Biofilms are microbial communities developing at the interface between two phases, usually solidliquid, where the micro-organisms are nested in a self-secreted polymer matrix. The biofilm mode of growth is predominant in nature (for e.g. the slimy matter forming on rocks at river bottoms, the viscous deposit in water pipes or even dental plaque) and confers a suitable environment for the development of the micro-organisms. This is particularly the case for porous media which provide favourable substrates given their significant surface to volume ratio. The multi-physical framework of biofilms in porous media is highly complex where the mechanical, chemical and biological aspects interacting at different scales are poorly understood and very partially controlled. An example is the feedback mechanism between flow, spatial distribution of the micro-organisms and the transport of nutrient (by diffusion and advection). Biofilms developing in porous media are a key process of many engineering applications, for example biofilters, soil bio-remediation, CO2 storage and medical issues like infections. Progress in this domain is substantially hindered by the limitations of experimental techniques in metrology and imaging in opaques structures. The main objective of this thesis is to propose robust and reproducible experimental methodologies for the investigation of biofilms in porous media. An experimental workbench under controlled physical and biological conditions is proposed along with a validated 3D imaging protocol based on X-ray micro-tomography (XR MT) using a novel contrast agent (barium sulfate and agarose gel) to quantify the spatial distribution of the biofilm. At first, the XR MT-based methodology is compared to a commonly used techniques for biofilm observation: one or multiple photon excitation fluorescence microscopy, here two-photon laser scanning microscopy (TPLSM). This comparison is performed on Pseudomonas Aeruginosa biofilms grown in transparent glass capillaries which allows for the use of both imaging modalities. Then, the study of uncertainty associated to different metrics namely volume, 3D surface area and thickness, is achieved via an imaging phantom and three different segmentation algorithms. The quantitative analysis show that the protocol enables a visualisation of the biofilm with an uncertainty of approximately 17% which is comparable to TPLSM (14%). The reproducibility and robustness of the XR MT-based methodology is demonstrated. The last step of this work is the achievement of a novel bioreactor elaborated by additive manufacturing and controlled by a high-performance micro-fluidic system. The experimental workbench that we have designed enables to monitor in real-time the evolution of transport properties (effective permeability), O2 concentrations and biofilm detachment by spectrophotometry, all under controlled hydrodynamical conditions. Our methodology allows to investigate the influence of biophysical parameters on the colonisation of the porous medium, for example, the influence of flow rate or nutrient concentration on the temporal development of the biofilm. In conclusion, the thesis work proposes a robust and reproducible experimental methodology for the controlled growth and 3D imaging of biofilms in porous media; while providing versatility in the control of the substrate’s micro-architecture as well as on the flow and biochemical culture conditions. To our knowledge, the scientific approach followed, along with the experimental apparatus, form the most complete methodology, at this time, for the study of biofilms in porous media.

