Développement et caractérisation d’une méthode photonique pour créer des distributions spatiales de protéines

Les cellules sont capables de détecter les distributions spatiales de protéines et ainsi de migrer ou s’étendre dans la direction appropriée. Une compréhension de la réponse cellulaire aux modifications de ces distributions spatiales de protéines est essentielle pour l’avancement des connaissances dans plusieurs domaines de recherches tels que le développement, l’immunologie ou l’oncologie. Un exemple particulièrement complexe est le guidage d’axones se déroulant pendant le développement du système nerveux. Ce dernier nécessite la présence de plusieurs distributions de molécules de guidages étant attractives ou répulsives pour connecter correctement ce réseau complexe qu’est le système nerveux. Puisque plusieurs indices de guidage collaborent, il est particulièrement difficile d’identifier la contribution individuelle ou la voie de signalisation qui est déclenchée in vivo, il est donc nécessaire d’utiliser des méthodes pour reproduire ces distributions de protéines in vitro. Plusieurs méthodes existent pour produire des gradients de protéines solubles ou liées aux substrats. Quelques méthodes pour produire des gradients solubles sont déjà couramment utilisées dans plusieurs laboratoires, mais elles limitent l’étude aux distributions de protéines qui sont normalement sécrétées in vivo. Les méthodes permettant de produire des distributions liées au substrat sont particulièrement complexes, ce qui restreint leur utilisation à quelques laboratoires. Premièrement, nous présentons une méthode simple qui exploite le photoblanchiment de molécules fluorescentes pour créer des motifs de protéines liées au substrat : Laser-assisted protein adsorption by photobleaching (LAPAP). Cette méthode permet de produire des motifs de protéines complexes d’une résolution micrométrique et d’une grande portée dynamique. Une caractérisation de la technique a été faite et en tant que preuve de fonctionnalité, des axones de neurones du ganglion spinal ont été guidés sur des gradients d’un peptide provenant de la laminine. Deuxièmement, LAPAP a été amélioré de manière à pouvoir fabriquer des motifs avec plusieurs composantes grâce à l’utilisation de lasers à différentes longueurs d’onde et d’anticorps conjugués à des fluorophores correspondants à ces longueurs d’onde. De plus, pour accélérer et simplifier le processus de fabrication, nous avons développé LAPAP à illumination à champ large qui utilise un modulateur spatial de lumière, une diode électroluminescente et un microscope standard pour imprimer directement un motif de protéines. Cette méthode est particulièrement simple comparativement à la version originale de LAPAP puisqu’elle n’implique pas le contrôle de la puissance laser et de platines motorisées, mais seulement d’envoyer l’image du motif désiré au modulateur spatial. Finalement, nous avons utilisé LAPAP pour démontrer que notre technique peut être utilisée dans des analyses de haut contenu pour quantifier les changements morphologiques résultant de la croissance neuronale sur des gradients de protéines de guidage. Nous avons produit des milliers de gradients de laminin-1 ayant différentes pentes et analysé les variations au niveau du guidage de neurites provenant d’une lignée cellulaire neuronale (RGC-5). Un algorithme pour analyser les images des cellules sur les gradients a été développé pour détecter chaque cellule et quantifier la position du centroïde du soma ainsi que les angles d’initiation, final et de braquage de chaque neurite. Ces données ont démontré que les gradients de laminine influencent l’angle d’initiation des neurites des RGC-5, mais n’influencent pas leur braquage. Nous croyons que les résultats présentés dans cette thèse faciliteront l’utilisation de motifs de protéines liées au substrat dans les laboratoires des sciences de la vie, puisque LAPAP peut