Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. und engl. Sprache |
| Beschreibung: | 167 Bl. Ill., graph. Darst. |
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| 100 | 1 | |a Blanco Redondo, Beatriz |e Verfasser |0 (DE-588)104997235X |4 aut | |
| 245 | 1 | 0 | |a Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster |c von Beatriz Blanco Redondo |
| 246 | 1 | 3 | |a Untersuchungen der Phosphorylierung von Synapsin und Charakterisierung monoklonaler Antikörper der Würzburg Hybridoma Library in Drosophila melanogaster |
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| 502 | |a Würzburg, Univ., Diss., 2014 | ||
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Datensatz im Suchindex
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| adam_text |
STUDIES OF SYNAPSIN PHOSPHORYLATION AND
CHARACTERIZATION OF MONOCLONAL ANTIBODIES FROM
THE WIIRZBURG HYBRIDOMA LIBRARY IN DROSOPHILA
MELA.NOGASTER
UNTERSUCHUNGEN DER PHOSPHORYLIERUNG VON
SYNAPSIN UND CHARAKTERISIERUNG MONOKLONALER
ANTIKORPER DER WIIRZBURG HYBRIDOMA LIBRARY IN
DROSOPHILA MELANOGATER
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE GRADUATE SCHOOL OF LIFE SCIENCES,
JULIUS-MAXIMILIANS-UNIVCRSITAL WIIRZBURG,
SECTION NEUROSCIENCE
SUBMITTED BY
BEATRIZ BLANCO REDONDO
FROM
BARCELONA, SPAIN
WIIRZBURG, 2014
HTTP://D-NB.INFO/1052912095
TABLE OF CONTENT
I. SUMMARY 1
II. ZUSAMMENFASSUNG 3
1. INTRODUCTION 6
1.1 THE SYNAPSE 6
1.1.1 SYNAPTIC VESICLES 9
1.2 CYCLIC AMP-DEPENDENT PROTEIN KINASE A (PKA) 12
1.2.1 THE CATALYTIC SUBUNIT 13
1.2.2 THE REGULATOR)' SUBUNIT 13
1.2.3 DROSPHILAPKA 14
1.3 CALCIUM/CALMODULIN-DEPENDENL PROTEIN KINASE II (CAMKII) 14
1.3.1 DROSOPHILA CAMKII 16
1.4 SYNAPSINS 17
1.4.1 CELLULAR AND SUBCELLULAR DISTRIBUTION OF VERTEBRATES SYNAPSINS 17
1.4.2 INTERACTION WITH SYNAPTIC VESICLES AND CYTOSKELETAL COMPONENTS 18
1.4.3 MAMMALIAN SYNAPSINS 19
1.4.3.1 SYNAPSIN DOMAIN STRUCTURE 20
1.4.3.2 SYNAPSIN TARGETING 20
1.4.3.3 PHOSPHORYLATION OF MAMMALIAN SYNAPSINS 20
1.4.3.4 SYNAPSINS AND HUMAN DISORDERS 22
1.4.4 DROSOPHILA SYNAPSINS 22
1.4.4.1 PHOSPHORYLATION OF DROSOPHILA SYNAPSINS 23
1.4.4.2 SYNAPSIN NULL MUTANTS 24
1.5 SYNAPSE-ASSOCIATED PROTEIN OF 47 KDA (SAP47) 25
1.6 OLFACTORY' RESPONSE IN DROSOPHILA 26
1.7 GAL4/UAS SYSTEM IN DROSOPHILA MELANOGASTER 27
1.8. MONOCLONAL ANTIBODIES 27
1.9 GOALS OF THE THESIS 29
2. MATERIAL 30
2.1 FLY STRAINS 30
2.2 BUFFERS AND REAGENTS 31
2.2.1 DNA AND RNA ANALYSIS 31
2.2.1.1 PRIMERS 31
* 2.2.1.2 REAGENTS FOR GENOMIC DNA ISOLATION 32
2.2.2 PROTEIN ANALYSIS 33
"'2.2.2.1 SDS-PAGE 33
2.2.2.2 WESTERN BLOT 34
2.2.2.3 SILVER STAINING (MODIFIED AFTER BLUM ET AL., FOR MASS
SPECTROMETRY). 35
2.2.2.4 TWO DIMENSIONAL SDS-PAGE 35
2.2.3 IMMUNOCHEMISTRY 36
2.2.3.1 IMMUNOPRECIPITATION 36
2.2.3.2 COVALENT COUPLING OF ANTIBODIES TO PROTEIN-G-AGAROSE FOR IP 37
2.2.4 IMMUNOHISTOCHEMISTRY 37
2.2.4.1 ADULT AND LARVA BRAIN WHOLE MOUNTS 37
2.2.4.2 LARVA NMJ DISSECTIONS 38
2.2.4.3 IMMUNOSTAINING OF CRYOSECTIONS 39
2.2.5 HYBRIDOMA CELL CULTURE 39
2.2.6 CHEMICALS 40
2.3 ANTIBODIES 40
2.3.1 PRIMARY ANTIBODIES 40
2.3.2 SECONDARY ANTIBODIES 41
2.4 LADDERS 41
2.4.1 PAGERULER* PRE-STAINCD PROTEIN LADDER (FERMENTAS) 41
2.4.2 GENERULER* 100 BP DNA LADDER (FERMENTAS) 41
