La membrane plasmique constitue la première entité séparant la cellule de son environnement. A ce rôle de barrière s'ajoute celui de réguler la. Par conséquent, la membrane plasmique est une zone privilégiée pour le passage d'information. Cependant, son étude reste difficile, ne serait-ce que par l'extraordinaire complexité d'organisation de cet assemblage supramoléculaire.Mon projet de thèse vise à développer de nouvelles approches expérimentales pour explorer plus spécifiquement l'organisation et le rôle de la membrane plasmique d'une cellule dans les mécanismes de transduction de l'information. Deux axes ont été privilégiés : le premier, concerne la description de la dynamique d'organisation de la membrane ; le deuxième concerne l'inter-connectivité et la transmission du signal d'une cellule avec d'autres partenaires.Ce manuscrit se compose de plusieurs parties. Le premier chapitre introduira succinctement les questions biologiques. Dans le second chapitre, je présenterai des méthodes utilisées pour l'étude de la membrane. J'y présenterai aussi une série d'observation que j'ai réalisée sur la diffusion de l'EGFR. Le troisième chapitre sera consacré à la technique de corrélation croisée de fluorescence depuis le montage jusqu'à l'étude du modèle EGFR. Dans la quatrième partie, nous verrons comment les collaborations à l'interface biophysique ont permis des développements innovants sur un système de pinces optiques holographiques. J'y présenterai les applications de ce système à différent modèles d'intérêt biologique. Enfin, je conclurai ce document par une brève discussion autour des résultats obtenus aussi bien d'un point de vue méthodologique que biologique. / The plasma membrane separates the cell from its environment. But it is more than a barrier any cell has to communicate with the outside world. Therefore the plasma membrane plays a prime role in transferring and exchanging information. However, the biological study of the plasma membrane remains difficult due to the extraordinary complexity of it organization.My thesis is a part of an effort to develop new experimental approaches to explore more specifically the organization and the role of the plasma membrane in the signal transduction mechanisms. Two major aspects were followed: the first one concerns the description of the dynamics of membrane organization and of molecular interactions, the second concerns the inter-connectivity and signal transduction between a cell and other biological partners.This manuscript is composed of several parts. The first chapter briefly introduces the biological questions that I tried to answer. In the second chapter, I present the methods commonly used to study the membrane with a dynamic perspective. Additionally, I include a series of observations that I made on the EGF receptor diffusion. The third chapter is devoted to the fluorescence cross-correlation technique to study the assembly of the EGFR. In the fourth part, I demonstrate how scientific collaborations at the interface between biology and physics have led to the development of innovative solutions on a holographic optical tweezers system. I present applications of this system in different biological models. Finally, I conclude this thesis with a brief discussion about my technological and biological results.

Binding-sites which facilitate the transport of substrates across membranes are ubiquitous in membrane proteins. To understand this fundamental process in cells, we build up a synthetic membrane system consisting of microfluidic channels and colloidal particles. Holographic optical tweezers are used to modulate the potential energy landscape in those channels. We show how to extract the underlying energy potential by analysing local transition probabilities. Our method is applicable both to equilibrium systems and non-equilibrium steady states. Our method offers improved robustness when dealing with fragmented trajectories or small ensembles of data compared to other established approaches, such as probability density function and splitting probability. Meanwhile, we utilise the intensity distribution of the optical traps generated by holographic optical tweezers to estimate energy landscapes featuring high energy barriers where transitions rarely occur. We use this newly developed experimental system to mimic the functionality of membrane protein transporters that are known to alternate their substrate-binding sites between the extracellular and cytosolic side of the membrane. We study particle transport through a channel coupled with an energy well that oscillates its position between the two entrances of the channel deterministically and stochastically. Optimised particle transport across the channel is obtained by adjusting the oscillation frequency. At the optimal oscillation frequency, the translocation rate of particles through the channel is a hundred times higher with respect to free diffusion across the channel. Our findings reveal the effect of time dependent potentials on particle transport across a channel. This work adds a new tool for the investigation of highly controlled membrane transport processes at the micron scale. Our results are relevant for improving our understanding of membrane transport especially for microfluidics application.

In a time when microorganisms are controlling the world, research in biology is more relevant than ever and this requires some powerful instruments. Optical tweezers use a focused laser beam to manipulate and probe objects on the nano- and microscale. This allows for the exploration of a miniature world at the border between biology, chemistry and physics. New methods for biophysical and physicochemical measurements are continuously being developed and at Umeå University there is a need for a new system that combines several of these methods. This would truly be the new Swiss army knife of biophysical and biomechanical research, extending their reach in the world of optical tweezing. My ambition with this project is to design and construct a robust system that incorporates optical trapping with high-precision force measurements and Raman spectroscopy, as well as introducing the possibility of generating multiple traps by using a spatial light modulator (SLM). The proposed design incorporates four different lasers and a novel combination of signal detection techniques. To allow for precise control of the systems components and laser beams, I designed and constructed motorized opto-mechanical components. These are controlled by an in-house developed software that handles data processing and signal analysis, while also providing a user interface for the system. The components include, motorized beam blockers and optical attenuators, which were developed using commonly available 3D printing techniques and electronic controllers. By designing the system from scratch, I could eliminate the known weaknesses of conventional systems and allow for a modular design where components can be added easily. The system is divided into two parts, a laser breadboard and a main breadboard. The former contains all the equipment needed to generate and control the laser beams, which are then coupled through optical fibers to the latter. This contains the components needed to move the optical trap inside the sample chamber, while performing measurements and providing user feedback. Construction and testing was done for one sub-system at a time, while the lack of time required a postponement for the implementation of Raman and SLM. The system performance was verified through Allan variance stability tests and the results were compared with other optical tweezers setups. The results show that the system follows the thermal limit for averaging times (τ) up to ~1 s when disturbances had been eliminated, which is similar to other systems. However, we could also show a decrease in variance all the way to τ = 2000 s, which is exceptionally good and not found in conventional systems. The force-resolution was determined to be on the order of femtonewtons, which is also exceptionally good. Thus, I conclude that this optical tweezers setup could lie as a solid foundation for future development and research in biological science at Umeå University for years to come.