Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn
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| 100 | 1 | |a Schmitt, Dominique |d 1986- |e Verfasser |0 (DE-588)1134987722 |4 aut | |
| 245 | 1 | 0 | |a Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain |b = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn |c submitted by Dominique Schmitt from Lichtenfels |
| 246 | 1 | 1 | |a Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn |
| 264 | 1 | |a Würzburg |c January 2017 | |
| 300 | |a XII, 156 Blätter |b Illustrationen | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Julius-Maximilians-Universität Würzburg |d 2017 | ||
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| 546 | |a Zusammenfassung in deutscher und englischer Sprache | ||
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| 653 | |a Syap1 | ||
| 653 | |a Sap47 | ||
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Datensatz im Suchindex
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| adam_text | INITIAL CHARACTERIZATION OF MOUSE SYAPL IN THE NERVOUS SYSTEM:
SEARCH FOR INTERACTION PARTNERS, EFFECTS OF GENE KNOCKDOWN AND
KNOCKOUT, AND TISSUE DISTRIBUTION WITH FOCUS ON THE ADULT BRAIN
ERSTE CHARAKTERISIERUNG DES MAUS-SYAPL IM NERVENSYSTEM:
SUCHE NACH INTERAKTIONSPARTNERN, AUSWIRKUNGEN VON GEN-
KNOCKDOWN UND-KNOCKOUT SOWIE UNTERSUCHUNGEN UEBER DIE
VERTEILUNG IM GEWEBE MIT FOKUS AUF DAS ADULTE GEHIRN
DOCTORAL THESIS FOR A DOCTORAL DEGREE AT THE INSTITUTE FOR CLINICAL
NEUROBIOLOGY,
UNIVERSITY HOSPITAL WUERZBURG AND GRADUATE SCHOOL OF LIFE SCIENCES,
JULIUS-MAXIMILIAN-UNIVERSITY WUERZBURG
SECTION: NEUROSCIENCE
SUBMITTED BY
DOMINIQUE SCHMITT
FROM
LICHTENFELS
WUERZBURG, JANUARY 2017
TABLE OF CONTENTS
TABLE O F
CONTENTS................................................................................................................................I
LL
LIST O F
FIGURES.......................................................................................................................................VI
LIST OF
TABLES.....................................................................................................................................
VIII
ABBREVIATIONS......................................................................................................................................IX
ZUSAMMENFASSUNG.............................................................................................................................
13
ABSTRACT.................................................................................................................................................
15
1.
INTRODUCTION................................................................................................................................17
1.1 SAP47 - THE
DROSOPHILA HOMOLOGUE OF MAMMALIAN SYAPL
..........................................
17
1.2 THE MAMMALIAN SYNAPSE ASSOCIATED PROTEIN
1............................................................18
1.3 AKT/PROTEIN KINASE B - A MULTIFUNCTIONAL CELLULAR INTERFACE
........................................
19
1.4 TBCE AND ITS IMPORTANT FUNCTION IN TUBULIN FOLDING
..................................................
21
1.5 VESICULAR TRAFFICKING AND PROTEIN SORTING
....................................................................
23
1.6 THE
SYAPL KNOCKOUT MOUSE: STEM CELL SELECTION AND MOUSE PRODUCTION
..................
27
1.7 AIMS OF THE
THESIS.........................................................................................................29
2. MATERIALS AND M
ETHODS.............................................................................................................30
2.1
MATERIALS.....................................................................................................................
30
2.1.1
ANIMALS................................................................................................................30
2.1.2 CELL
LINES:..............................................................................................................
30
2.1.3 BUFFERS AND
SOLUTIONS............................................................................................32
2.1.4 SUPPLIERS FOR MEDIA, SOLUTIONS AND SUPPLEMENTS
.................................................
34
2.1.5
KITS.......................................................................................................................
35
2.1.6
ANTIBODIES............................................................................................................
35
2.1.7
PROGRAMS..............................................................................................................37
2.2
METHODS.....................................................................................................................
38
2.2.1 SYAPL KNOCKOUT MOUSE GENOTYPING
.....................................................................
38
2.2.2 DNA PURIFICATION FROM AGAROSE GEL
.....................................................................
39
2.2.3 DNA
SEQUENCING..................................................................................................39
2.2.4 RNA
ISOLATION......................................................................................................
39
2.2.5 CDNA
SYNTHESIS....................................................................................................40
2.2.6
RT-PCR...............................................................................................................
40
2.2.7 PRODUCTION OF SYAPL KNOCKDOWN AND CONTROL LENTIVIRUSES
.................................