3. METHODS 42
3.1 HANDLING DROSOPHIHI MCLANOGASLCR. 42
3.2 DNA ANALYSIS 43
3.2.1 ISOLATION AND PURIFICATION OF GENOMIC DNA 43
3.2.2 POLYMERASE CHAIN REACTION (PCR) 43
3.2.3 PCR LOR SEQUENCING 44
3.2.4 PURIFICATION OF DNA FROM PCR FOR SEQUENCING 45
3.3 PROTEIN ANALYSIS 46
3.3.1 1D-SDS-PAGE ANALYSIS 46
3.3.1.1 NON-PRE-CAST SDS-PAGE SYSTEM 46
3.3.1.2 PRE-CAST SDS-PAGE SYSTEM (FROM INVITROGEN) 47
3.3.2 WESTERN BLOTTING 47
3.3.2.1 TRANSFER 48
3.3.2.2 BLOCKING 49
3.3.2.3 DETECTION 49
3.3.2.4 ALKALINE PHOSPHATASE TREATMENT 50
3.3.3 2D-SDS-PAGE ANALYSIS 50
3.3.4 BLUM SILVER STAINING 53
3.4 IMMUNOCHEMISTRY PROCEDURES 54
3.4.1 IMMUNOPRECIPITATION 54
3.4.2 COVALENT COUPLING OF ANTIBODIES TO PROTEIN-G-AGAROSE FOR
IMMUNOPRECIPITATION 55
3.4.2.1 PREPARATION OF PROTEIN-G-AGAROSE 55
. 3.4.2.2 COUPLING PROCEDURE 55
3.5 IMMUNOHISTOCHEMISTRY PROCEDURES 55
3.5.1 ADULT BRAIN WHOLE MOUNTS 56
3.5.2 LARVA BRAIN WHOLE MOUNTS 56
3.5.3 LARVA NMJ DISSECTIONS 57
3.5.4 IMMUNOSTAINING OF CRYOSECTIONS 57
3.6 HYBRIDOMA CELL CULTURE 58
3.7 * BEHAVIORAL ASSAYS 59
3J-1 SHORT-TERM OLFACTORY HABITUATION 59
3.8 ANTISERA GENERATION 60
4. RESULTS 61
4.1 STUDIES OF SYNAPSIN PHOSPHORYLATION 61
4.1.1 SPECIFICITY OF THE ANTISERA 61
4.1.2 TRANSGENIC SYNAPSIN MUTANTS 66
4.1.3 THE ROLE OFPKA 70
4.1.4 THE ROLE OR CAMKII 72
4.1.5. AGE STUDIES 76
4.1.6 BEHAVIORAL ANALYSIS 77
4.1.7 DISTRIBUTION OF SYNAPSIN PHOSPHORYLATED AT S6 SITE IN THE
DROSOPHILA BRAIN
.'. 81
4.1.7.1 WILD-TYPE HIES 81
4.1.7.2 SYNAPSIN MUTANTS 82
4.1.7.3 SYNAPSIN TRANSGENIC HIES 84
4.1.7.4 RING NEURONS SPECIFIC (C232-GAL4 LINE) 85
4.1.8 LILE EXPECTANCY 86
4.1.9 INTERACTION BETWEEN SYNAPSIN AND SAP47 87
4.1.10 VARIABILITY OF SYNAPSIN PROTEIN 90
4.1.11 ALKALINE PHOSPHATASE TREATMENT 91
4.2 CHARACTERIZATION OF MONOCLONAL ANTIBODIES FROM THE WURZBURG
HYBRIDOMA
LIBRARY AGAINST DROSOPLIILA BRAIN 92
4.2.1 ANTIBODY DESCRIPTION 92
4.2.2 CO-LOCALIZATION OF NBL68 AND PDF 94
4.2.3 NBL69 ANTIBODY 95
4.2.4 COMPARISON BETWEEN JANELIA FARM GAL4 LINES AND THE MONOCLONAL
ANTIBODIES
98
4.2.5 COMPARISON BETWEEN ANION GAL4 LINES AND THE MONOCLONAL
ANTIBODIES. 101
4.2.5.1 ANION GAL4 AND NBL68. DIFFERENCES BETWEEN LARVA AND ADULT HIES
. 103
5. DISCUSSION 105
5.1 STUDIES OF SYNAPSIN PHOSPHORYLATION 105
5.1.1 SPECIFICITY OF THE ANTISERA 106
5.1.2 PKA PHOSPHORYLATES SYNAPSIN AT S6 SITE 109
5.1.3 NOT ONLY PKA PHOSPHORYLATES SYNAPSIN AT S6 SITE 111
5.1.4.CAMKII PHOSPHORYLATES SYNAPSIN AT S6 SITE BUT NOT AT S464 SITE 112
5.1.5 SYNAPSIN AND THE AGE-RELATED MEMORY IMPAIRMENT 114
5.1.6 SYNAPSIN NULL MUTANTS DO NOT SHOW SHORTER LIFESPAN 115
5.1.7 SYNAPSIN AND SAP47 DO NOT INTERACT WITH EACH OTHER DIRECTLY 115
5.1.8 VARIABILITY OF SYNAPSIN PROTEIN 117
5.2 IDENTILICATION AND STRUCTURAL CHARACTERIZATION OF INTEMEURONS OF THE
DROSOPHILA
BRAIN BY MONOCLONAL ANTIBODIES OF THE WURZBURG HYBRIDOMA LIBRARY 119
5.2.1 ANTIBODY DESCRIPTION 119
"5.2.2 THE MAB NBL68 CO-LOCALIZES WITH PDF 120
5.2.3 JANELIA GAL4 COLLECTION 120
5.2.4 ANION GAL4 121
5.2.5 THE NBL69 ANTIBODY 122
6. REFERENCES 123
7. APPENDIX 144
7.1 SYNAPSIN GENE 144
7.2 SYNAPSIN GENSPAN 144
7^3 LIST OF TABLES 154
7.4 LIST OF FIGURES 155
7.5 LIST OF ABBREVIATIONS 158
7.6 AFFIDAVIT/ EIDESSTATTLICHE ERKLARUNG 161
