64 research outputs found
Towards the next Generation in Human-machine-interfacing:Controlling Wearable Robots via Neuromusculoskeletal Modelling
Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.
BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades.
RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage.
CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements
DYNAMIC SINGLE CELL MEASUREMENTS OF SIGNALLING CASCADE ACTIVITY
All organisms are composed of a multitude of cell types, having each a specific task to accom- plish. This cell-to-cell heterogeneity results from a complex process of differentiation initiated by the interpretation of intra and extracellular cues by signalling cascades. Therefore, it becomes fundamental to study the signal processing driven by signalling cascades, in order to understand how cells organise during development, but also to comprehend how and why signals are some- times misinterpreted and lead to diseases such as cancer. MAPK pathways belong to a very well-conserved family of signalling cascades, known to be mostly implicated in the adaptation to environmental stimuli and to regulate various key physiological processes. The various com- ponents of the MAPK pathways are well defined. However, we still need to understand how signalling cascades cross-communicate with each other and how this leads to different cell-fate decision making. The limitation is mostly technical, as such investigation would require to dy- namically and simultaneously investigate the activity state of MAPK components in live single cells. The aim of this thesis was to build a new kind of assay, enabling to extract these inform- ation. Here, I described the design of the synthetic protein SKARS, that relocates from the nucleus to the cytoplasm following specific phosphorylation. This synthetic protein is composed of a docking site (DS), that enables a specific interaction with a MAP kinase, a nuclear local- isation sequence (NLS), that promotes an active nuclear importation, and a fluorescent protein (FP), that allows to determine its sub-cellular localisation. Once the cell activates the MAPK, the NLS gets inhibited by phosphorylation, which leads to the cytoplasmic accumulation of the fluorescent protein. This nucleus-to-cytoplasm shuttling is used as a proxy for MAPK activity. In order to demonstrate this, we applied this technology in the mating pathway study of the model organism Saccharomyces cerevisiae. By combining time-lapse fluorescent microscopy with com- putational analysis, we extracted the dynamic MAPK activity in hundreds of cells, and observed that when cells are exposed to exogenous pheromone, the MAPK is differentially activated from one cell to another. Using the Whi5 transcription factor as a sensor for the cell cycle progression, we showed that these different signal patterns are specific to the cell-cycle stage of the cell. The MAPK is fully activated in G1, inhibited in S, G2 and M and transiently activated during the transition between G1 and S. Moreover, single cell analysis of mutants let us conclude that the model describing how the cell cycle regulates the mating pathway is incomplete. To conclude, we showed that the parallel measurement of multiple signalling cascades is a valuable strategy enlighten very subtle pathway cross-regulation events. Thanks to the straightforward structure of the SKARS, crosstalk mechanisms between signalling cascades can be assessed, by combining multiple sensors in the same cell. We are currently applying this technology to mammalian cells, and strongly believe that this technique can be an advantage in fields such as development or cancer research.
