Méthodes spectroscopiques et optiques pour le suivi de la croissance des biofilms en milieu microfluidique

Les biofilms sont largement répandus dans la plupart des écosystèmes terrestres. Ils peuvent être formés par la plupart des microorganismes. Dans le cadre de cette thèse, les biofilms bactériens, plus spécifiquement ceux formés par la bactérie Pseudomonas sp. CT07, ont été étudiés. Ils ont plusieurs rôles, utiles ou nuisibles, pour la santé humaine, l’agriculture et l’industrie. Contrairement aux bactéries planctoniques qui peuvent nager librement, les bactéries sessiles s’attachent aux surfaces où elles peuvent former des biofilms. Pendant ce processus, elles produisent une matrice extracellulaire faite de, mais pas exclusivement, polysaccharides, de protéines, d’ADN et d’ARN. Les propriétés mécaniques de la matrice rendent le biofilm très résilient à son environnement. Elle est viscoélastique et les bactéries peuvent modifier de manière dynamique les propriétés mécaniques du biofilm. La grande variété de groupements fonctionnels disponibles grâce aux différentes biomolécules qui sont présentes dans la matrice permettent de piéger des molécules organiques et les ions dissous. Cela est responsable de plusieurs mécanismes de résistance bactérienne aux antibiotiques. L’objectif principal de cette thèse est de concevoir de nouvelles méthodologies analytiques pour étudier les biofilms et obtenir plus d’informations sur leur structure et ce qui peut les influencer. La spectroscopie infrarouge et la microscopie optique ont été utilisées dans des canaux microfluidiques pour suivre la croissance des biofilms. La combinaison des deux techniques permet l’obtention d’informations sur la composition en différentes biomacromolécules et sur la structure du biofilm. Ces méthodes ont permis d’évaluer l’efficacité de l’inoculation directe et de l’inoculation par un biofilm en amont. L’utilisation de microcanaux avec un faible rapport d’aspect a conduit en des différences importantes dans les conditions hydrodynamiques entre le centre du microcanal et ses coins. Dans cette configuration, les biofilms ont tendance à croître à partir des murs de côté qui sont plus courts. À cet endroit, les forces de cisaillement sont les plus faibles. La microscopie confocale intermittente montre la présence de canaux d’eau exempts de bactéries à l’intérieur d’un biofilmà proximité du coin du microcanal. Nous émettons l’hypothèse que ce canal a un rôle important dans le transfert de masse à l’intérieur du biofilm lorsqu’il devient plus épais. Lorsque les biofilms des bactéries Pseudomonas croissent, leur structure peut être influencée par le type de milieu de culture. Les biofilms croissant dans les milieux complexes obtenus à partir d’extraits de levure peuvent former des structures allongées nommées streamers qui ont été analysées par microscopie confocale à balayage laser. L’imagerie en trois dimensions de ces structures dans des microcanaux droits est rapportée pour la première fois. Dans un milieu de culture minimal avec le citrate de sodium comme seule source de carbone, nous avons observé et quantifié des patrons fractals à la base du biofilm dans le temps. Nous avons aussi conçu un dispositif microfluidique pour l’étude in situ par spectroscopie Raman exaltée par les surfaces (SERS). Cette méthode permet d’avoir un signal Raman rehaussé et une sensibilité élevée pour le citrate de sodium, une source de carbone commune pour les bactéries, à faible concentration. Les différentes méthodologies développées dans le cadre de cette thèse peuvent être appliquées à d’autre systèmes plus complexes dans le futur. La combinaison de la microfluidique pour le contrôle précis de l’écoulement ainsi que les mesures multiplexées dans des microcanaux en parallèle est la clé pour obtenir des indices importants et statistiquement pertinents sur la croissance des biofilms et les méthodes pour les contrôler.Biofilms are widely spread among most of earth ecosystems. They can be formed by a variety of microorganisms. In the scope of this thesis, bacterial biofilms, more specifically those formed by the bacterium Pseudomonas sp. CT07, have been studied. They have many roles, useful and harmful, for the human health, agriculture and industry. As opposed to planktonic bacteria that can swim freely, sessile bacteria are attached to surfaces where they can form biofilms. During this process, they produce an extracellular matrix made of, but not exclusively, polysaccharides, proteins, DNA and RNA. The mechanical properties of the matrix make the biofilm very resilient to its surrounding environment. It is viscoelastic, and the bacteria can dynamically modify the mechanical properties of the biofilm. The high variety of functional groups available due to the different biomolecules present allows trapping of organic molecules and dissolved ions by the matrix. This is responsible for multiple mechanism of resistance to antimicrobial by bacteria. The main objective of this thesis is to develop new analytical methodologies to study biofilms and obtain more insights on the structure of biofilms and what can influence them. Infrared spectroscopy and optical microscopy were used in microfluidic channel to follow biofilm growth. The combination of the two techniques enabled acquisition of information on the composition in biomacromolecules and biofilm structure. These methods allowed to assess the efficiency of direct inoculation and inoculation from an upstream biofilm. The use of low aspect ratio channels resulted in strong differences in hydrodynamic conditions between the middle of the channel and the channel corners. In this configuration biofilms tended to grow from the short side-walls of microchannels where shear stress was lowest. Time-lapse confocal microscopy showed the presence of a biochannel inside the biofilm in the corner positions. It is hypothesized that this channel has an important role in mass transfer in biofilm as it grows thicker. As the biofilms of Pseudomonas bacteria grew, their structure could be influenced by the type of growth media. Biofilms grown in complex media obtained from yeast cell extract could form elongated structures called streamers which were analysed with confocal laser scanning microscopy. Three-dimensional imaging of these structures in regular straight microchannels is the first of its kind. In a minimal medium with citrate as the sole carbon source, we observe and quantify time-dependant fractal patterns at the biofilm base. We have also developed a microfluidic device for in situ study by surface enhanced Raman spectroscopy (SERS). This method allows having enhanced Raman signal and high sensitivity of sodium citrate, a common carbon source for bacteria, at low concentration. The different methodology developed in this thesis can be applied to more complex systems in the future. Combination of microfluidic for precise flow control and multiplexed measurement in massively parallelized channels is key to get deeper, statistically relevant insights in biofilm growth and methods to control them

