Molecular mechanisms involved in the induction of pluripotency:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | Nebentitel: Molekulare Mechanismen im Prozess der Zellreprogrammierung |
| Beschreibung: | [12], 158 Bl. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Molecular mechanisms involved in the induction of pluripotency |c by Guifré Ruiz Acero |
| 246 | 1 | 3 | |a Molekulare Mechanismen im Prozess der Zellreprogrammierung |
| 264 | 1 | |c 2012 | |
| 300 | |a [12], 158 Bl. |b Ill., graph. Darst. | ||
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| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Nebentitel: Molekulare Mechanismen im Prozess der Zellreprogrammierung | ||
| 502 | |a Berlin, Freie Univ., Diss., 2012 | ||
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Datensatz im Suchindex
| _version_ | 1804149484463063040 |
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| adam_text | IMAGE 1
CONTENTS
1. ABSTRACT 1
2. ZUSAMMENFASSUNG 2
3. INTRODUCTION 3
3.1. FROM EMBRYONIC STEM CEILS TO INDUCED PLURIPOTENCY 3
3.2. REGULATORY NETWORK IN EMBRYONIC STEM CELLS 4
3.2.1. CORE TRANSCRIPTIONAL MACHINERY 5
3.2.1.1. OCT4 6
3.2.1.2. SOX2 6
3.2.1.3. KLF4 7
3.2.1.4. C-MYC 7
3.2.2. TRANSCRIPTIONAL REGULATION 8
3.2.3. EPIGENETIC REGULATION 9
3.3. INDUCED PLURIPOTENCY WITH DEFINED FACTORS 10
3.3.1. TECHNICAL CONSIDERATIONS IN THE INDUCTION OF PLURIPOTENCY 11
3.3.1.1. VIRAL DELIVERY SYSTEM 12
3.3.1.2. NON-INTEGRATIVE APPROACHES 12
3.3.2. PATHWAYS INVOLVED IN REPROGRAMMING 14
3.4. MATHEMATICAL MODELLING 15
3.5. AIMS OF THE PROJECT 17
4. MATERIAL AND METHODS 19
4.1. MATERIAL 19
4.1.1. REAGENTS AND KITS 19
4.1.2. BUFFERS AND SOLUTIONS 21
4.1.3. EQUIPMENT 21
4.1.4. CELL LINES 23
4.1.5. CELL CULTURE MEDIA 23
4.1.6. BACTERIAL STRAINS 24
4.1.7. ENZYMES 24
4.1.8. PRIMARY ANTIBODIES 24
4.1.9. SECONDARY ANTIBODIES 25
4.1.10. OLIGONUCLEOTIDES 25
4.1.11. PLASMIDS 28
HTTP://D-NB.INFO/1030500738
IMAGE 2
4.1.11.1. PMXS-HOCT3/4, PMXS-HS0X2, PMXS-HKLF4, PMXS-HC-MYC 28
4.1.11.2. PLENTI-GFP 30
4.1.11.3. PUMVC3-GAG-POL 31
4.1.11.4. PCMV-VSV-G 32
4.1.11.5. PMKO.1 PURO P53-SH-RNA 33
4.1.11.6. PLKO.1 PURO SCRAMBLE-SHRNA 34
4.2. METHODS 35
4.2.1. THAWING AND MAINTAINING FIBROBLASTS 35
4.2.2. CRYOPRESERVATION OF FIBROBLASTS 35
4.2.3. PREPARATION OF IPS CULTURE PLATES 35
4.2.4. INACTIVATION OF FIBROBLASTS (FEEDERS PREPARATION) 36
4.2.5. CONDITIONED MEDIA PREPARATION 36
4.2.6. PASSAGE OF HUMAN IPS CELLS ON MATRIGEL 36
4.2.7. TRANSFECTION OF PACKAGING CELLS 37
4.2.8. INFECTION OF FIBROBLASTS 37
4.2.9. DERIVATION OF P53-KNOCK-DOWN CELL LINES 38
4.2.10. INHIBITION OF TGF(3 SIGNALLING PATHWAY RECEPTORS 38
4.3. PROTEIN-BASED METHODS 39
4.3.1. IMMUNOCYTOCHEMISTRY 39
4.4. RNA/DNA PROTOCOLS 39
4.4.1. RNA ISOLATION 39
4.4.2. REVERSE TRANSCRIPTION PCR 39
4.4.3. RNA QUANTIFICATION AND QUALITY CONTROL 40
4.4.4. QUANTITATIVE REAL-TIME PCR 41
4.4.5. PLASMID ISOLATION 42
4.4.6. RNA AND DNA QUANTIFICATION 43
4.4.7. AGAROSE GEL ELECTROPHORESIS 43
4.4.8. ILLUMINA 8-SAMPLE BEADCHIP HYBRIDISATION 43
4.5. DATA ANALYSIS 43
4.5.1. INITIAL ANALYSIS OF THE MICROARRAY DATA 43
4.5.2. PATHWAY ANALYSIS 44
4.5.3. META-ANALYSIS OF PUBLIC DATASETS 44
4.6. MODELLING 45
5. RESULTS 47
5.1. INFECTION OF HUMAN FIBROBLASTS 47
5.1.1. CONFIRMATIONS OF TRANSDUCED FIBROBLASTS 48
5.1.2. EXPRESSION ANALYSIS OF THE MICROARRAY DATA 52
IMAGE 3
5.2. PATHWAY ANALYSIS OF THE MICROARRAY DATA 56
5.2.1. PATHWAYS COMMONLY REGULATED 57
5.2.2. PATHWAYS DIFFERENTIALLY EXPRESSED AFTER KLF4 TRANSDUCTION 59
