON 9 FOUND IN A PATIENT IN OUR HOSPITAL
10
1.4.2. CALRETICULIN MUTATIONS IN THE MARIMO CELL LINE 10
2. AIM 11
3. MATERIALS AND METHODS 12
3.1. MATERIALS 12
3.1.1. STANDARD EQUIPMENT 12
3.1.2. REAGENTS 13
3.1.3. SOLUTIONS AND BUFFERS 15
3.1.4. ENZYMES 17
3.1.5. KITS 18
3.1.6. SOFTWARE AND DATABASES 18
3.2. MOLECULAR BIOLOGICAL METHODS 19
3.2.1. PATIENT MATERIAL 19
3.2.2. RNA ISOLATION 19
3.2.3. CDNA SYNTHESIS 20
3.2.4. PRIMER DESIGN AND POLYMERASE CHAIN REACTION 20
3.2.5. AGAROSE GEL ELECTROPHORESIS AND DNA GEL EXTRACTION 23
3.2.6. RESTRICTION DIGEST AND LIGATION 23
3.2.7. GATEWAY CLONING AND PLASMIDS 25
3.2.8. TRANSFORMATION 28
3.2.9. MINIPREPARATION OF PLASMID DNA 28
3.2.10. MAXIPREPARATION OF PLASMID DNA 29
3.3. CELL-CULTURE TECHNIQUES 30
3.3.1. CELL LINES 30
3.3.2. CELL CULTURE 30
3.3.3. CELL COUNTING 32
3.3.4. TRANSIENT TRANSFECTION 32
3.3.5. GENERATION OF RETROVIRAL SUPERNATANTS 32
3.3.6. RETROVIRAL TRANSDUCTION OF 32D CELLS 33
3.3.7. GENERATION AND INDUCTION OF TETRACYCLINE-INDUCIBLE T-REX* HELA
CELLS 33
3.3.8. FLOW CYTOMETRY AND FLUORESCENCE ACTIVATED CELL SORTING 34
3.4. ANALYSIS OF SIGNALING AND JUNCTION 35
3.4.1. PROLIFERATION ASSAY 3 5
3.4.2. WESTERN BLOT 35
3.4.3. QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION 38
3.4.4. APOPTOSIS ASSAY 39
3.4.5. MTT ASSAY 40
3.4.6. STATISTICAL ANALYSIS 40
4. RESULTS 42
4.1. REVIEW OF THE EXPRESSION VECTORS AND ANALYSIS OF THE SEQUENCING
DATA 42
4.2. ANALYSIS OF PROLIFERATION AND INTRACELLULAR SIGNALING 43
4.2.1. ASSESSMENT OF PROLIFERATION RATES 43
4.2.2. ASSESSMENT OF INTRACELLULAR TYROSINE PHOSPHORYLATION 44
4.2.3. ASSESSMENT OF STAT5 AND ERK PROTEIN LEVELS AND PHOSPHORYLATION 47
4.2.4. DETERMINATION OF STAT5 MRNA LEVELS 50
4.3. ANALYSIS OF GFP EXPRESSION, CELL VIABILITY AND APOPTOSIS 52
4.3.1. DETERMINATION OF GFP+ CELL LEVELS 52
4.3.2. RESULTS OF THE MTT ASSAY 53
4.3.3. RESULTS OF THE APOPTOSIS ASSAY 55
4.4. ANALYSIS OF EXOGENOUS CALRETICULIN PROTEIN EXPRESSION 58
4.4.1. ASSESSMENT OF PROTEIN EXPRESSION IN TRANSFECTED 293T CELLS 58
4.4.2. ASSESSMENT OF PROTEIN EXPRESSION IN TETRACYCLINE-INDUCIBLE
T-REX HELA CELLS 59
4.4.3. ASSESSMENT OF PROTEIN EXPRESSION IN RETROVIRALLY TRANSDUCED 32D
CELLS 59
4.4.4. DETERMINATION OF EXOGENOUS CALRETICULIN MRNA LEVELS IN
RETROVIRALLY
TRANSDUCED 32D CELLS 60
5. DISCUSSION 62
5.1. ANALYSIS OF CALRDEL52 REGARDING ITS ONCOGENIC EFFECT 63
5.1.1. 32D CELLS EXPRESSING CALRDEL52 ARE GROWTH FACTOR-DEPENDENT 63
5.1.2. 32D CELLS EXPRESSING THE CALRDEL52 VARIANT SHOW NO CONSTITUTIVE
ACTIVATION OF INTRACELLULAR SIGNALING 63
5.2. 32D CELLS EXPRESSING THE CALRDEL52 VARIANT SHOW INFERIOR CELL
VIABILITY
AND ENHANCED TENDENCY TO UNDERGO APOPTOSIS 66
5.3. EXPRESSION OF CALRDEL9 SHOWS NO ONCOGENIC FEATURES AND IS UNLIKELY
TO
CAUSE DISEASE EVEN WITH FORCED OVER-EXPRESSION OF MPL 68
5.4. EXPRESSION OF CALRDEL52 IS ASSOCIATED WITH REDUCED ABUNDANCE AND
IMPAIRED DETECTABILITY OF THE EXOGENOUS CALRETICULIN PROTEIN 70
6. SUMMARY 73
7. REFERENCES 74
8. APPENDIX 81
8.1. ACKNOWLEDGEMENTS 81
8.2. ERKLAERUNG § 5 ABS. 1 ZUR DATENAUFBEWAHRUNG 82
8.3. EIDESSTATTLICHE ERKLAERUNG GEMAESS § 5 ABS. (1) UND § 11 ABS. (3) 12.
83
8.4. CURRICULUM VITAE
84
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| any_adam_object | 1 |
| author | Köhler, Johanna |
| author_GND | (DE-588)1156957117 |
| author_facet | Köhler, Johanna |
| author_role | aut |
| author_sort | Köhler, Johanna |
| author_variant | j k jk |
| building | Verbundindex |
| bvnumber | BV045423109 |
| ctrlnum | (OCoLC)1136189919 (DE-599)DNB1162281502 |
| discipline | Medizin |
| format | Thesis Book |
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| spelling | Köhler, Johanna Verfasser (DE-588)1156957117 aut Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL vorgelegt von Johanna Köhler Aachen [2018] 84 Seiten Illustrationen 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation RWTH Aachen 2018 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1162281502/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030808957&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Köhler, Johanna Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL |
| subject_GND | (DE-588)4113937-9 |
| title | Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL |
| title_auth | Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL |
| title_exact_search | Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL |
| title_full | Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL vorgelegt von Johanna Köhler |
| title_fullStr | Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL vorgelegt von Johanna Köhler |
| title_full_unstemmed | Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL vorgelegt von Johanna Köhler |
| title_short | Functional in vitro analysis of CALR mutations in 32D myeloid progenitor cells without forced over-expression of MPL |
| title_sort | functional in vitro analysis of calr mutations in 32d myeloid progenitor cells without forced over expression of mpl |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1162281502/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030808957&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT kohlerjohanna functionalinvitroanalysisofcalrmutationsin32dmyeloidprogenitorcellswithoutforcedoverexpressionofmpl |
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