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
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English German |
| Veröffentlicht: |
Erlangen ; Nürnberg
[2018]
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | Zusammenfassung in deutscher Sprache |
| Beschreibung: | IV, 117 Seiten Illustrationen, Diagramme 21 cm |
Internformat
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| 100 | 1 | |a De Maria, Monica |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Effect of membrane cholesterol manipulation on the G protein coupled chemokine receptor type 4 signaling and homodimerization |b = Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4 |c vorgelegt von Monica De Maria |
| 246 | 1 | 1 | |a Effekt der Membran-Cholesterin Manipulation auf den Signalweg und die Homodimerisierung des G-Protein gekoppelten Chemokinrezeptors Typ 4 |
| 264 | 1 | |a Erlangen ; Nürnberg |c [2018] | |
| 300 | |a IV, 117 Seiten |b Illustrationen, Diagramme |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Zusammenfassung in deutscher Sprache | ||
| 502 | |b Dissertation |c Friedrich-Alexander-Universität Erlangen-Nürnberg |d 2018 | ||
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Datensatz im Suchindex
| _version_ | 1804178907037958144 |
|---|---|
| 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
|
| any_adam_object | 1 |
| author | De Maria, Monica |
| author_facet | De Maria, Monica |
| author_role | aut |
| author_sort | De Maria, Monica |
| author_variant | m m d mm mmd |
| building | Verbundindex |
| bvnumber | BV045201835 |
| ctrlnum | (OCoLC)1099483565 (DE-599)DNB1164190555 |
| discipline | Biologie |
| format | Thesis Book |
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| id | DE-604.BV045201835 |
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| indexdate | 2024-07-10T08:11:25Z |
| institution | BVB |
| language | English German |
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| physical | IV, 117 Seiten Illustrationen, Diagramme 21 cm |
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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 |
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