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Tous les organismes sont composĂ©s dâune multitude de cellules ayant chacune une tĂąche spĂ©ci- fique Ă accomplir. Cette hĂ©tĂ©rogĂ©nĂ©itĂ© rĂ©sulte dâun processus complexe de diffĂ©renciation initiĂ© par lâanalyse de signaux intracellulaires et extracellulaires par des cascades de signalisation. Par consĂ©quent, il devient fondamental dâĂ©tudier le traitement du signal par les voies de signalisation pour comprendre comment les cellules sâorganisent pendant le dĂ©veloppement, mais aussi pour comprendre comment et pourquoi ces signaux peuvent parfois ĂȘtre mal interprĂ©tĂ©s et entraĂźner lâapparition de maladies telles que le cancer. Les voies impliquant les MAP Kinases (MAPK) appartiennent Ă une famille trĂšs bien conservĂ©e de cascades de signalisation connues pour leurs rĂŽles dans lâadaptation aux stimuli environnementaux et dans la rĂ©gulation de divers processus physiologiques clĂ©s. Les diffĂ©rents composants des voies impliquant les MAPK commencent Ă ĂȘtre bien connus. Cependant, nous ne comprenons toujours pas comment les cascades de signalisa- tion sâautorĂ©gulent et comment cela conduit Ă divers comportements cellulaires. La limitation est principalement technique, car une telle recherche nĂ©cessiterait de sonder lâĂ©tat dâactivitĂ© des com- posants des cascades de MAPK, dynamiquement et simultanĂ©ment dans chaque cellule vivante. Lâobjectif de cette thĂšse fut de construire un nouveau type de senseur permettant dâextraire ces informations. Dans ce manuscrit, nous dĂ©crivons le design, ainsi quâune application de la protĂ©ine synthĂ©tique SKARS (Synthetic Kinase Activity Relocation Sensor). Elle a Ă©tĂ© conçue pour se dĂ©placer du noyau vers le cytoplasme aprĂšs avoir Ă©tĂ© spĂ©cifiquement phosphorylĂ©e par la kinase dâintĂ©rĂȘt. La protĂ©ine est composĂ©e dâun domaine de docking (DS), qui permet une interaction spĂ©cifique avec une MAPK, un signal de localisation nuclĂ©aire (NLS), qui favorise une importation active dans le noyau et une protĂ©ine fluorescente pour observer sa position dans les compartiments cellulaires. Une fois que la cellule active la MAPK, le NLS est inhibĂ© aprĂšs phosphorylation ce qui conduit Ă lâaccumulation de la protĂ©ine fluorescente dans le cytoplasme. Le passage du noyau au cytoplasme est utilisĂ© comme mesure de lâactivitĂ© MAPK. Comme preuve de concept, nous avons appliquĂ© cette technologie dans lâĂ©tude de la voie dâaccouplement chez lâorganisme modĂšle Saccharomyces cerevisiae. En combinant la microscopie Ă fluorescence en temps rĂ©el avec une analyse informatique, nous avons extrait lâactivitĂ© dynamique de la MAPK dans des centaines de cellules et avons observĂ© que lorsque les cellules sont exposĂ©es Ă de la phĂ©romone exogĂšne, la MAPK sâactive de maniĂšre diffĂ©rente dâune cellule Ă lâautre. En utilisant le facteur de transcription Whi5 comme senseur de la progression du cycle cellulaire, nous avons montrĂ© que ces diffĂ©rents types de trace dâactivitĂ© de MAPK sont spĂ©cifiques Ă chaque phase du cycle cellulaire dans laquelle se trouve la cellule. La MAPK est entiĂšrement activĂ©e en G1, inhibĂ©e en S, G2 et M et activĂ©e de maniĂšre transitoire Ă lâinteraction entre G1 et S. De plus, lâanalyse de cellule mutantes individuellement nous a permis de conclure que le modĂšle dĂ©crivant le mĂ©canisme de rĂ©gulation de la voie dâaccouplement par le cycle cellulaire est incomplet. Pour conclure, la mesure en parallĂšle de plusieurs cascades de signalisation est une stratĂ©gie intĂ©res- sante pour dĂ©tecter de fins Ă©vĂ©nements de rĂ©gulation entre les voies de signalisation. GrĂące Ă sa structure modulable, plusieurs SKARS peuvent ĂȘtre construits et combinĂ©s dans le but dâĂ©tudier les mĂ©canismes de rĂ©gulation croisĂ©s entre les cascades de signalisation. Nous appliquons actuel- lement cette technologie aux cellules mammifĂšres et nous sommes fermement convaincus que cette technique pourrait bĂ©nĂ©ficier Ă divers domaines tels que la recherche sur le dĂ©veloppement ou la recherche sur le cancer.