Bacillus anthracis, l'agent infectieux responsable de la maladie du charbon, est un agent pathogène majeur du risque biologique provoqué, notamment en raison de la sévérité de la forme respiratoire de la maladie. Celle-ci résulte de l'inhalation de spores dont les mécanismes de pénétration au niveau pulmonaire sont mal connus à l'heure actuelle. Cette thèse présente les apports des microscopies confocale et biphotonique à l'étude de ces mécanismes de pénétration des spores inhalées. Le modèle murin CX3CR1+/gfp, dont la sous-population CD11b+ de cellules dendritiques (DCs) exprime constitutivement la protéine de fluorescence verte (GFP), a été utilisé dans ces travaux. Une première partie présente le développement d'une méthode automatisée de discrimination des DCs parmi d'autres populations cellulaires exprimant le même fluorophore, en se basant sur le calcul d'un coefficient morphologique. Cette méthode a permis d'étudier dans un deuxième temps le comportement spécifique de la sous-population de DCs CD11b, après infection par des spores de B. anthracis. L'étude microscopique a été d'abord effectuée in situ, c'est-à-dire sur des explants pulmonaires maintenus dans des conditions favorables à la préservation de l'activité cellulaire, puis in vivo, sur des souris anesthésiées et ventilées. Le protocole d'imagerie tire profit d'une stratégie d'acquisition et de traitement a posteriori des données permettant de surmonter, sans contrainte mécanique appliquée à l'organe, les problèmes de focalisation liés aux mouvements thoraciques durant la ventilation de l'animal. Cette stratégie originale utilise un sur-échantillonnage de l'acquisition et profite du signal de seconde harmonique généré par le collagène comme référence spatiale ; elle a permis l'observation in vivo d'interactions entre DCs et macrophages au niveau pulmonaire. Ces interactions, de type synapse immunologique, sont favorisées par l'infection et présentent donc un rôle fonctionnel qui reste à définir. La formation de synapses immunologiques entre macrophages et DCs pourrait non seulement représenter un chaînon manquant à l'explication de la pénétration des spores de B. anthracis au niveau pulmonaire, mais pourrait aussi constituer un enjeu crucial dans la compréhension de la réponse immunitaire associée aux infections pulmonaires. / Bacillus anthracis, the causative agent of anthrax, is a major bioterrorism pathogen mainly because it can lead to a severe respiratory form of the disease. This form results from inhalation of spores, whose ways of entry into the lungs are not fully understood. This thesis reports the contribution of confocal and two-photon microscopy to the study of the penetration mechanisms of inhaled spores. The animal model utilized was CX3CR1+/gfp mouse, which constitutively expresses the green fluorescent protein (GFP) on CD11b+ dendritic cells (DCs). First, we present an automated method allowing discrimination of DCs among other GFP expressing cells, based on a morphologic coefficient. This method was then applied to the study of the specific behavior of CD11b DCs, after infection by B. anthracis spores. The microscopic study was first performed in situ, i.e. on explanted organs kept in conditions favorable to cell dynamics, then in vivo, i.e. on anesthetized and ventilated mice. In this case the imaging protocol profits from both acquisition and post-processing strategies, and allowed overcoming the focalization pitfalls coming from chest movements during ventilation. This novel strategy is based on an over-sampling of frame acquisition and utilizes second harmonic generation signal from alveolar collagen as a spatial reference. It led to the first ever in vivo observation of interactions between DCs and macrophages at the lung level. These immunological synapse-like structures are promoted by infection and thus display a functional role unknown until now. The formation of macrophages-DCs immunological synapses not only could represent a missing-link in figuring out the B. anthracis spore penetration mechanisms at the lung level, but more importantly could lead to a better understanding of the immune response associated with pulmonary infections.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22376号 / 医博第4617号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 林 康紀, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20245号 / 医博第4204号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 羽賀 博典, 教授 鈴木 茂彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Fluorescence microscopy has played a pivotal role in the realm of biological and biomedical research, allowing researchers to delve into the intricacies of living organisms at the cellular and molecular levels. By using fluorescent probes, one can visualize specific molecules and structures within cells, fundamentally transforming our comprehension of biology and medicine. However, fluorescence microscopy faces its own set of challenges, namely, photobleaching and photodamage. Photobleaching involves the irreversible loss of fluorescence signal during imaging, while photodamage results in harmful effects on cells. Both severely limit fluorescence signal and observation time. Although remedies exist to mitigate these problems, most of them rely on chemical approaches. In this dissertation, to address these issues, I investigated two optical approaches that exploit control of light either in space or time. Firstly, I developed multiline scanning confocal microscopy (mLS) with a digital micro-mirror device. This method provides programmable patterns of the illumination beam as well as the detection slit. Through experimental results and optical simulations, I assessed the depth discrimination of mLS under different optical parameters and compared it with a multipoint system such as spinning disk confocal microscopy (SDCM). Surprisingly, under the same illumination duty cycle, I found that mLS offers better optical sectioning than SDCM. Importantly, the parallelized line illumination showed a much lower photobleaching rate compared to single-line scanning microscopy, while their optical sectioning capabilities remained similar. I applied this technique to visualize heterogeneous mouse epiblast stem cells, a challenging task in imaging. Secondly, I delved into low photobleaching rate two-photon microscopy (2PM). 2PM inherently provides excellent optical sectioning due to its nonlinear effects, making it suitable for high-resolution imaging within biological tissues. However, the high peak power of ultrafast pulses has always been associated with severe photobleaching, posing a longstanding challenge. I found that controlling the repetition rates of ultrafast lasers is a potential strategy to enhance photostability. Specifically, I used repetition rates lower or higher than 80 MHz in 2PM and conducted systematic experiments to investigate how optical parameters such as wavelengths, excitation powers, and pulse schemes can influence the photobleaching kinetics of fluorescent proteins and organic fluorophores. This thesis embarks on a journey to explore innovative strategies and methodologies aimed at reducing photobleaching while maintaining high-quality imaging in the realm of fluorescence microscopy.