40
2.2.8 PROTEIN EXTRACTION FROM MOUSE
TISSUE..................................................................41
2.2.9 PROTEIN ISOLATION FROM MOUSE
FAT.........................................................................42
2.2.10 CRUDE SUBCELLULAR FRACTIONATION OF NSC34
CELLS..................................................42
2.2.11 SYNAPTIC VESICLE
ISOLATION....................................................................................
42
2.2.12
IMMUNOCYTOCHEMISTRY........................................................................................43
2.2.13 IMMUNOHISTOCHEMISTRY OF VIBRATOME
SECTIONS...................................................44
2.2.14 IMMUNOHISTOCHEMISTRY OF NEUROMUSCULAR JUNCTIONS
.........................................
45
2.2.15 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS AND WESTERN BLOT ANALYSIS
..................
45
2.2.16 WESTERN
BLOTTING.................................................................................................
47
2.2.17 CELL
CULTURE.........................................................................................................
47
2.2.18 MICROTUBULE FRACTIONATION
..................................................................................
50
2.2.19 STIMULATION
EXPERIMENTS.....................................................................................
51
2.2.20 BREFELDIN A TREATMENT OF CULTURED PRIMARY MOTONEURONS
..................................
51
2.2.21
CO-IMMUNOPRECIPITATION...................................................................................
52
2.2.22 PROTEIN
CROSS-LINKING...........................................................................................53
2.2.23
MICROSCOPY.........................................................................................................
53
2.2.24 STATISTICAL
ANALYSIS...............................................................................................
53
3.
RESULTS.........................................................................................................................................54
3.1 SYAP 1 ANTIBODY SPECIFICITY
TEST...................................................................................
54
3.1.1 VALIDATION OF ANTIBODY SPECIFICITY FOR BIOCHEMICAL ANALYSIS
................................
54
3.1.2 VALIDATION OF ANTIBODY SPECIFICITY FOR IMMUNOFLUORESCENCE ANALYSIS
..................
56
3.2 SUBCELLULAR LOCALIZATION OF SYAP 1
PROTEIN...................................................................58
3.2.1 SYAPL ACCUMULATES IN CLOSE PROXIMITY TO ORGANELLES OF THE
SECRETORY PATHWAY.... 58
3.2.2 BREFELDIN A TREATMENT DISRUPTS GOLGI ASSOCIATION OF SYAPL
.................................
66
3.2.3 SYAPL IS FOUND IN CYTOSOLIC AND MEMBRANOUS FRACTIONS
......................................
68
3.2.3 SYAPL IS PRESENT IN AXONAL GROWTH CONES AND SYNAPTIC VESICLES
....
.
......................69
3.3 SYAPL PROTEIN IS NOT RESTRICTED TO
SYNAPSES................................................................73
3.4 FUNCTIONAL ANALYSIS OF SYAPL
PROTEIN.........................................................................74
3.5 SYAPL KNOCKDOWN DOES NOT AFFECT AKT PHOSPHORYLATION IN PRIMARY
MOTONEURONS... 79
3.6 POSSIBLE INTERACTION PARTNERS OF
SYAPL......................................................................
87
3.6.1 POSSIBLE INTERACTION OF SYAPL AND TBCE
PROTEIN................................................. 87
3.6.2 POSSIBLE INTERACTION OF SYAPL AND AKT
PROTEIN.....................................................91
3.7 INITIAL CHARACTERIZATION OF THE FIRST
SYAPL KNOCKOUT MOUSE.......................................93
3.7.1 CHANGING THE GENETIC
BACKGROUND.....................................................................
93
3.7.2 ESTABLISHMENT OF A GENOTYPING
PROTOCOL..............................................................93
3.7.3 PHENOTYPIC CHARACTERIZATION OF THE SYAPL KNOCKOUT MOUSE
...............................
95
3.7.4 SYAPL PROTEIN DISTRIBUTION IN
VIVO.......................................................................
98
3.8 VERIFICATION OF SYAPL KNOCKDOWN DATA AND FUNCTIONAL ANALYSIS FROM
ISOLATED SYAPL
MOTONEURONS.......................................................................................................................
116
3.8.1 SUBCELLULAR DISTRIBUTION OF SYAPL PROTEIN IN SYAPLA MOTONEURONS
...................
116
3.8.2 MORPHOLOGICAL AND FUNCTIONAL ANALYSIS OF SYAPL PROTEIN IN SYAPL 1
CULTURED
MOTONEURONS....................................................................................................................118
3.8.3 AKT-PHOSPHORYLATION IS NOT ALTERED IN SYAPL A MOTONEURONS AND IN
VIVO..........120
4.