être effectué à l’aide d’un microscope confocal ou d’un microscope standard légèrement modifié. Cela pourrait contribuer à l’augmentation du nombre de laboratoires travaillant sur le guidage avec des gradients liés au substrat afin d’atteindre la masse critique nécessaire à des percées majeures en neuroscience.Cells are able to sense spatial distribution of proteins and accordingly migrate or extend in the appropriate direction. Understanding cellular responses to modifications in molecular spatial distributions is essential for advances in several fields such as development, immunology and oncology. A particularly complex example is axonal guidance that occurs during the development of the nervous system, which relies on distributions of attractive and repulsive guidance molecules to correctly wire this intricate network. Since several guidance cues collaborate to development of the nervous system, it is particularly difficult to assess the individual contribution of each cue and the signaling cascade each trigger in vivo; therefore methods to reproduce those distributions individually in vitro are necessary to study in detail the effect of each guidance cue. Several methods exist to produce graded distributions of protein that are either soluble or substrate-bound. A few methods making solution gradients are already widely used in several laboratories to perform experiments with the guidance cues that are normally diffusing in vivo. However, current methods allowing the fabrication of substrate-bound gradients are quite complex, which restrict their use to a few laboratories. First, we present a straightforward method exploiting photobleaching of a fluorescently tagged molecule using a visible laser to generating substrate-bound protein patterns: Laser-assisted protein adsorption by photobleaching (LAPAP). This method allows producing complex patterns of protein with micron spatial resolution and high dynamic range. An extensive characterization of the technique was performed and as proof of functionality, axons from dorsal root ganglions cells were guided on laminin peptide gradients. Secondly, LAPAP was improved in order to produce multicomponent patterns by using lasers at different wavelengths and antibodies conjugated to fluorophores corresponding to these wavelengths. Moreover, to speed-up the fabrication process and simplify the device, we developed widefield illumination LAPAP which uses a spatial light modulator, a light emitting diode and a standard microscope to directly print patterns. This patterning method is relatively simple compared to the original LAPAP setup, since it does not involve controlling the laser power or a motorized stage, but only sends an image of the desired pattern to a spatial light modulator. Finally, we used LAPAP to show how it could be used in automated high-content screening assays to quantify the morphological changes resulting from axon growth on gradients of guidance proteins. We produced thousands of laminin-1 gradients of different slopes and analyzed the variations in neurite guidance of neuron-like cells (RGC-5). An image analysis algorithm was developed to process bright field microscopy images, detecting each cell and quantifying the soma centroid and the initiation, terminal and turning angles of the maximal neurite. This data showed that laminin gradients influence the initiation angle of neurite extension of RGC-5, but does not contribute to its turning. We believe that the results presented in this thesis will facilitate the use of substrate- bound protein patterning in typical life science laboratories, since a confocal microscope or a slightly modified standard microscope is the only specialized equipment needed to fabricate patterns by LAPAP. This could increase the number of laboratories working with substrate-bound protein patterns in order to reach the critical mass necessary for major advances in neuroscience