Le paysage, entre art et politique

Depuis ces temps lointains où le jardin royal symbolisait le royaume jusqu’à l’actualité de l’image numérique qui, à force de se multiplier, substitue au monde son propre spectacle, les représentations paysagères, aussi artistiques soient-elles, ont toujours conservé un pouvoir mobilisateur qui leur confère une réelle valeur politique. C’est pourquoi le paysage a très tôt fait l’objet de conventions, que relaient désormais les normes édictées par le législateur, la bureaucratie et le marché. Cette puissance politique des représentations paysagères, qui n’est pas sans lien avec leur valeur artistique, n’est en rien diminuée depuis que le paysage, déjà accaparé par le savoir technique, a été élevé au rang d’objet scientifique. On peut même croire que s’est ainsi nouée une nouvelle alliance de l’art et de la politique. Les textes de huit auteurs québécois et français réunis dans ce volume traitent diversement du paysage, des formes qu’il prend et qu’il a prises, des règles de sa composition et des raisons qui justifieraient sa conservation, sa mise en valeur, son enchantement. Il y est aussi question de ses usages et des fonctions qu’on lui a attribuées, non seulement à l’époque contemporaine, mais aussi dans le passé, proche ou plus lointain. Se pose de plus le problème du lien entre la réalité concrète du paysage, inscrite dans l’espace géographique et ses représentations, ancrées dans l’esprit humain.

Native spider silk as a biological optical fiber.

International audienceIn this study, we demonstrate the use of eco-friendly native spider silk as an efficient optical fiber in air, highly bent fibers, and physiological liquid. We also integrated the silk filament in a photonic chip made of polymer microstructures fabricated by UV lithography. The molding process is non-destructive for silk and leads to an efficient micro-optical coupling between silk and synthetic optical structures. These optical performances combined with the unique biocompatibility, bioresorbability, flexibility, and tensile strength of silk filaments pave the way for new applications in biological media and for original biophotonic purposes

A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons

IntroductionHuman induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process.MethodsIn this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter.ResultsThe retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types.DiscussionOur results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures

Low-value clinical practices in injury care: a scoping review and expert consultation survey

