A conditional mouse model to elucidate the role of the CLUH protein in vivo:
Gespeichert in:
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| Format: | Abschlussarbeit Mikrofilm Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Ausgabe: | [Mikrofiche-Ausg.] |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 115 Bl. Ill., graph. Darst. |
Internformat
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| 100 | 1 | |a Hansen, Henriette Elena |d 1981- |e Verfasser |0 (DE-588)1053745729 |4 aut | |
| 245 | 1 | 0 | |a A conditional mouse model to elucidate the role of the CLUH protein in vivo |c vorgelegt von Henriette Elena Hansen |
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| 264 | 1 | |c 2014 | |
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| 776 | 0 | 8 | |i Reproduktion von |a Hansen, Henriette Elena, 1981- |t A conditional mouse model to elucidate the role of the CLUH protein in vivo |d 2014 |
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Datensatz im Suchindex
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| adam_text | 1. TABLE OF CONTENTS
1. TABLE OF CONTENTS 5
2. ABSTRACT 9
3. INTRODUCTION 10
3.1 MITOCHONDRIA AND THEIR DISTRIBUTION AND DYNAMICS 10
3.2 THE CLUH PROTEIN 12
3.2.1 THE CLUH PROTEINS IN DIFFERENT SPECIES 12
3.2.2 THE DOMAINS OF THE MURINE CLUH PROTEIN 15
3.3 STRESS GRANULES 16
3.4 THE MTOR PATHWAY 17
3.4.1 THE MTOKCI AND MTORC2 17
3.4.2 MTORCL ACTIVATION VIA INSULIN SIGNALING 18
3.4.3 AMINO ACID SENSING BY MTORCL 19
3.5 AUTOPHAGY 20
3.5.1 AUTOPHAGOSOME FORMATION 20
3.5.2 THE SIGNALING CASCADES DURING AUTOPHAGOSOME FORMATION 20
3.5.3 SELECTIVE AUTOPHAGY IS MEDIATED BY P62 21
3.5.4 METABOLIC CHANGES AT BIRTH - IMPORTANCE OF AUTOPHAGY AFTER BIRTH
22
4. AIM 24
5. RESULTS 25
5.1 CLUH IS A CYTOSOLIC PROTEIN THAT PARTIALLY CO-LOCALIZES WITH THE
EUKARYOTIC
TRANSLATION INITIATION FACTOR 4G2 25
5.2 CLUH DOES NOT GO TO STRESS GRANULES BUT RESIDES IN TIA-1-POSITIVE
GRANULES UNDER
GLUCOSE DEPRIVATION CONDITIONS 27
5.3 GENERATION OF A CONDITIONAL CLUH KNOCK OUT MOUSE MODEL 29
5.3.1 THE TARGETING STRATEGY 29
5.3.2 RAISING OF THE MOUSE MODEL 31
5.3.3 FULL BODY KNOCK OUT AND PURKINJE NEURONS SPECIFIC KNOCK OUT OF
CLUH GENE 34
5.4 ANALYSIS OF THE FULL BODY KNOCK OUT 35
5.4.1 CHARACTERIZATION OF CLUH
1
MEFS ON MRNA LEVEL SHOWS REDUCED LEVELS OF
CLUH MRNA IN THE CLUH
1
MEFS 35
5.4.2 CHARACTERIZATION OF CLUH
1
MEFS ON PROTEIN LEVEL SHOWS THE COMPLETE
DEPLETION OF THE PROTEIN 35
5.4.3 THE CLUSTERING PHENOTYPE OF THE MITOCHONDRIA IS NOT OBSERVED IN
CLUH
1
MEFS 38
5.4.4 STEADY STATE LEVELS OF SOME NUCLEAR ENCODED MITOCHONDRIAL PROTEINS
ARE
DECREASED IN CLUH
1
MEFS 40
5
HTTP://D-NB.INFO/1058237012
5.4.5 THE RESPIRATORY FUNCTION OF THE MITOCHONDRIA IS NOT IMPAIRED IN
CLUH
1
MEFS AND ATP LEVELS ARE NORMAL 42
5.4.6 NEWBORNS OF THE FULL BODY KNOCK OUT DO NOT SURVIVE THE FIRST DAY
OF LIFE
BUT DO NOT SHOW DEVELOPMENTAL DEFECTS 45
5.4.7 CLUH
1
MICE HAVE PROBLEMS TO SUSTAIN THEIR BLOOD GLUCOSE LEVELS UNDER