DISCUSSION..................................................................................................................................123
4.1 GENERATION OF THE FIRST SYAPL KNOCKOUT
MOUSE.........................................................123
4.2 SYAPL ANTIBODY
SPECIFICITY.........................................................................................
124
4.3 SUBCELLULAR DISTRIBUTION OF SYAPL
IN
VITRO.................................................................
125
4.4 SYAPL
EXPRESSION.......................................................................................................127
4.5 SYAPL DISTRIBUTION IN THE MOUSE NERVOUS
SYSTEM..................................................... 128
4.6 SYAPL AND ITS INTERACTION
PARTNERS............................................................................
129
4.7 FUNCTIONAL
ANALYSIS...................................................................................................
131
4.8
CONCLUSION................................................................................................................
135
5.
REFERENCES..................................................................................................................................136
6. A
PPENDIX...................................................................................................................................150
6.1 SYAPL OVEREXPRESSION
VECTOR....................................................................................
150
6.2 GENOTYPING AND QRT-PGR PRIMER SEQUENCES
..........................................................
150
6.3 NUCLEAR/CYTOSOLIC FRACTIONATION OF NSC34
CELLS........................................................150
6.4 SH-SYAPL SEQUENCE
BLAST........................................................................................
151
AFFIDAVIT/EIDESSTATTLICHE
ERKLAERUNG..............................................................................................152
CURRICULUM
VITAE..............................................................................................................................
153
ACKNOWLEDGEMENTS..........................................................................................................................
155
|
| any_adam_object | 1 |
| author | Schmitt, Dominique 1986- |
| author_GND | (DE-588)1134987722 |
| author_facet | Schmitt, Dominique 1986- |
| author_role | aut |
| author_sort | Schmitt, Dominique 1986- |
| author_variant | d s ds |
| building | Verbundindex |
| bvnumber | BV044344751 |
| collection | ebook |
| ctrlnum | (OCoLC)1159314281 (DE-599)DNB1130588033 |
| dewey-full | 610 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 610 - Medicine and health |
| dewey-raw | 610 |
| dewey-search | 610 |
| dewey-sort | 3610 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| format | Thesis Book |
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| id | DE-604.BV044344751 |
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| index_date | 2024-09-20T13:29:36Z |
| indexdate | 2024-11-25T17:51:13Z |
| institution | BVB |
| language | English |
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| physical | XII, 156 Blätter Illustrationen |
| psigel | ebook |
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| publishDateSort | 2017 |
| record_format | marc |
| spellingShingle | Schmitt, Dominique 1986- Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn Synapse (DE-588)4184224-8 gnd Motoneuron (DE-588)4135747-4 gnd Nervensystem (DE-588)4041643-4 gnd Golgi-Apparat (DE-588)4192795-3 gnd |
| subject_GND | (DE-588)4184224-8 (DE-588)4135747-4 (DE-588)4041643-4 (DE-588)4192795-3 (DE-588)4113937-9 |
| title | Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn |
| title_alt | Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn |
| title_auth | Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn |
| title_exact_search | Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn |
| title_full | Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn submitted by Dominique Schmitt from Lichtenfels |
| title_fullStr | Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn submitted by Dominique Schmitt from Lichtenfels |
| title_full_unstemmed | Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn submitted by Dominique Schmitt from Lichtenfels |
| title_short | Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain |
| title_sort | initial characterization of mouse syap1 in the nervous system search for interaction partners effects of gene knockdown and knockout and tissue distribution with focus on the adult brain erste charakterisierung des maus syap1 im nervensystem suche nach interaktionspartnern auswirkungen von gen knockdown und knockout sowie untersuchungen uber die verteilung im gewebe mit fokus auf das adulte gehirn |
| title_sub | = Erste Charakterisierung des Maus-Syap1 im Nervensystem: Suche nach Interaktionspartnern, Auswirkungen von Gen-Knockdown und-Knockout sowie Untersuchungen über die Verteilung im Gewebe mit Fokus auf das adulte Gehirn |
| topic | Synapse (DE-588)4184224-8 gnd Motoneuron (DE-588)4135747-4 gnd Nervensystem (DE-588)4041643-4 gnd Golgi-Apparat (DE-588)4192795-3 gnd |
| topic_facet | Synapse Motoneuron Nervensystem Golgi-Apparat Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-147319 http://d-nb.info/1130588033/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029747719&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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