Neurogenesis is the process of generation of neurons during embryonic development and adulthood. The focus of this doctoral work is the study of the cell biological aspects of neurogenesis and the mechanisms regulating the switch of neural stem cells from proliferation to differentiation. During embryonic development neurogenic divisions occur at the apical or basal side of the pseudostratified epithelium that forms the wall of the neural tube, the neuroepithelium. Apical asymmetric neurogenic divisions (AP) give rise to a neuron and a progenitor cell, while basal symmetric neurogenic divisions (BP) give rise to two neurons. The first part of this thesis is focused on the study of some cell biological aspects of BPs. We first validated the use of the Tis21-GFP knock in mouse line, previously generated in our laboratory. We found that the totality of neurogenic progenitors is marked by the expression of a nuclear GFP. We calculated the abundance of BPs overtime since the onset of neurogenesis showing that BPs overcome APs over development. We studied the loss of apical contact of the basal dividing cells. We found that both neurogenic and non-neurogenic basally dividing progenitors miss the apical contact; which is lost prior mitosis. We generated and characterized a second mouse line, the Tubb3-GFP line expressing a plasma membrane-localized GFP in neurons. These two lines were crossed to obtain a new line (TisTubb-GFP) allowing detection of neurogenic divisions and tracking daughter cells. Using this model: (i) we imaged symmetric neurogenic divisions of BPs, identifying daughter cells as neurons, during imaging; (ii) we compared the kinetics of betaIII-tubulin-GFP appearance after apical or basal mitosis, showing that daughters of BPs express betaIII-tubulin-GFP faster than daughters coming from apical divisions; (iii) we imaged neuronal migration and localization of the Golgi apparatus. Neurogenesis in the adult is confined to two specific regions in the telencephalon: the sub ependymal zone, lining the ventricle, and dentate gyrus of the hippocampus. The second part of this thesis focuses on the adult neurogenic progenitors lineage. Tis21-GFP expression was found and characterized in the two adult neurogenic regions from early postnatal to adulthood. Using a panel of markers for the adult neurogenic cell lineage and confocal imaging, we characterized Tis21-GFP expression, in the dentate gyrus. Tis21-GFP is first expressed in the neurogenic subpopulation of doublecortin positive cells. Tis21-GFP is inherited by the neurons and eventually degraded. Moreover, our data suggest that mitotic Tis21-GFP cells are an indicator of the levels of neurogenesis more accurate than doublecortin positive cells, in the early postnatal mouse. (Anlage Quick time movies 77,88 MB)

The roles of the vascular architecture and blood flow in response to neurovascular diseases are important in predicting physiological outcomes. Observing these parameters chronically with optical imaging techniques provides insight into the neurovascular recovery process. We develop and deploy optical imaging systems for monitoring the progression of vascular structure, perfusion, and functional blood response after ischemic stroke in a chronic rodent model to observe vascular dynamics of the cortex under normal and diseased pathologies. Specifically, we monitor the progression of the vascular structure and cerebral blood flow (CBF) over a chronic period in the rodent cortex after photo-thrombotic occlusion. Multi-Exposure Speckle Imaging (MESI) provides surface measurements of microvascular flow dynamics while Two-Photon Fluorescence Microscopy offers direct visualization of the microvascular structure. We observe the occurrence of vascular reorientation in the sub-surface microvascular structure over a 35 day post-occlusion period. We also correlate MESI flow estimates in the parenchyma with sub-surface microvascular volume fractions from two-photon microscopy to assess how vascular density influences the surface-integrated MESI measurements. Next, we develop and validate a MESI