STARVATION CONDITIONS 49
5.4.8 LIVERS OF STARVED CLUH PUPS DO NOT SHOW AUTOPHAGOSOMES 50
5.4.9 ANALYSIS OF CLUHMEFS UNDERLINES THE HYPOTHESIS OF IMPAIRED
AUTOPHAGY 51
5.4.10 CLUH PUPS SHOW REDUCED LC3 PROTEIN LEVELS 51
5.4.11 THE MTOR PATHWAY SEEMS TO BE CONSTITUTIVELY ACTIVE 53
5.4.12 STRESS GRANULE FORMATION IN CLUH
1
MEFS 54
5.4.13 STEADY STATE LEVELS OF NUCLEAR ENCODED PROTEINS ARE DECREASED IN
HEART
AND LIVER OF THE FULL BODY KNOCK OUT 56
5.5 KNOCK OUT OF CLUH IN PURKINJE NEURONS, HIGHLY POLARIZED CELLS, DOES
NOT LEAD TO
NEURODEGENERATION UP TO THE AGE OF 20 WEEKS 57
5.5.1 STEADY STALE LEVELS OF CLUH IS HIGHER IN CORTICAL ASTROCYTES THAN
IN CORTICAL
NEURONS 60
MATERIAL AND METHODS 61
6.1 REAGENTS 61
6.2 MOLECULAR BIOLOGICAL METHODS 61
6.2.1 CACH-COMPETENT E. COLI 61
6.2.2 TRANSFORMATION OF E. COLI 61
6.2.3 PICKING E. COLI CLONES FROM LB-PLATES 62
6.2.4 EXTRACTION OF PLASMIDS FROM E.COLI (MIDI-PREP) 62
6.2.5 PHENOL/CHLOROFORM EXTRACTION OF DNA 63
6.2.6 DNA QUANTIFICATION 63
6.2.7 AGAROSE GEL ELECTROPHORESIS 63
6.2.8 GENERAL CLONING PROCEDURE 64
6.2.9 CI I TREATMENT 64
6.2.10 POLYMERASE CHAIN REACTIONS (PCR) FOR CLONING 65
6.2.11 CLONING OF THE TARGETING VECTOR PCLUH 65
6.2.12 SOUTHERN BLOT ANALYSIS OF GENOMIC DNA 66
6.2.13 PREPARATION OF THE PROBES FOR SOUTHERN BLOT ANALYSIS 67
6.2.14 RNA EXTRACTION 67
6.2.15 CDNA SYNTHESIS 68
6.2.16 ANALYSIS OF CDNA BY PCR 68
6.2.17 QUANTITATIVE REAL TIME PCR (QRT-PCR) 69
6.2.18 DNA EXTRACTION FROM MOUSE TAILS 70
6.2.19 GENOTYPING OF THE MOUSE LINES 70
6.3 BIOCHEMICAL METHODS 71
6.3.1 PROTEIN ISOLATION FROM CELLS 71
6.3.2 PROTEIN ISOLATION FROM MOUSE TISSUE 71
6.3.3 PROTEIN QUANTIFICATION 71
6.3.4 SDS-PAGE AND WESTERN BLOTTING 71
6.3.5 MEASURING THE OXYGEN CONSUMPTION OF PRIMARY MHFS 72
6.3.6 DETERMINATION OF CELLULAR ATP CONTENT 73
6.4 CELL CULTURE METHODS 73
6.4.1 CULTURING CELLS 73
6.4.2 TRANSFECTION OF CELLS 74
6.4.3 STRESS GRANULES EXPERIMENT 74
6.4.4 STRESS GRANULES EXPERIMENT FOR QUANTIFICATION IN MEF CELLS 74
6.4.5 MITOTRACKER TREATMENT 75
6.4.6 IMMUNOFLUORESCENCE ON CELLS 75
6.4.7 PREPARING PRIMARY MEF CELLS 75
6.4.8 MTOR ACTIVATION EXPERIMENT (MODIFIED FROM (THEDIECK ET AL., 2013))
76
6.4.9 LC3 EXPERIMENT 76
6.4.10 PREPARING FEEDER CELLS FOR CULTURING MURINE ES CELLS 76
6.4.11 CULTURING EMBRYONIC STEM CELLS 77
6.4.12 TARGETING EMBRYONIC STEM CELLS 77
6.4.13 PICKING ES CLONES 77
6.4.14 EXPANDING POSITIVE ES CLONES 78
6.5 MOUSE MANAGEMENT 79
6.5.1 PERMISSION FOR ANIMAL EXPERIMENTS 79
6.5.2 MOUSE LINES 79
6.5.3 C-SECTION OFA PREGNANT MOUSE 81
6.5.4 PHENOTYPE ANALYSIS OF CLUH ~ PUPS 81
6.5.5 PERFUSION OF MICE 81
6.6 HISTOLOGICAL METHODS 82
6.6.1 VIBRATOME SECTIONS 82
6.6.2 CRYOSTAT SECTIONS 82
6.6.3 IMMUNOFLUORESCENCE ON VIBRATOME SECTIONS 82
6.6.4 HEMATOXYLIN EOSIN STAINING 83
6.6.5 COLLECTING SAMPLES FOR EM 83
6.7 LISTS 84
6.8 MACHINES AND DEVICES 91
7. DISCUSSION 92
7.1 THE FULL BODY KNOCK OUT OF CLUH REVEALS A FUNCTION OF CLUH IN
AUTOPHAGY 92