5.2.3. PATHWAYS DIFFERENTIALLY EXPRESSED AFTER C-MYC TRANSDUCTION 61
5.2.4. PATHWAYS DIFFERENTIALLY EXPRESSED AFTER SIMULTANOUS
OVER-EXPRESSION OF
OCT4, SOX2, AND KLF4 64
5.2.5. PATHWAYS DIFFERENTIALLY EXPRESSED AFTER SIMULTANOUS
OVER-EXPRESSION OF
OCT4, SOX2, KLF4, AND C-MYC 67
5.3. VALIDATION OF MICROARRAY DATA BY QRT-PCR 69
5.3.1. P53 SIGNALLING PATHWAY REGULATION AFTER TRANSDUCTION 70
5.3.2. EXPRESSION CHANGES IN APOPTOTIC SIGNALLING 71
5.3.3. EXPRESSION CHANGES IN CELL CYCLE COMPONENTS 74
5.3.4. TGFP SIGNALLING PATHWAY REGULATION AFTER TRANSDUCTION 77
5.3.5. DNA DAMAGE RESPONSE - HOMOLOGOUS RECOMBINATION 79
5.3.6. COMPARISON BETWEEN QRT-PCR AND MICROARRAY RESULTS 80
5.4. MODELLING 81
5.4.1. ASSEMBLY OF THE MODEL 82
5.4.1.1. P53 SIGNALLING PATHWAY 83
5.4.1.2. APOPTOSIS 85
5.4.1.3. CELL CYCLE 88
5.4.1.4. TGF(3 SIGNALLING PATHWAY 90
5.4.1.5. DNA DAMAGE RESPONSE - HOMOLOGOUS RECOMBINATION 93
5.4.2. SIMULATIONS - REPROGRAMMING 94
5.4.2.1. OVER-EXPRESSION OF THE REPROGRAMMING FACTORS 94
5.4.2.2. INHIBITION OF SENESCENCE AND APOPTOSIS 96
5.4.3. VALIDATION OF IN SILICO PREDICTIONS 99
5.4.3.1. P53 KNOCK-DOWN DURING THE REPROGRAMMING PROCESS 99
5.4.3.2. INHIBITION OF TGF SIGNALLING DURING THE REPROGRAMMING PROCESS
102
5.4.4. EVASION OF SENESCENCE AND APOPTOSIS-CANCER CELL LINES 104
5.4.4.1. COMPARISON OF CANCER CELL LINES AND TRANSDUCED CELLS BY
QRT-PCR. 108
6. DISCUSSION 110
6.1. VIRUS-MEDIATED OVER-EXPRESSION 110
6.2. FACTOR-SPECIFIC REGULATION 111
6.3. COMBINED EXPRESSION: OCT4, SOX2, AND KLF4 113
6.4. COMBINED EXPRESSION: OCT4, SOX2, KLF4, AND C-MYC 116
6.5. MATHEMATICAL MODELLING 119
6.6. REPROGRAMMING AND CANCER 121
IMAGE 4
6.7. REPROGRAMMING AND OTHER DISEASES 123
6.8. NOVELTY AND IMPACT OF THE WORK 124
7. ABBREVIATIONS 125
8. SEMANTICS 127
9. REFERENCES 128
10. APPENDIX 127
|
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| spelling | Ruiz Acero, Guifré Verfasser (DE-588)102609593X aut Molecular mechanisms involved in the induction of pluripotency by Guifré Ruiz Acero Molekulare Mechanismen im Prozess der Zellreprogrammierung 2012 [12], 158 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Nebentitel: Molekulare Mechanismen im Prozess der Zellreprogrammierung Berlin, Freie Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:kobv:188-fudissthesis000000038962-0 http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000038962 kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025277030&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ruiz Acero, Guifré Molecular mechanisms involved in the induction of pluripotency |
| subject_GND | (DE-588)4113937-9 |
| title | Molecular mechanisms involved in the induction of pluripotency |
| title_alt | Molekulare Mechanismen im Prozess der Zellreprogrammierung |
| title_auth | Molecular mechanisms involved in the induction of pluripotency |
| title_exact_search | Molecular mechanisms involved in the induction of pluripotency |
| title_full | Molecular mechanisms involved in the induction of pluripotency by Guifré Ruiz Acero |
| title_fullStr | Molecular mechanisms involved in the induction of pluripotency by Guifré Ruiz Acero |
| title_full_unstemmed | Molecular mechanisms involved in the induction of pluripotency by Guifré Ruiz Acero |
| title_short | Molecular mechanisms involved in the induction of pluripotency |
| title_sort | molecular mechanisms involved in the induction of pluripotency |
| topic_facet | Hochschulschrift |
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