Weather effects on autumn nocturnal migration of passerines on opposite shores of the St. Lawrence estuary

We modeled migration intensity as a function of weather, using nightly migration measurements from Doppler surveillance weather radar during autumn migration on the north (Côte-Nord) and south (Gaspésie) shores of the St. Lawrence estuary, Québec, Canada. The radar had negative elevation angles, an uncommon characteristic among weather radars, which allowed simultaneous low-altitude monitoring of bird migration on each side of the estuary. Precipitation and wind both had strong effects on the intensity of migration. Very few birds migrated when >40% of the area had precipitation, especially when winds were strong. Light winds were associated with the strongest migration intensity, regardless of wind direction; in stronger winds, migration was likely only when winds were predominantly from the north. Days immediately after adverse weather events, which are assumed to lead to an accumulation of migrants, were associated with an increase in the intensity of migration in Côte-Nord, but not in Gaspésie. Time since the passage of a cold front had no effect in either region. Bird flight direction and behavior in relation to wind differed on each side of the estuary. On Côte-Nord, birds tended to migrate in a southwesterly direction along the St. Lawrence north coast, in a direction relatively unaffected by wind direction; they compensated or overcompensated for wind drift by following the coast. By contrast, birds in Gaspésie tended to fly in a more southerly direction. They migrated partially or almost fully downwind with only limited compensation, their flight direction often changing with wind direction. Nous avons modélisé l'intensité migratoire automnale en fonction de la météo, en utilisant des mesures de la migration nocturne prises à la fois sur la rive nord (Côte-Nord) et la rive sud (Gaspésie) de l'estuaire du Saint-Laurent, Québec, Canada, à l'aide d'un radar Doppler de surveillance météorologique. Ce radar effectue des balayages à des angles négatifs, une caractéristique rare chez les radars météo qui permet entre autres, de relever des données de migration d'oiseaux à basse altitude et simultanément de chaque côté de l'estuaire. Nos résultats montrent que les précipitations et le vent avaient de forts effets sur l'intensité migratoire. Peu d'oiseaux migraient quand 40% ou plus du territoire était affecté par des précipitations, particulièrement en combinaison avec des vents forts. Les plus fortes intensités migratoires étaient associés avec des vents légers, peu importe la direction du vent; par vents forts, la migration était plus probable quand les vents avaient une composante nord. Un événement de conditions météo adverses à la migration s'ensuivait d'une augmentation de l'intensité migratoire sur la Côte-Nord, mais pas en Gaspésie. Le passage d'un front froid n'avait aucun effet sur la migration dans l'une ou l'autre des deux régions. La direction de vol des oiseaux et leur comportement relativement au vent différaient de chaque côté de l'estuaire. Sur la Côte-Nord, les oiseaux avaient tendance à migrer vers Ie sud-ouest, Ie long de la rive nord du Saint-Laurent; cette direction de vol était peu affectée par la direction du vent et les oiseaux suivaient la côte en compensant ou surcompensant pour la dérive par les vents. En opposition, les oiseaux en Gaspésie avaient tendance à voler vers Ie sud; leur migration se faisait en suivant partiellement ou pleinement le vent avec peu de compensation et avec une direction de vol qui changeait souvent avec la direction du vent

Quasi-analyticity and determinacy of the full moment problem from finite to infinite dimensions

This paper is aimed to show the essential role played by the theory of quasi-analytic functions in the study of the determinacy of the moment problem on finite and infinite-dimensional spaces. In particular, the quasi-analytic criterion of self-adjointness of operators and their commutativity are crucial to establish whether or not a measure is uniquely determined by its moments. Our main goal is to point out that this is a common feature of the determinacy question in both the finite and the infinite-dimensional moment problem, by reviewing some of the most known determinacy results from this perspective. We also collect some properties of independent interest concerning the characterization of quasi-analytic classes associated to log-convex sequences.Comment: 28 pages, Stochastic and Infinite Dimensional Analysis, Chapter 9, Trends in Mathematics, Birkh\"auser Basel, 201

Explicit formulae in probability and in statistical physics

We consider two aspects of Marc Yor's work that have had an impact in statistical physics: firstly, his results on the windings of planar Brownian motion and their implications for the study of polymers; secondly, his theory of exponential functionals of Levy processes and its connections with disordered systems. Particular emphasis is placed on techniques leading to explicit calculations.Comment: 14 pages, 2 figures. To appear in Seminaire de Probabilites, Special Issue Marc Yo

Defining a self-evaluation digital literacy framework for secondary educators: the DigiLit Leicester project

Despite the growing interest in digital literacy within educational policy, guidance for secondary educators in terms of how digital literacy translates into the classroom is lacking. As a result, many teachers feel ill-prepared to support their learners in using technology effectively. The DigiLit Leicester project created an infrastructure for holistic, integrated change, by supporting staff development in the area of digital literacy for secondary school teachers and teaching support staff. The purpose of this article is to demonstrate how the critique of existing digital literacy frameworks enabled a self-evaluation framework for practitioners to be developed. Crucially, this framework enables a co-operative, partnership approach to be taken to pedagogic innovation. Moreover, it enables social and ethical issues to underpin a focus on teacher-agency and radical collegiality inside the domain of digital literacy. Thus, the authors argue that the shared development framework constitutes a new model for implementing digital literacy aimed at transforming the provision of secondary education across a city

