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Table of contents
1 Introduction
1.1 Generalities
1.1.1 Magneuron project
1.1.2 RhoGTPases during cell migration
1.1.2.1 The Rho family
1.1.2.2 Intersectin: A GEF for Cdc42
1.1.2.3 Tiam1: A GEF for Rac1
1.1.2.4 GEF regulation
1.1.2.5 PI3K: Phosphoinositol regulation of GEF activity
1.1.3 Intracellular pathways
1.2 Perturbation of intracellular pathways
1.2.1 Manipulation of cell functions: Multicellular approach;
1.2.1.1 Functional genomics
1.2.1.2 Drug induced dimerization
1.2.2 Single cell manipulations
1.2.2.1 Light based signaling manipulation
1.2.2.2 Magnetic control
1.3 Developping manipulation of intracellular magnetic objects
1.3.0.1 Diffusion of particles in the cytoplasm
1.3.0.2 Limitation to diffusion in the cytoplasm
1.3.0.3 Measuring diffusion in intracellular space
1.3.1 Particles stability
1.3.1.1 Colloidal stability
1.3.1.2 Passivation
1.3.2 Magnetic forces on nanoscale magnetic objects
1.3.2.1 Magnetic particles characteristic and Magnetic field
1.3.2.2 Manipulation of proteins inside the cytoplasm
1.3.3 Specific targeting of proteins
1.3.4 Final words
2 Material and methods
2.1 Cellular biology
2.1.1 Cell lines
2.1.1.1 Hela CCL2 (Hela)
2.1.1.2 Retinoid Pigmental Epithelium hTERT (RPE1)
2.1.1.3 human Mesenchymal Stem Cells (hMSC)
2.1.1.4 SH-SY5Y cells
2.1.2 Medium
2.1.2.1 DMEM medium
2.1.2.2 DMEM/F12 (1:1) medium
2.1.3 Transient transfection
2.1.4 Stable cell lines
2.1.5 Coverslip coating
2.2 Micropatterning
2.2.1 Coverslip cleaning
2.2.2 Passivating molecule: PLL-g-PEG
2.2.3 Passivating molecule: PAcrAm-g-(PMOXA,NH2,Si)
2.2.4 Micropatterning: Quartz mask approach
2.2.5 Micropatterning: photopattering approach
2.2.6 Incubation with protein
2.2.7 Cell platting
2.3 Molecular biology
2.3.1 Gene cloning
2.3.2 Plasmid list
2.4 Microscopy
2.4.1 Olympus inverted microscopes
2.4.2 Nikon inverted microscope
2.4.3 Optogenetics: DMD equiped microscope
2.4.4 Heating system
2.5 Particles
2.5.1 Silica particles
2.5.2 Ferritin
2.5.2.1 Ferritin: Protein purification
2.5.2.2 Protein expression
2.5.2.3 Purification : protocol
2.5.2.4 Purification : Histag
2.5.2.5 Core synthesis
2.5.3 Quantum Dots
2.5.4 Other particles
2.6 Magnetic configuration
2.6.1 Iron tips
2.6.2 Micro array
2.7 Internalization of particles
2.7.1 Microinjection
2.7.2 Electroporation
2.7.3 Pinocytic loading
2.8 Micromanipulation: attraction of the particles
2.8.1 Magnetic tips
2.8.2 Micromagnets
2.9 Analysis and quantifications
2.9.1 Intensity measurements
2.9.2 Protrusion growth
2.9.3 Single Particle tracking
2.9.4 SPT: analysis
2.9.5 Cell mapping
3 Results
3.1 Exploration of cytoplasm environment and particles diffusion properties
3.1.1 Behaviors of particles below pore size
3.1.2 Zwitterionic Quantum dots
3.1.2.1 Diffusion of zwitterionic coated particles in the cytoplasm
3.1.2.2 Efficient targeting using Biotin-Streptavidin strategy
3.1.3 Exploration of intracellular cytoplasm
3.1.3.1 Diffusion in full cells
3.1.3.2 Exploring intracellular space: what’s next?
3.2 Stability of magnetic particles
3.2.1 Behaviors of magnetic particles
3.2.1.1 Si-MNPs
3.2.1.2 fMNPs
3.2.2 Parallelization
3.2.2.1 Note on the development of parallelized manipulation
3.3 Magnetic manipulation of intracellular signals
3.3.1 Manipulation of ITSN1 DH-PH domain
3.3.1.1 State of the technic: a successful manipulation
3.3.1.2 About the dynamics of the observed events
3.4 Exploring the limits of magnetic intracellular manipulation
3.4.1 Low affinity
3.4.2 Upstream signal activation: Ish2
3.4.3 Crowding
3.4.4 Discussion on magnetic manipulation
4 Conclusions and perspectives
4.1 Manipulation of intracellular mechanisms with the magneto-molecular approach
4.1.1 Magnetic particles
4.1.2 Magnetic manipulation
4.1.3 Signaling proteins
4.1.4 A clearer view on the cytoplasm
4.2 Final words: Controlling cells mechanisms
