The role of raf-mediated signalling pathways for motoneuron: development and survival
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2000
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2000 |
| Beschreibung: | IV, 138, 2 Bl. Ill. graph. Darst. |
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| 100 | 1 | |a Pei, Geng |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a The role of raf-mediated signalling pathways for motoneuron |b development and survival |c von Geng Pei |
| 264 | 1 | |c 2000 | |
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Datensatz im Suchindex
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CONTENTS
1. ABSTRACT
1
2. INTRODUCTION 3
2.1. CELLULAR FUNCTION OF THE RAS-MAPK SIGNALLING PATHWAY 3
2.2. MEMBERS OF THE
RAF GENE FAMILY AND THEIR ROLE IN INTRACELLULAR SIGNAL
TRANSDUCTION 4
2.3. RAF PROTEIN STRUCTURE AND EXPRESSION 5
2.4. MECHANISMS OF RAF ACTIVATION 6
2.5. RAF-MAPK SIGNALLING PATHWAY IN THE CNS 7
2.6. EXPRESSION OF MEMBERS OF THE
RAF FAMILY IN THE CNS 8
2.7. RAF KINASES AND APOPTOSIS 9
2.7.1. GENERAL FACTORS OF APOPTOSIS. 9
2.7.2. MECHANISMS OF APOPTOSIS 11
2.7.2.1. MITOCHONDRIA AND APOPTOSIS 11
2.12.2.
A TRIGGER IN APOPTOSIS: CYTOCHROME C RELEASE FROM
MITOCHONDRIA 14
2.7.2.3. THE BCL-2 PROTEIN FAMILY AND APOPTOSIS 15
2.7.2.4. THE DOWNSTREAM MEDIATORS OF APOPTOSIS: CASPASES
AND THEIR INHIBITORS 17
2.7.3. ACTIVATION OF RAF KINASES AND APOPTOSIS 19
2.8. THE OBJECTIVE OF THIS STUDY 21
2.9. BRIEF SUMMARY OF THE RESULTS 22
3. MATERIALS AND METHODS
24
3.1. EXPERIMENTAL ANIMALS AND TISSUE PREPARATION 24
3.2. ANTIBODIES 25
3.3. WESTERN BLOTTING 26
3.4. RAF SINGLE, RAF AND MITOCHONDRIAL MARKER DOUBLE IMMUNOFLUORESCENCE
IN VIVO AND IMMUNOFLUORESCENCE CONFOCAL MICROSCOPY 27
3.5. CYTOCHROME C IMMUNOFLUORESCENCE 28
3.6. PREADSORPTION CONTROLS FOR IMMUNOFLUORESCENCE STAINING 29
3.7. MOUSE MOTONEURON CULTURES AND INDUCTION OF RAF ACTIVATION IN MOUSE
EMBRYONIC MOTONEURONS IN VITRO 29
3.8. IMMUNOSTAINING OF CULTURED MOTONEURONS: DETECTION OF MEMBERS OF
THE RAF FAMILY 31
3.9. DETERMINATION OF MOUSE MOTONEURON SURVIVAL IN WILD-TYPE AND EL 3
B-RAF/+ AND B-RAF' MICE 32
3.10. IMMUNODOUBLELABELLING OF CULTURED E12.5
B-RAF MUTANT MOTONEURONS
WITH B-RAF AND NEUROFILAMENT 32
I
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/959865209
3.11. IMMUNOHISTOFLUORESCENCE FOR RAFS, P75
NTR AND ISLET-1 IN E13
B-RAF AND E14 C-RAF-1 MUTANT MOUSE SPINAL CORDS IN VIVO 33
3.12. CALCULATION AND STATISTICS 34
3.13. GENOTYPING OF
P75S
YY
', B-RAF AND C-RAF-1 MICE 34
3.14. TRANSECTION OF THE FACIAL NERVE AND DETERMINATION OF FACIAL
MOTONEURON NUMBERS IN BRAINSTEM SERIAL SECTIONS 36
3.15. VECTOR CONSTRUCTION 37
3.16. PRIMARY NEURONAL CULTURE 38
3.17. TRANSFECTION BY TRITURATION 39
3.18. STAINING FOR LACZ EXPRESSION 39
3.19. IMMUNOSTAINING AND FLUORESCENCE MICROSCOPY OF CULTURED SENSORY
NEURONS 40
3.20. WESTERN BLOT ANALYSIS AND SEMIQUANTITATIVE ANALYSIS OF ITA
EXPRESSION 41
3.21. RT-PCR ANALYSIS 42
4. RESULTS
43
4.1. EFFECTS OF NEUROTROPHIC FACTORS ON ISOLATED MOTONEURONS 43