7.2 AUTOPHAGOSOME FORMATION SEEMS TO BE INHIBITED IN CLUH MEFS 93
7
7.3 THE ROLE OF CLUH IN SGS 95
7.4 THE ROLE OL CLUH IN ENERGY SUPPLY OFTHE CELL 97
7.5 THE FUNCTION OF CLUH IN MITOCHONDRIAL DISTRIBUTION 98
7.6 CONCLUSION 99
8. BIBLIOGRAPHY. 101
|
| any_adam_object | 1 |
| author | Hansen, Henriette Elena 1981- |
| author_GND | (DE-588)1053745729 |
| author_facet | Hansen, Henriette Elena 1981- |
| author_role | aut |
| author_sort | Hansen, Henriette Elena 1981- |
| author_variant | h e h he heh |
| building | Verbundindex |
| bvnumber | BV042153408 |
| ctrlnum | (OCoLC)896290027 (DE-599)DNB1058237012 |
| dewey-full | 572.6919353 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.6919353 |
| dewey-search | 572.6919353 |
| dewey-sort | 3572.6919353 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| edition | [Mikrofiche-Ausg.] |
| format | Thesis Microfilm Book |
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| institution | BVB |
| language | English |
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| physical | 115 Bl. Ill., graph. Darst. |
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| spelling | Hansen, Henriette Elena 1981- Verfasser (DE-588)1053745729 aut A conditional mouse model to elucidate the role of the CLUH protein in vivo vorgelegt von Henriette Elena Hansen [Mikrofiche-Ausg.] 2014 115 Bl. Ill., graph. Darst. txt rdacontent h rdamedia he rdacarrier Köln, Univ., Diss., 2014 Mikroform-Ausgabe Mikrofiche-Ausg.: 2 Mikrofiches : 24x (DE-588)4113937-9 Hochschulschrift gnd-content Reproduktion von Hansen, Henriette Elena, 1981- A conditional mouse model to elucidate the role of the CLUH protein in vivo 2014 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027593186&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hansen, Henriette Elena 1981- A conditional mouse model to elucidate the role of the CLUH protein in vivo |
| subject_GND | (DE-588)4113937-9 |
| title | A conditional mouse model to elucidate the role of the CLUH protein in vivo |
| title_auth | A conditional mouse model to elucidate the role of the CLUH protein in vivo |
| title_exact_search | A conditional mouse model to elucidate the role of the CLUH protein in vivo |
| title_full | A conditional mouse model to elucidate the role of the CLUH protein in vivo vorgelegt von Henriette Elena Hansen |
| title_fullStr | A conditional mouse model to elucidate the role of the CLUH protein in vivo vorgelegt von Henriette Elena Hansen |
| title_full_unstemmed | A conditional mouse model to elucidate the role of the CLUH protein in vivo vorgelegt von Henriette Elena Hansen |
| title_short | A conditional mouse model to elucidate the role of the CLUH protein in vivo |
| title_sort | a conditional mouse model to elucidate the role of the cluh protein in vivo |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027593186&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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