000126134 001__ 126134
000126134 005__ 20190717172516.0
000126134 0247_ $$2doi$$a10.5075/epfl-thesis-4209
000126134 02470 $$2urn$$aurn:nbn:ch:bel-epfl-thesis4209-3
000126134 02471 $$2nebis$$a5652607
000126134 037__ $$aTHESIS
000126134 041__ $$aeng
000126134 088__ $$a4209
000126134 245__ $$aMiniaturized bioanalytics to probe the function of membrane proteins
000126134 269__ $$a2008
000126134 260__ $$bEPFL$$c2008$$aLausanne
000126134 300__ $$a186
000126134 336__ $$aTheses
000126134 520__ $$aG-protein coupled receptors (GPCRs) are the most abundant class of proteins in the cell body. Such receptors are of major interest as potential therapeutic targets. Downscaling and parallelization of bioanalytics opens novel routes to rapidly screen and identify potential drugs with a decrease in regard to the costs, and elucidate novel functions of signaling networks under physiological conditions. Native vesicles are small autonomous biological containers, which are efficiently produced from all cell lines. They are composed of their mother cell plasma membrane and enclose part of their cytoplasm. Membrane receptors are then exposed at their surface and already demonstrate to induce cellular signaling when exposed to receptor ligands. Native vesicles were investigated in this present work, as novel possibilities to downscale receptor investigation in live cells, using neurokinin 1 receptor (NK1R) as a representative model. Here native vesicle production and purification was optimized. The influence of the cell cycle on the production efficiency was demonstrated. Biological proteins were downregulated in order to produce blebbing cells. Native vesicle characterization was achieved. The receptors also demonstrate to be efficiently labeled by agonist and antagonist, allowing to access information about the binding kinetics as well as KD values. The results are in agreement with those obtained in live cells. Native vesicles also demonstrate to internalize agonist after application, demonstrating receptor desensitization and signaling performance similar as live cells. Confocal microscopy shows that cells expressing the NK1R-CFP have two binding affinities for their main agonist, substance P. Similar results could be observed with flow cytometry. The high affinity binding is related to cholesterol content in the cell membrane and was abolished by cholesterol depletion with methyl-β-cyclodextrin. Micro-contact printing (µCP) was used to (bio)functionalize surfaces with proteins, polymers or functionalized nanoparticles. Precise sample positioning by micro-contact printing shows improves nuclear magnetic resonance excitation and detection, when performed with a planar microcoil probe. µCP was used to produce native vesicle arrays by two procedures, and fluorescent binding assay shows the binding of fluorescent ligands to the receptor. Laser tweezers allow manipulating cell membranes without requiring the use of polystyrene beads. From pulled membranes, native vesicles were produced. In addition from pulled membranes, large tethers were produced and artificial connections were established with neighboring cells. Intercellular communication was investigated by whole-cell patch clamp in dissociated primary dorsal root ganglion neurons after optical induced connection, as well as in HEK cells expressing Cx36. The lab-on-chip assay development demonstrates: the high production of native vesicles in microchannels; the efficient purification obtained by negative dielectrophoresis depletion in MEMS chips; perfect trapping and thus immobilization of native vesicles in a new optical multi-tweezer array. Fluorescence labeling was performed with native vesicles trapped in a multi-tweezer array inside microfluidic channel in the presence of two laminar flows. Optical multi-tweezer array setup shows to be the fastest and more efficient technique in order to perform immobilization and labeling of native vesicles in the microfluidic channel. It is presently the only technique to perform fluorescence measurements when maintaining objects trapped.
000126134 6531_ $$aNative Vesicles
000126134 6531_ $$aG-protein coupled receptor (GPCR)
000126134 6531_ $$aNK1R
000126134 6531_ $$aSP
000126134 6531_ $$aDorsal Root Ganglions (DRGs)
000126134 6531_ $$aPrimary cells
000126134 6531_ $$aCx36
000126134 6531_ $$aLigand affinity
000126134 6531_ $$aCholesterol
000126134 6531_ $$aPlanar Microcoil Confocal Microscopy
000126134 6531_ $$aFlow Cytometry
000126134 6531_ $$aNanoparticle
000126134 6531_ $$aPatch Clamp
000126134 6531_ $$aLaser Tweezer
000126134 6531_ $$aNMR
000126134 6531_ $$aOptical Multi-Tweezer Array
000126134 6531_ $$aDielectrophoresis, MEMS
000126134 6531_ $$aMicro-Contact Printing (µCP)
000126134 6531_ $$aMicrofluidics
000126134 6531_ $$aVésicules Natives
000126134 6531_ $$aRécepteurs Couplés aux Protéines G (RCPGs)
000126134 6531_ $$aR-NK1
000126134 6531_ $$aSP
000126134 6531_ $$aGanglions Dorsaux (DRGs)
000126134 6531_ $$aCellules Primaires
000126134 6531_ $$aCx36
000126134 6531_ $$aAffinité du Ligand
000126134 6531_ $$aCholestérol
000126134 6531_ $$aMicrobobine Planaire
000126134 6531_ $$aMicroscopie Confocale
000126134 6531_ $$aCytométrie en Flux
000126134 6531_ $$aNanoparticules
000126134 6531_ $$aPatch Clamp
000126134 6531_ $$aPince Optique
000126134 6531_ $$aRMN
000126134 6531_ $$aRéseau de Pinces Optiques
000126134 6531_ $$aDiélectrophorèse
000126134 6531_ $$aMEMS
000126134 6531_ $$aImpression par Microcontact (µCP)
000126134 6531_ $$aMicrofluidique
000126134 700__ $$aPascoal, Pedro
000126134 720_2 $$aVogel, Horst$$edir.$$g106666$$0240645
000126134 8564_ $$zTexte intégral / Full text$$yTexte intégral / Full text$$uhttps://infoscience.epfl.ch/record/126134/files/EPFL_TH4209.pdf$$s36111297
000126134 909C0 $$xU10170$$pLCPPM$$0252153
000126134 909CO $$pthesis-bn2018$$pDOI$$pSB$$ooai:infoscience.tind.io:126134$$qDOI2$$qGLOBAL_SET$$pthesis
000126134 918__ $$bSB-SCGC$$cISIC$$aSB
000126134 919__ $$aLCPPM
000126134 920__ $$b2008
000126134 970__ $$a4209/THESES
000126134 973__ $$sPUBLISHED$$aEPFL
000126134 980__ $$aTHESIS