Verfügbarkeit: Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization

Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization: = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4

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Bibliographische Detailangaben
1. Verfasser:	De Maria, Monica (VerfasserIn)
Format:	Abschlussarbeit Buch
Sprache:	English German
Veröffentlicht:	Erlangen ; Nürnberg [2018]
Schlagworte:	Membranlipide Signaltransduktion Cholesterin Hochschulschrift
Online-Zugang:	Inhaltsverzeichnis Inhaltsverzeichnis
Beschreibung:	Zusammenfassung in deutscher Sprache
Beschreibung:	IV, 117 Seiten Illustrationen, Diagramme 21 cm

Internformat

MARC


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Datensatz im Suchindex

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adam_text	TABLE OF CONTENT SUMMARY.................................................................................................................I ZUSAMMENFASSUNG.............................................................................................. ILL 1. INTRODUCTION..................................................................................................... 1 1.1. THE G-PROTEIN COUPLED RECEPTORS AND THEIR MAIN FEATURES .................................................... 1 1.1.1. GPCRS SIGNALING PATHWAYS................................................................................................ 2 1.2. THE EFFECT OF MEMBRANE LIPIDS ON GPCR FUNCTION.................................................................... 4 1.2.1. ROLE OF CHOLESTEROL IN GPCR ACTIVITY................................................................................ 4 1.3. THE CHEMOKINES AND THEIR RECEPTORS.............................................................................................6 1.4. THE HCXCR4 RECEPTOR: BIOLOGY, FUNCTIONS AND CHOLESTEROL INFLUENCE...................................8 1.4.1. BIOLOGY AND FUNCTION OF THE HCXCR4 RECEPTOR................................................................8 1.4.2. THE ROLE OF CHOLESTEROL IN HCXCR4 RECEPTOR..................................................................10 2. AIM OF THE PROJECT..........................................................................................11 3. MATERIALS AND METHODS.................................................................................12 3.1. CELL BASED ASSAYS........................................................................................................................ 12 3.1.1. CELLS GROWING AND MAINTENANCE.....................................................................................12 3.1.2. CELLS COUNTING..................................................................................................................12 3.1.3. TRANSIENT TRANSFECTION OF HEK CELLS WITH TRANSLT-293 AND BRANCHED POLYETHYLENIMINE (PEI)............................................................................................................. 12 3.1.4. TRANSIENT TRANSFECTION OF HEK 293-T CELLS WITH LINEAR POLYETHYLENIMINE (PEI) .......... 13 3.1.5. TRANSIENT TRANSFECTION O F HEK 293-T CELLS WITH ROTIFECT...........................................13 3.1.6. TRANSIENT TRANSFECTION OF XTC WITH VIAFECT.................................................................14 3.1.7. BIOLUMINESCENCE RESONANCE ENERGY TRANSFER (BRET)-BASED CAMP ASSAY ON HEK CELLS 15 3.1.8. CAMP BIOLUMINESCENCE RESONANCE ENERGY TRANSFER (BRET) ASSAY ON XTC CELLS ....... 16 3.1.9. BIOLUMINESCENCE COMPLEMENTATION ASSAY IN HEK 293-T AND XTC CELLS ...................... 16 3.1.10. IMMUNOHISTOCHEMISTRY (IHC) ON HEK AND XTC ........................................................... 17 3.1.11. BCA ASSAY......................................................................................................................17 3.1.12. IM M UNOBLOTTING........................................................................................................... 18 3.1.13. COOMASSIE STAINING........................................................................................................21 3.1.14. CHOLESTEROL DEPLETION....................................................................................................21 3.1.15. CHOLESTEROL ASSAY..........................................................................................................22 3.2. MOLECULAR BIOLOGY............................................................................................................................ 23 3.2.1. CLONING PCR..................................................................................................................... 23 3.2.2. DNA EXTRACTION IN AGAROSE G E L....................................................................................... 24 3.2.3. RESTRICTION ENZYMES DIGESTS........................................................................................... 24 3.2.4. VECTOR-DEPHOSPHORYLATION............................................................................................. 24 3.2.5. DNA LIGATION.................................................................................................................... 25 3.2.6. TRANSFORMATION OF PLASMIDS INTO COMPETENT BACTERIAL CELLS ......................................... 25 3.2.7. MINI-SCALE PLASMID PREPARATION.....................................................................................25 3.2.8. MIDI-SCALE PLASMID PREPARATION.....................................................................................27 3.2.9. MAXI-SCALE PLASMID PREPARATION.....................................................................................27 3.3. PROTEIN PURIFICATION AND RECONSTITUTION.....................................................................................29 3.3.1. PROTEIN EXPRESSION AND PURIFICATION...............................................................................29 3.3.2. BRADFORD ASSAY................................................................................................................. 29 3.3.3. PROTEIN RECONSTITUTION (DONE IN ISABEL ALVES S LABORATORY) ........................................... 30 3.4. SOFTWARE U SED .................................................................................................................................. 