High temporal resolution analysis of GPCR-mediated signalling:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2016
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| Beschreibung: | XVII, 128 Seiten Illustrationen, Diagramme 21 cm |
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Datensatz im Suchindex
| _version_ | 1804176757966766080 |
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| adam_text | Titel: High temporal resolution analysis of GPCR-mediated signalling
Autor: Gruteser, Nadine
Jahr: 2016
Content
Zusammenfassung................................................................................................I
Abstract................................................................................................................Ill
Content..................................................................................................................V
List of Abbreviations.........................................................................................XIII
1 Introduction..................................................................................................1
1.1 G protein-coupled receptors (GPCRs)...............................................................1
1.1.1 Biogenic amine receptors......................................................................................2
1.1.2 GPCR-mediated signalling......................................................................................3
1.2 Second messenger detection.............................................................................5
1.2.1 Analysis of cAMP signals........................................................................................5
1.2.2 Analysis of Ca2+signals...........................................................................................8
1.3 Adeno-associated virus (AAV)-mediated gene transfer................................11
1.3.1 AAV capsid and genome organization.................................................................12
1.3.2 AAV infection.......................................................................................................13
1.4 Aim.......................................................................................................................14
2 Materials and Methods..............................................................................17
2.1 Materials..............................................................................................................17
2.1.1 Chemicals, kits and consumables........................................................................17
2.1.2 Primers, oligonucleotides and sequencing..........................................................18
2.1.3 Size markers.........................................................................................................18
2.1.3.1 DNA ladder...........................................................................................................18
2.1.3.2 Protein marker.............................................19
2.1.4 Plasmids...............................................................................................................19
2.2 Processing of nucleic acids.................................................20
2.2.1 Separation and isolation of nucleic acids.............................................................20
2.2.1.1 Agarose gel electrophoresis.................................................................................20
2.2.1.2 DNA quantification...............................................................................................20
2.2.1.3 DNA extraction and purification..........................................................................20
2.2.2 Polymerase chain reaction (PCR).........................................................................20
2.2.2.1 Amplification of nucleic acids..............................................................................21
2.2.2.2 Mutagenesis of nucleic acids...............................................................................21
2.2.3 Modification of nucleic acids...............................................................................22
2.2.3.1 Restriction digest of DNA.....................................................................................22
2.2.3.2 Dephosphorylation of vector backbones.............................................................22
2.2.3.3 Ligation.................................................................................................................22
2.3 Escherichia coli (E.coli) cell culture.................................................................23
2.3.1 Bacteria strains.....................................................................................................23
2.3.2 Media and cultivation..........................................................................................23
2.3.3 Competent E. coli cells.........................................................................................24
2.3.4 Transformation of competent E. coli cells...........................................................24
2.4 Plasm id DNA preparation..................................................................................24
2.4.1 Quick Mini-preparation........................................................................................25
2.4.2 Mini preparation..................................................................................................25
2.4.3 Max! preparation.................................................................................................25
2.4.4 Endotoxin free plasmid preparation....................................................................Z5
2.5 Protein expression, purification, and analysis...............................................26
2.5.1 Expression of recombinant proteins in £. coli......................................................26
2.5.2 Protein purification..............................................................................................26
2.5.2.1 Ni-NTA purification of 6x His-fusion proteins......................................................26
2.5.2.2 Size exclusion chromatography (SEC)..................................................................27
2.5.3 Separation of proteins via gel electrophoresis....................................................28
2.5.4 Staining of SDS polyacrylamide gels....................................................................28
2.5.5 Protein quantification..........................................................................................29
2.6 Mammalian cell culture......................................................................................29
2.6.1 General cell culture..............................................................................................29
2.6.1.1 Cell culture handling............................................................................................29
2.6.1.2 Determination of cell numbers............................................................................29
2.6.1.3 Freezing and thawing of cells...............................................................................30
2.6.2 Human embryonic kidney 293 (HEK293) cell culture..........................................31
2.6.2.1 HEK293 cell lines..................................................................................................31
2.6.2.2 HEK293 medium and cultivation..........................................................................31
2.6.2.3 Transient transection of HEK293 cells................................................................32
2.6.2.4 Stable transection of HEK293 cells.....................................................................33
2.6.3 Primary hippocampal neuron (PHN) cell culture.................................................33
2.6.3.1 PHN medium and cultivation...............................................................................33
2.6.3.2 PHN preparation..................................................................................................34
2.6.4 Generation of recombinant adeno-associated virus (rAAV) vectors...................34
2.6.4.1 HEK29B cell cultivation.............................................34
2.6.4.2 Transfection, harvest and cell lysis for generation of rAAV particles..................35
2.6.4.3 Density gradient purification of rAAV particles...................................................36
2.6.4.4 Titer determination of rAAV suspensions............................................................36
2.6.4.5 Transduction of cells with rAAVs.........................................................................39
2.7 Immunostaining..................................................................................................39