4.1.1. ISOLATION OF MOTONEURONS FROM EMBRYONIC MICE 43
4.1.2. THE EFFECT OF BDNF ON CULTURED MOTONEURONS FROM CONTROL AND
P75NTR /' MICE 46
4.1.3. CHARACTERIZATION OF THE NEUROTROPHIN EFFECTS IN CULTURED
P75NM-
DEFICIENT MOTONEURONS 47
4.1.4. NGF REDUCES FACIAL MOTONEURON SURVIVAL AFTE AXOTOMY: ROLE OF
P75NTR IN VIVO 53
4.2. THE ROLE OF BCL-2 FOR MEDIATION OF NEUROTROPHIC FACTOR SIGNALS ON
MOTONEURON SURVIVAL 54
4.2.1. EXPRESSION AND CELLULAR LOCALIZATION OF RAF KINASES IN
BCL-2
DEFICIENT MOTONEURONS 54
4.3. THE ROLE OF CYTOCHROME C: DEVELOPMENTAL EXPRESSION IN MOUSE SPINAL
CORD AND IMMUNOLOCALIZATION IN MOTONEURONS IN THE PRESENCE AND
ABSENCE OF BCL-2 GENE 55
4.3.1. DEVELOPMENTAL EXPRESSION OF CYTOCHROME C IN NORMAL MOUSE
SPINAL CORD AND ITS EXPRESSION IN BCL-2* * AND BCL-2 MOUSE
SPINAL CORDS BY WESTERN BLOT ANALYSIS 55
4.3.2. CYTOCHROME C IMMUNOSTAINING IN
BCL-2*'* AND BCL-2 '' MOUSE
SPINAL AND FACIAL MOTONEURONS 57
4.4. EXPRESSION AND LOCALIZATION OF RAFS IN THE DEVELOPING SPINAL CORD
AND IN
MOTONEURONS 58
4.4.1. DEVELOPMENT OF TECHNIQUES FOR IMMUNOLOCALIZATION OF RAFS IN
MOTONEURONS: SPECIFICITY OF RAF ANTIBODIES FOR THEIR CORRESPONDING
RAF ISOFORMS 58
4.4.2. EXPRESSION OF A-RAF, B-RAF AND C-RAF-1 PROTEINS IN MOUSE SPINAL
CORD AT DIFFERENT EMBRYONIC AND POSTNATAL STAGES 61
4.4.3. DETECTION OF THE DISTRIBUTION OF A-RAF, B-RAF AND C-RAF-1 PROTEIN
KINASES IN MOUSE MOTONEURONS WITHIN THE VENTRAL HORN OF THE
SPINAL CORD AND THE FACIAL NUCLEUS 63
4.4.4. B-RAF AND C-RAF-1 ARE COLOCALIZED WITH A-6403 MITOCHONDRIAL
MARKER CYTOCHROME C OXIDASE SUBUNIT I IN MOUSE MOTONEURONS 66
II
4.4.5. REDISTRIBUTION OF RAF PROTEIN KINASES TO THE PERINUCLEAR SPACE
AND
TO THE NUCLEUS OF MOTONEURONS AFTER ADDITION OF NEUROTROPHIC
FACTORS IN VITRO 69
4.4.6. INTRACELLULAR INVOLVEMENT OF B-RAF KINASE SIGNALLING IN
ANTI-APOPTOTIC
EFFECTS VIA NEUROTROPHIC FACTORS IN MOTONEURONS 71
4.4.6.1. IMMUNOHISTOCHEMICAL DETECTION OF A-RAF, B-RAF AND
C-RAF-1 PROTEIN KINASES IN E13 B-RAF* AND B-RAF
MOUSE SPINAL MOTONEURONS, AND IN E14 C-RAF-L*'* AND
C-RAF-L' MOUSE SPINAL MOTONEURONS 71
4.4.6.1.1. EXPRESSION IN E13 B-RAF* AND B-RAF MOUSE SPINAL
MOTONEURONS 71
4.4.6.1.2. EXPRESSION IN E14 C-RAF-L*'* AND C-RAF-L ' MOUSE
SPINAL MOTONEURONS 72
4.4.6.2. ISLET-1 AND P75
NTR IMMUNOREACTIVITIES IN MOTONEURONS
ARE DECREASED IN B-RAF MOUSE BUT NO DIFFERENCES ARE
DETECTABLE BETWEEN C-RAF-L*'* AND C-RAF-L/ MICE 75
4.4.6.2.1. EXPRESSION IN E13
B-RAF* AND B-RAF MOUSE SPINAL
MOTONEURONS 75
4.4.6.2.2. EXPRESSION IN E14 C-RAF-L*'* AND C-RAF-L'' MOUSE
SPINAL MOTONEURONS 75
4.4.6.3. ABSENCE OF
B-RAF GENE BUT NOT C-RAF-L LEADS TO LOSS OF
VIABILITY OF MOUSE MOTONEURONS IN VITRO 78
4.4.6.4. TRANSFECTION OF
B-RAF EXPRESSION PLASMIDS TO B-RAF
MOTONEURONS AND SENSORY NEURONS RESCUES NEUROTROPHIC
FACTOR-MEDIATED SURVIVAL 83
4.4.6.4.1. DEVELOPMENT OF TECHNIQUES FOR PLASMID TRANSFECTION
OF PRIMARY SENSORY AND SYMPATHETIC NEURONS 83
A. NGF UPREGULATES ITA EXPRESSION VIA PI-3
PATHWAYS IN CULTURED NEURONS 83
B. OVEREXPRESSION OF ITA PROMOTES NEURONAL
SURVIVAL IN PRIMARY SENSORY AND SYMPATHETIC
NEURONS 87
C.