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La cellule est lâunitĂ© biologique structurelle et fonctionnelle fondamentale de tous les ĂȘtres vi- vants. Chez lâhomme, les cellules sâassemblent pour former des structures complexes telles que des organes, des muscles ou des os. Bien que la plupart des cellules qui composent notre corps possĂšdent exactement le mĂȘme code gĂ©nĂ©tique, chacune dâentre elles se diffĂ©rencie, entre autres, par sa fonction. Cette diversitĂ© Ă©merge dâune interprĂ©tation diffĂ©rente des changements envi- ronnementaux. Lâanalyse de ces signaux se fait Ă lâintĂ©rieur de la cellule par un grand rĂ©seau de protĂ©ines. Ce rĂ©seau sâapparente Ă un circuit Ă©lectrique, oĂč chaque interrupteur est reliĂ© Ă un ou plusieurs autres interrupteurs. Au niveau cellulaire, Ă chaque interrupteur correspond une protĂ©ine faisant circuler le signal dans une branche prĂ©cise du rĂ©seau en contrĂŽlant son Ă©tat âON/OFFâ. Suivant le type de chemin parcouru, le signal induira un type de rĂ©ponse ou un autre. Ce processus est communĂ©ment appelĂ© transduction du signal. Si la transduction du signal dâune cellule Ă©tait mesurĂ©e, sa voie de diffĂ©renciation cellulaire serait anticipĂ©e. Cette thĂšse prĂ©sente une technique permettant de mesurer la transduction du signal dans des cellules vivantes et en temps rĂ©el. La technique se base sur une protĂ©ine synthĂ©tique appelĂ©e SKARS qui va agir comme une sonde pour lâactivitĂ© dâun nĆud du circuit de signalisation. Lorsque le nĆud nâest pas actif, la protĂ©ine est dans le noyau de la cellule et lorsque le nĆud sâactive, la protĂ©ine sâaccumule en dehors. Afin de visualiser ce processus, la sonde Ă Ă©tĂ© marquĂ©e avec une protĂ©ine fluorescente. En utilisant un microscope Ă fluorescence, nous pouvons suivre la position de la sonde au cours du temps et en dĂ©duire le degrĂ© dâactivation du nĆud mesurĂ©. Nous avons appliquĂ© cette mĂ©thode dans lâĂ©tude du processus dâaccouplement chez la levure Saccharomyces cerevisiae. Deux sondes ont Ă©tĂ© utilisĂ©es pour mesurer en parallĂšle lâactivitĂ© du nĆud de lâaccouplement et du nĆud impliquĂ© dans le dĂ©clenchement de la division cellulaire. Nos rĂ©sultats montrent que la voie de lâaccouplement ne peut ĂȘtre activĂ©e quâĂ condition que les cellules sentent de la phĂ©romone et que la voie de la division cellulaire soit Ă©teinte. Par consĂ©quent, la cellule ne peut choisir que lâune de ces deux voies de diffĂ©renciation. GrĂące Ă cette mĂ©thode, nous avons aussi dĂ©montrĂ© que le modĂšle dĂ©crivant le mĂ©canisme dâinhibition croisĂ© de ces deux voies Ă©tait incomplet. En conclu- sion, la sonde SKARS est outil extrĂȘmement utile pour dĂ©finir comment la cellule interprĂšte les signaux de lâenvironnement et ainsi comprendre pourquoi certaines voies de diffĂ©renciation sont choisies plus que dâautres. Cette sonde a Ă©tĂ© utilisĂ©e chez un organisme trĂšs simplifiĂ©, mais nous sommes convaincus que cette technique permettra de rĂ©pondre Ă dâimportantes questions dans les domaines de recherche sur le dĂ©veloppement et sur le cancer
Traitement des signaux EMG et son application pour commander un exosquelette
Le vieillissement de la population dans notre socieÌteÌ moderne va entraiÌner de nouveaux besoins pour lâassistance aux personnes aÌgeÌes et pour la reÌhabilitation. Les exosquelettes sont une piste de recherche prenant de plus en plus dâimportance pour reÌpondre aÌ ces nouveaux challenges. Deux de ces challenges sont, la reÌalisation dâun controÌle naturel pour lâutilisateur et la seÌcuriteÌ.
Cette maiÌtrise cherche aÌ reÌpondre aÌ ces deux probleÌmatiques. Nous avons donc deÌveloppeÌ un outil de travail informatique utilisant les deÌcharges eÌlectriques produites par les neurones moteurs pour contracter les muscles et un modeÌle des os et des muscles du bras.