2.7.1 Immunocytochemistry (ICC)................................................................................39
2.7.2 Immunocytochemistry for super-resolution microscopy....................................40
2.8 Spectroscopic analysis of Epacl -camps variants..........................................41
2.8.1 Spectroscopic analysis in cuvette experiments...................................................41
2.8.2 Spectroscopic analysis in multi-well plates..........................................................42
2.9 Calcium fluorimetry............................................................................................42
2.10 Fluorescence microscopy.................................................................................43
2.10.1 Confocal laser scanning microscopy (CLSM)........................................................43
2.10.2 Super-resolution microscopy...............................................................................43
2.10.3 Live cell imaging...................................................................................................44
2.11 Stopped-flow measurements............................................................................46
2.11.1 Principle of stopped-flow measurements............................................................46
2.11.2 Stopped-flow setup..............................................................................................46
2.11.3 Fluorescence measurements...............................................................................49
2.11.3.1 Caged compounds................................................................................................50
2.11.4 Data analysis.......................................51
2.12 Preparation of membrane proteins and analysis of adenylyl cyclase (AC)
activity.................................................................................................................52
2.13 Software..............................................................................................................53
3 Results........................................................................................................55
3.1 Construction and characterization of targeted Epacl -camps variants........55
3.1.1 Detection of cAMP signals with Epacl-camps.....................................................56
3.1.2 Construction of targeted Epacl-camps variants..................................................57
3.1.3 In vitro characterization of Epacl-camps variants...............................................59
3.1.3.1 Expression and purification of Epacl-camps variants.........................................60
3.1.3.2 Spectroscopic characterization of Epacl-camps variants....................................62
3.1.3.3 Fast-processing in vitro characterization of Epacl-camps...................................64
3.1.4 Characterization of Epacl-camps variants in HEK293 cells.................................65
3.1.4.1 Subcellular localization of Epacl-camps variants in HEK293 cells.......................65
3.1.4.2 Functionality of Epacl-camps variants in HEK293 cells.......................................67
3.1.5 Summary results part 1........................................................................................70
3.2 Kinetic measurements of GPCR-mediated signalling....................................71
3.2.1 Receptor composition of HEK293 cell lines for kinetic measurements...............71
3.2.2 Microscopic characterization of DmOctalB and DmOctpl cell lines..................72
3.2.3 Fluorimetric characterization of DmOctalB and DmOctfil cell lines..................73
3.2.4 Stopped-flow measurements using DmOctalB and DmOctpl cell lines............76
3.2.4.1 Calibration of the stopped-flow system..............................................................76
3.2.4.2 Characterization of DmOctalB and DmOctpl cell lines in stopped-flow
experiments.........................................................................................................77
3.2.5 Time-resolved measurements ofcAMP signals in FlpTM-DmOctfil cells with
caged compounds...........................................84
3.2.5.1 Specific activation of the DmOctpi receptor with caged octopamine................85
3.2.5.2 Specific activation of the CNGA2 channel with caged cAMP...............................87
3.2.5.3 Activation of membrane-bound ACs with a precursor of caged forskolin..........88
3.2.6 Summary results part 2........................................................................................90
3.3 Recombinant adeno-associated virus (rAAV)-mediated expression of
Epacl-camps in primary hippocampal neurons (PHN)..................................81
3.3.1 Generation of rAAV vectors.................................................................................81
3.3.2 Subcellular localization of Epacl-camps variants in PHNs...................................83
3.3.3 Summary results part 3........................................................................................86
4 Discussion..................................................................................................97
4.1 Construction and characterization of targeted Epacl -camps variants........97
4.1.1 Recombinant adeno-associated virus (rAAV)-mediated expression of Epacl-
camps in primary hippocampal neurons (PHN).................................................100
4.2 Kinetic measurements of GPCR-mediated signalling..................................101
4.2.1 Stopped-flow measurements of stably transfected HEK293 cells.....................101
4.2.2 Time-resolved measurements of GPCR-mediated signalling.............................104
4.3 Outlook..............................................................................................................108
5 References................................................................................................109
6 Appendix...................................................................................................122
6.1 Sequences......................................................................................................122
6.1.1 Localization sequences fused to the N-terminus of Epacl-camps....................122
6.1.2 Sequence of Epacl-camps...................................122
6.1.3 Sequence of Epacl-campsNuc.............................................................................123
6.1.4 Sequence of Epacl-campsPM..............................................................................124
6.1.5 Sequence of Epacl-campsR279E...........................................................................125
6.2 Emission spectra of Epad-camps at different cAMP concentrations.......126
6.3 Super-resolution microscopy of Epact -campsPM in HEK293 celts.............127
6.4 Fluorimetric measurements of FlpTM-DmOctpi cells.................................127
6.5 DEACM-cANIP photolysis in HEK293 cells....................................................128
6.6 Structure of 6-AEC-forskolin...........................................................................128
Danksagung......................................................................................................129
Erklarung...........................................................................................................130
|
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| spelling | Gruteser, Nadine Verfasser (DE-588)1102541451 aut High temporal resolution analysis of GPCR-mediated signalling vorgelegt von Nadine Gruteser Köln 2016 XVII, 128 Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität zu Köln 2016 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1108812546/04 Inhaltsverzeichnis HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029280144&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Gruteser, Nadine High temporal resolution analysis of GPCR-mediated signalling |
| subject_GND | (DE-588)4113937-9 |
| title | High temporal resolution analysis of GPCR-mediated signalling |
| title_auth | High temporal resolution analysis of GPCR-mediated signalling |
| title_exact_search | High temporal resolution analysis of GPCR-mediated signalling |
| title_full | High temporal resolution analysis of GPCR-mediated signalling vorgelegt von Nadine Gruteser |
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| title_full_unstemmed | High temporal resolution analysis of GPCR-mediated signalling vorgelegt von Nadine Gruteser |
| title_short | High temporal resolution analysis of GPCR-mediated signalling |
| title_sort | high temporal resolution analysis of gpcr mediated signalling |
| topic_facet | Hochschulschrift |
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