ITA ANTISENSE EXPRESSION ABOLISHES NGF-MEDIATCD
SURVIVAL IN PRIMARY SENSORY AND SYMPATHETIC
NEURONS 93
4.4.6.4.2. TRANSFECTION OF
B-RAF EXPRESSION PLASMIDS TO B-RAF
MOTONEURONS AND SENSORY NEURONS RESCUES NEUROTROPHIC
FACTOR-MEDIATED SURVIVAL 95
5. DISCUSSION 99
5.1. EXPRESSION OF RAF PROTEIN KINASES DURING EMBRYONIC AND POSTNATAL
DEVELOPMENT 99
5.2. IMMUNOHISTOCHEMICAL DISTRIBUTION OF THREE RAF PROTEIN KINASES IN
MOUSE
MOTONEURONS WITHIN THE SPINAL VENTRAL HORN AND THE FACIAL NUCLEUS 101
5.3. SUBCELLULAR LOCALIZATION OF RAF PROTEIN KINASES: COLOCALIZATION
WITH
THE A-6403 MITOCHONDRIAL MARKER IN MOUSE MOTONEURONS 102
5.4. INTRACELLULLAR REDISTRIBUTION OF RAF PROTEIN KINASES IN MOTONEURONS
III
AFTER ACTIVATION BY NEUROTROPHIC FACTORS
IN VITRO 104
5.5. B-RAF KINASE AND APOPTOSIS IN MOTONEURONS 106
6. REFERENCES I J 3
7. ABBREVIATIONS I3
6
IV |
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| indexdate | 2024-10-09T18:10:54Z |
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| language | English |
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| physical | IV, 138, 2 Bl. Ill. graph. Darst. |
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| spelling | Pei, Geng Verfasser aut The role of raf-mediated signalling pathways for motoneuron development and survival von Geng Pei 2000 IV, 138, 2 Bl. Ill. graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2000 Zelldifferenzierung (DE-588)4067536-1 gnd rswk-swf Raf-Kinasen (DE-588)4422659-7 gnd rswk-swf Maus (DE-588)4169148-9 gnd rswk-swf Raf Biochemie (DE-588)4534420-6 gnd rswk-swf Motoneuron (DE-588)4135747-4 gnd rswk-swf Signaltransduktion (DE-588)4318717-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Motoneuron (DE-588)4135747-4 s Zelldifferenzierung (DE-588)4067536-1 s Raf Biochemie (DE-588)4534420-6 s DE-604 Maus (DE-588)4169148-9 s Signaltransduktion (DE-588)4318717-1 s Raf-Kinasen (DE-588)4422659-7 s DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009065325&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Pei, Geng The role of raf-mediated signalling pathways for motoneuron development and survival Zelldifferenzierung (DE-588)4067536-1 gnd Raf-Kinasen (DE-588)4422659-7 gnd Maus (DE-588)4169148-9 gnd Raf Biochemie (DE-588)4534420-6 gnd Motoneuron (DE-588)4135747-4 gnd Signaltransduktion (DE-588)4318717-1 gnd |
| subject_GND | (DE-588)4067536-1 (DE-588)4422659-7 (DE-588)4169148-9 (DE-588)4534420-6 (DE-588)4135747-4 (DE-588)4318717-1 (DE-588)4113937-9 |
| title | The role of raf-mediated signalling pathways for motoneuron development and survival |
| title_auth | The role of raf-mediated signalling pathways for motoneuron development and survival |
| title_exact_search | The role of raf-mediated signalling pathways for motoneuron development and survival |
| title_full | The role of raf-mediated signalling pathways for motoneuron development and survival von Geng Pei |
| title_fullStr | The role of raf-mediated signalling pathways for motoneuron development and survival von Geng Pei |
| title_full_unstemmed | The role of raf-mediated signalling pathways for motoneuron development and survival von Geng Pei |
| title_short | The role of raf-mediated signalling pathways for motoneuron |
| title_sort | the role of raf mediated signalling pathways for motoneuron development and survival |
| title_sub | development and survival |
| topic | Zelldifferenzierung (DE-588)4067536-1 gnd Raf-Kinasen (DE-588)4422659-7 gnd Maus (DE-588)4169148-9 gnd Raf Biochemie (DE-588)4534420-6 gnd Motoneuron (DE-588)4135747-4 gnd Signaltransduktion (DE-588)4318717-1 gnd |
| topic_facet | Zelldifferenzierung Raf-Kinasen Maus Raf Biochemie Motoneuron Signaltransduktion Hochschulschrift |
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