Employing the Metabolic “Branch Point Effect” to Generate an All-or-None, Digital-like Response in Enzymatic Outputs and Enzyme-Based Sensors

Here, we demonstrate a strategy to convert the graded Michaelis−Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the “branch point effect”, a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter’s response. Above an arbitrarily selected “threshold” substrate concentration, the nonsignaling HK enzyme saturates leading to a “sudden” activation of the first signaling GOx enzyme and a far steeper dose−response curve than that observed for simple Michaelis−Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications

Spatiotemporal Patterns in Nest Box Occupancy by Tree Swallows Across North America

Data from the North American Breeding Bird Survey (BBS) suggest that populations of aerial insectivorous birds are declining, particularly in northeastern regions of the continent, and particularly since the mid-1980s. Species that use nest boxes, such as Tree Swallows (Tachycineta bicolor), may provide researchers with large data sets that better reveal finer-scale geographical patterns in population trends. We analyzed trends in occupancy rates for ca. 40,000 Tree Swallow nest-box-years from 16 sites across North America. The earliest site has been studied intensively since 1969 and the latest site since 2004. Nest box occupancy rates declined significantly at five of six (83%) sites east of -78° W longitude, whereas occupancy rates increased significantly at four of ten sites (40%) west of -78° W longitude. Decreasing box occupancy trends from the northeast were broadly consistent with aspects of a previous analysis of BBS data for Tree Swallows, but our finding of instances of increases in other parts of the continent are novel. Several questions remain, particularly with respect to causes of these broad-scale geographic changes in population densities of Tree Swallows. The broad geographic patterns are consistent with a hypothesis of widespread changes in climate on wintering, migratory, or breeding areas that in turn may differentially affect populations of aerial insects, but other explanations are possible. It is also unclear whether these changes in occupancy rates reflect an increase or decrease in overall populations of Tree Swallows. Regardless, important conservation steps will be to unravel causes of changing populations of aerial insectivores in North America

Malaria pigment crystals as magnetic micro-rotors: Key for high-sensitivity diagnosis

The need to develop new methods for the high-sensitivity diagnosis of malaria has initiated a global activity in medical and interdisciplinary sciences. Most of the diverse variety of emerging techniques are based on research-grade instruments, sophisticated reagent-based assays or rely on expertise. Here, we suggest an alternative optical methodology with an easy-to- use and cost-effective instrumentation based on unique properties of malaria pigment reported previously and determined quantitatively in the present study. Malaria pigment, also called hemozoin, is an insoluble microcrystalline form of heme. These crystallites show remarkable magnetic and optical anisotropy distinctly from any other components of blood. As a consequence, they can simultaneously act as magnetically driven micro-rotors and spinning polarizers in suspensions. These properties can gain importance not only in malaria diagnosis and therapies, where hemozoin is considered as drug target or immune modulator, but also in the magnetic manipulation of cells and tissues on the microscopic scale

FactSage thermochemical software and databases, 2010–2016

The FactSage computer package consists of a series of information, calculation and manipulation modules that enable one to access and manipulate compound and solution databases. With the various modules running under Microsoft Windows® one can perform a wide variety of thermochemical calculations and generate tables, graphs and figures of interest to chemical and physical metallurgists, chemical engineers, corrosion engineers, inorganic chemists, geochemists, ceramists, electrochemists, environmentalists, etc. This paper presents a summary of the developments in the FactSage thermochemical software and databases during the last six years. Particular emphasis is placed on the new databases and developments in calculating and manipulating phase diagrams