Cet outil utilise la librairie informatique ROS et OpenSim. Elle permet de connaiÌtre la force et le mouvement deÌveloppeÌs par le coude. De plus, un autre outil informatique a eÌteÌ deÌveloppeÌ pour optimiser le modeÌle des os et des muscles du bras pour le personnaliser aÌ lâutilisateur pour un meilleur reÌsultat.
Une carte dâacquisition utilisant des eÌlectrodes de surface pouvant eÌtre relieÌes avec un ordinateur par USB et compatible avec ROS a eÌteÌ deÌveloppeÌe. Pour tester les algorithmes deÌveloppeÌs, un exosquelette pour le coude utilisant un actionneur compliant et controÌleÌ en force a eÌteÌ conçu. Pour compenser le poids de lâexosquelette et lâeffet dâamortissement passif de lâactionneur, une compensation de graviteÌ dynamique a eÌteÌ deÌveloppeÌe.
Finalement, des expeÌrimentations ont eÌteÌ meneÌes sur lâefficaciteÌ de lâoptimisation du modeÌle et sur lâexosquelette avec les diffeÌrents algorithmes
Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.
Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise
Robust simultaneous myoelectric control of multiple degrees of freedom in wrist-hand prostheses by real-time neuromusculoskeletal modeling
Objectives: Robotic prosthetic limbs promise to replace mechanical function of lost biological extremities and restore amputees' capacity of moving and interacting with the environment. Despite recent advances in biocompatible electrodes, surgical procedures, and mechatronics, the impact of current solutions is hampered by the lack of intuitive and robust man-machine interfaces. Approach: Based on authors' developments, this work presents a biomimetic interface that synthetizes the musculoskeletal function of an individual's phantom limb as controlled by neural surrogates, i.e. electromyography-derived neural activations. With respect to current approaches based on machine learning, our method employs explicit representations of the musculoskeletal system to reduce the space of feasible solutions in the translation of electromyograms into prosthesis control commands. Electromyograms are mapped onto mechanical forces that belong to a subspace contained within the broader operational space of an individual's musculoskeletal system. Results: Our results show that this constraint makes the approach applicable to real-world scenarios and robust to movement artefacts. This stems from the fact that any control command must always exist within the musculoskeletal model operational space and be therefore physiologically plausible. The approach was effective both on intact-limbed individuals and a transradial amputee displaying robust online control of multi-functional prostheses across a large repertoire of challenging tasks. Significance: The development and translation of man-machine interfaces that account for an individual's neuromusculoskeletal system creates unprecedented opportunities to understand how disrupted neuro-mechanical processes can be restored or replaced via biomimetic wearable assistive technologies
MyoSim:Fast and physiologically realistic MuJoCo models for musculoskeletal and exoskeletal studies
Owing to the restrictions of live experimentation, musculoskeletal simulation models play a key role in biological motor control studies and investigations. Successful results of which are then tried on live subjects to develop treatments as well as robot aided rehabilitation procedures for addressing neuromusculoskeletal anomalies ranging from limb loss, to tendinitis, from sarcopenia to brain and spinal injuries. Despite its significance, current musculoskeletal models are computationally expensive, and provide limited support for contact-rich interactions which are essential for studying motor behaviors in activities of daily living, during rehabilitation treatments, or in assistive robotic devices. To bridge this gap, this work proposes an automatic pipeline to generate physiologically accurate musculoskeletal, as well as hybrid musculoskeletal-exoskeletal models. Leveraging this pipeline we present MyoSim - a set of computationally efficient (over 2 orders of magnitude faster than state of the art) musculoskeletal models that support fully interactive contact rich simulation. We further extend MyoSim to support additional features that help simulate various real-life changes/diseases, such as muscle fatigue, and sarcopenia. To demonstrate the potential applications, several use cases, including interactive rehabilitation movements, tendon-reaffirmation, and the cosimulation with an exoskeleton, were developed and investigated for physiological correctness. Web-page: https://sites.google.com/view/myosuit
Multi-scale modelling of the human neuromuscular system for symbiotic human-machine motor interaction
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