BACKGROUND: Tests and treatments that are not supported by evidence and could expose patients to unnecessary harm, referred to here as low-value clinical practices, consume up to 30% of healthcare resources. Choosing Wisely and other organisations have published lists of clinical practices to be avoided. However, few apply to injury and most are based uniquely on expert consensus. We aimed to identify low-value clinical practices in acute injury care. METHODS: We conducted a scoping review targeting articles, reviews and guidelines that identified low-value clinical practices specific to injury populations. Thirty-six experts rated clinical practices on a 5-point Likert scale from clearly low-value to clearly beneficial. Clinical practices reported as low-value by at least one level I, II or III study and considered clearly or potentially low-value by at least 75% of experts were retained as candidates for low-value injury care. RESULTS: Of 50,695 citations, 815 studies were included and led to the identification of 150 clinical practices. Of these 63 were considered candidates for low-value injury care; 33 in the emergency room, 9 in trauma surgery, 15 in the intensive care unit and 5 in orthopaedics. We also identified 87 'grey zone' practices, which did not meet our criteria for low-value care. CONCLUSIONS: We identified 63 low-value clinical practices in acute injury care that are supported by empirical evidence and expert opinion. Conditional on future research, they represent potential targets for guidelines, overuse metrics and de-implementation interventions. We also identified 87 'grey zone' practices, which may be interesting targets for value-based decision-making. Our study represents an important step towards the de-implementation of low-value clinical practices in injury care. LEVEL OF EVIDENCE: III

Glass transitions in native silk fibres studied by dynamic mechanical thermal analysis

Silks are a family of semi-crystalline structural materials, spun naturally by insects, spiders and even crustaceans. Compared to the characteristic β-sheet crystalline structure in silks, the non-crystalline structure and its composition deserves more attention as it is equally critical to the filaments' high toughness and strength. Here we further unravel the structure-property relationship in silks using Dynamic Mechanical Thermal Analysis (DMTA). This technique allows us to examine the most important structural relaxation event of the disordered structure the disordered structure, the glass transition (GT), in native silk fibres of the lepidopteran Bombyx mori and Antheraea pernyi and the spider Nephila edulis. The measured glass transition temperature Tg, loss tangent tan δ and dynamic storage modulus are quantitatively modelled based on Group Interaction Modelling (GIM). The "variability" issue in native silks can be conveniently explained by the different degrees of structural disorder as revealed by DMTA. The new insights will facilitate a more comprehensive understanding of the structure-property relations for a wide range of biopolymers

Dendritic release of neurotransmitters

Release of neuroactive substances by exocytosis from dendrites is surprisingly widespread and is not confined to a particular class of transmitters: it occurs in multiple brain regions, and includes a range of neuropeptides, classical neurotransmitters and signaling molecules such as nitric oxide, carbon monoxide, ATP and arachidonic acid. This review is focused on hypothalamic neuroendocrine cells that release vasopressin and oxytocin and midbrain neurons that release dopamine. For these two model systems, the stimuli, mechanisms and physiological functions of dendritic release have been explored in greater detail than is yet available for other neurons and neuroactive substances

Mechanical behaviour and formation process of silkworm silk gut

High performance silk fibers were produced directly from the silk glands of silkworms ("Bombyx mori") following an alternative route to natural spinning. This route is based on a traditional procedure that consists of soaking the silk glands in a vinegar solution and stretching them by hand leading to the so called silkworm guts. Here we present, to the authors’ best knowledge, the first comprehensive study on the formation, properties and microstructure of silkworm gut fibers. Comparison of the tensile properties and microstructural organization of the silkworm guts with those of naturally spun fibers allows gain of a deeper insight into the mechanisms that lead to the formation of the fiber, as well as the relationship between the microstructure and properties of these materials. In this regard, it is proved that an acidic environment and subsequent application of tensile stress in the range of 1000 kPa are sufficient conditions for the formation of a silk fiber

Étude de la structure de fibres de soie et de la soie en solution par spectroscopie vibrationnelle

Les travaux présentés portent sur l'étude de la structure moléculaire des fibres de soie et de la soie en solution. La spectroscopie infrarouge à réflexion totale atténuée a permis d'étudier la deutération de la soie de l'araignée Nephila clavipes et du ver à soie Bombyx mori. Nous avons pu identifier la présence de structures amorphes, de feuillets β cristallins et de feuillets β interfaciaux dans les deux fibres en plus de calculer leurs proportions et leur orientation. Le dichroïsme circulaire vibrationnel et l'activité optique Raman ont été utilisés pour étudier la structure secondaire de la soie en solution puisque ces techniques sont très sensibles à la conformation des protéines. Les spectres des protéines recombinantes rMaSpI et rMaSpII, analogues aux protéines de la soie d'araignée, et de la soie d'araignée et de Bombyx mori indiquent que la soie forme des hélices 3i en solution dans l'eau et dans l'eau lourde