8. CURRICULUM VITAE 162
9. ACKNOWLEDGMENTS 165 |
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| spelling | Blanco Redondo, Beatriz Verfasser (DE-588)104997235X aut Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster von Beatriz Blanco Redondo Untersuchungen der Phosphorylierung von Synapsin und Charakterisierung monoklonaler Antikörper der Würzburg Hybridoma Library in Drosophila melanogaster 2014 167 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache Würzburg, Univ., Diss., 2014 Neurowissenschaften (DE-588)7555119-6 gnd rswk-swf Taufliege (DE-588)4078168-9 gnd rswk-swf Monoklonaler Antikörper (DE-588)4040094-3 gnd rswk-swf Synapsine (DE-588)4339319-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Taufliege (DE-588)4078168-9 s Synapsine (DE-588)4339319-6 s Monoklonaler Antikörper (DE-588)4040094-3 s Neurowissenschaften (DE-588)7555119-6 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-93766 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-93766 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027249935&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Blanco Redondo, Beatriz Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster Neurowissenschaften (DE-588)7555119-6 gnd Taufliege (DE-588)4078168-9 gnd Monoklonaler Antikörper (DE-588)4040094-3 gnd Synapsine (DE-588)4339319-6 gnd |
| subject_GND | (DE-588)7555119-6 (DE-588)4078168-9 (DE-588)4040094-3 (DE-588)4339319-6 (DE-588)4113937-9 |
| title | Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster |
| title_alt | Untersuchungen der Phosphorylierung von Synapsin und Charakterisierung monoklonaler Antikörper der Würzburg Hybridoma Library in Drosophila melanogaster |
| title_auth | Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster |
| title_exact_search | Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster |
| title_full | Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster von Beatriz Blanco Redondo |
| title_fullStr | Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster von Beatriz Blanco Redondo |
| title_full_unstemmed | Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster von Beatriz Blanco Redondo |
| title_short | Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the Würzburg Hybridoma Library in Drosophila melanogaster |
| title_sort | studies of synapsin phosphorylation and characterization of monoclonal antibodies from the wurzburg hybridoma library in drosophila melanogaster |
| topic | Neurowissenschaften (DE-588)7555119-6 gnd Taufliege (DE-588)4078168-9 gnd Monoklonaler Antikörper (DE-588)4040094-3 gnd Synapsine (DE-588)4339319-6 gnd |
| topic_facet | Neurowissenschaften Taufliege Monoklonaler Antikörper Synapsine Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-93766 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027249935&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT blancoredondobeatriz studiesofsynapsinphosphorylationandcharacterizationofmonoclonalantibodiesfromthewurzburghybridomalibraryindrosophilamelanogaster AT blancoredondobeatriz untersuchungenderphosphorylierungvonsynapsinundcharakterisierungmonoklonalerantikorperderwurzburghybridomalibraryindrosophilamelanogaster |