32 4. RESULTS..............................................................................................................33 4.1. CELL-BASED EXPERIMENTS.................................................................................................................. 33 4.1.1. ROLE OF CHOLESTEROL ON HCXCR4 CHEMOKINE RECEPTOR ACTIVITY IN HEK 293-T CELLS.......33 4.1.2. ROLE OF CHOLESTEROL AND LIPID ENVIRONMENT ON CXCR4 ACTIVITY IN XTC CELLS ................. 46 4.1.3. COMPARISON BETWEEN XCXCR4 AND HCXCR4..................................................................61 4.2. PROTEIN EXPRESSION AND PURIFICATION............................................................................................ 69 4.2.1. HCXCR4 EXPRESSION, SOLUBILISATION AND PURIFICATION.......................................................69 4.2.2. HCXCR4 RECONSTITUTION IN POPC SUVS TESTED BY POLARIZED ATR-FTIR............................73 4.2.3. LIGAND BINDING TO HCXCR4 RECONSTITUTED IN A POPC MEMBRANE PROBED BY PLASMON WAVEGUIDE SPECTROSCOPY (PWR).............................................................................................. 74 5. DISCUSSION........................................................................................................ 79 5.1. MSSCD TREATM ENT INDUCES A SIGNIFICANT LOSS IN CHOLESTEROL WITHOUT ALTERING BOTH HCXCR4-EXPRESSION PATTERN AND CELL HEALTH......................................................................................79 5.2. THE ROLE OF LIPID IN THE LIGAND BINDING ON GPCRS.................................................................... 83 5.3. CHOLESTEROL DEPLETION AFFECTS HCXCR4 CAMP PATHWAY IN BOTH DIRECT AND INDIRECT M ANNER....................................................................................................................................................... 84 5.4. CHOLESTEROL DEPLETION AFFECTS THE HOMODIMERIZATION BY INDUCING CONFIGURATIONAL CHANGES......................................................................................................................................................88 6. APPENDIX.........................................................................................................93 6.1. PCDNA3.1-HCXCR4 AND PCDNA 3.1-HCXCR5 (PURCHASED CONSTRUCTS)..................................93 6.2. PCDNA3.1-FLAG_HCXCR4 AND PFC27K HALOTAG CMV-NEO FLEXI VECTOR - FLAG_HCXCR4_HALOTAG......................................................................................................................... 94 6.3. PCDNA3.1-HCXCR4 WT-MVENUS (PURCHASED CONSTRUCT) .......................................................... 94 6.4. PB IT L.L-C [TK-LGBIT]-HCXCR4 W T AND PBIT2.1-C [TK-SMBIT]-HCXCR4WT ........................... 95 6.5. PMA-T-XCXCR4 W T (PURCHASED CONSTRUCT) ................................................................................ 95 6.6. PB IT L.L-C [TK-LGBIT]-XCXCR4 W T AND PBIT2.1-C [TK-SMBIT]-XCXCR4WT.............................96 6.7. COMPARISON BETWEEN XCXCR4 AND HCXCR4..............................................................................97 7. BIBLIOGRAPHY...................................................................................................98 8. ACKNOWLEDGEMENTS 117
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author	De Maria, Monica
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discipline	Biologie
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spelling	De Maria, Monica Verfasser aut Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4 vorgelegt von Monica De Maria Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4 Erlangen ; Nürnberg [2018] IV, 117 Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Zusammenfassung in deutscher Sprache Dissertation Friedrich-Alexander-Universität Erlangen-Nürnberg 2018 Membranlipide (DE-588)4203814-5 gnd rswk-swf Signaltransduktion (DE-588)4318717-1 gnd rswk-swf Cholesterin (DE-588)4010034-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Membranlipide (DE-588)4203814-5 s Cholesterin (DE-588)4010034-0 s Signaltransduktion (DE-588)4318717-1 s DE-604 B:DE-101 application/pdf http://d-nb.info/1164190555/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030590790&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	De Maria, Monica Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4 Membranlipide (DE-588)4203814-5 gnd Signaltransduktion (DE-588)4318717-1 gnd Cholesterin (DE-588)4010034-0 gnd
subject_GND	(DE-588)4203814-5 (DE-588)4318717-1 (DE-588)4010034-0 (DE-588)4113937-9
title	Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4
title_alt	Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4
title_auth	Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4
title_exact_search	Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4
title_full	Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4 vorgelegt von Monica De Maria
title_fullStr	Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4 vorgelegt von Monica De Maria
title_full_unstemmed	Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4 vorgelegt von Monica De Maria
title_short	Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization
title_sort	effect of membrane cholesterol manipulation on the g protein coupled chemokine receptor type 4 signaling and homodimerization effekt der membran cholesterin manipulation auf den signalweg und die homodimerisierung des g protein gekoppelten chemokinrezeptors typ 4
title_sub	= Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4
topic	Membranlipide (DE-588)4203814-5 gnd Signaltransduktion (DE-588)4318717-1 gnd Cholesterin (DE-588)4010034-0 gnd
topic_facet	Membranlipide Signaltransduktion Cholesterin Hochschulschrift
url	http://d-nb.info/1164190555/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030590790&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT demariamonica effectofmembranecholesterolmanipulationonthegproteincoupledchemokinereceptortype4signalingandhomodimerizationeffektdermembrancholesterinmanipulationaufdensignalwegunddiehomodimerisierungdesgproteingekoppeltenchemokinrezeptorstyp4 AT demariamonica effektdermembrancholesterinmanipulationaufdensignalwegunddiehomodimerisierungdesgproteingekoppeltenchemokinrezeptorstyp4

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