technique for measuring absolute changes of the functional blood flow response to forepaw stimulation in rodents, termed FA MESI. The optimal camera exposures for capturing the CBF response to forepaw stimulation are extracted from a training set of animal data and the feasibility of the technique is demonstrated in a testing animal set by comparing functional response results between new and existing techniques. We then deploy this system in a chronic study monitoring the progression of hemodynamic parameters after ischemic stroke within the functionally responding area of the cortex. The progression of the regional CBF perfusion and absolute changes in the magnitude of the functional blood flow response are monitored chronically after photo-thrombotic occlusion. We compare the differences between absolute and relative measurements of the functional blood flow responses, and validate FA MESI by comparing baseline measurements to 15-exposure MESI over the sampled flow distributions. We demonstrate the differences measured between the functional outcomes and the regional CBF perfusion over a three week post-occlusion time period. / text

Au cours du développement cérébral des vertébrés, le processus permettant à des neurones nouveaux-nés de s'incorporer dans des réseaux déjà établis est mal compris. En effet, la majorité des études ayant été réalisées à l'échelle de la cellule, une description détaillée de la dynamique des circuits au cours de ce phénomène est manquante. Pour l'étudier, j'ai développé une méthode innovante utilisant la larve de poisson zèbre comme modèle expérimental et une approche pluridisciplinaire combinant la génétique, la microscopie bi-photonique et l'optogénétique pour suivre le développement de l'activité de neurones nouveaux-nés et des réseaux matures voisins dans un vertébré intacte et non-anesthésié. En utilisant cette technique j'ai décrit pour la première fois, pendant plusieurs jours consécutifs, le développement des propriétés fonctionnelles de neurones nouveaux nés avant et pendant leur incorporation dans les circuits du toit optique, la structure cérébrale la plus complexe du poisson zèbre permettant l'intégration l'information visuelle. Les résultats obtenus suggèrent une séquence de développement durant laquelle les neurones morphologiquement immatures spontanément actifs se connectent en premier à la rétine. Dans un second temps, ces neurones s'incorporant graduellement au circuit mature en montrant des corrélations avec des neurones matures éparses. Troisièmement, l'organisation spatiale des corrélations entre les neurones nouveaux-nés est raffinée et devient plus dense. Ces résultats suggèrent que les neurones nouveaux-nés se connectent dans un premier temps a une population éparse de neurones matures avant que les connections a longue distance disparaissent permettant aux neurones en développement d'obtenir une signature fonctionnelle robuste (ex. réponses restreintes spatialement). Récemment, des traitements basés sur la transplantation des tissues neuronaux ont été développées pour certaines maladies neuro-dégénératives (ex. maladie de Parkinson). Cependant ces thérapies sont actuellement limitées par le faible taux de survie et l'incorporation des neurones injectés. Ces travaux apportent une meilleure compréhension des mécanismes à l’œuvre lors de la formation de circuits neuronaux et pourront peut-être permettre d'améliorer l'efficacité des traitements utilisant des cellules souches pour réparer le cerveau humain. / In the vertebrate brain, mechanisms leading to the incorporation of newborn neurons into already functional networks still remain poorly understood. Indeed, since most of the studies have been performed at the single-cell level, a detailed description of the circuit dynamics is lacking. To investigate this phenomenon, I have developed a pioneer methodology using the zebrafish larva as an experimental model and a multidisciplinary approach combining genetics, two-photon microscopy and optogenetics to monitor the developing activity of genetically targeted newborn neurons and the surrounding matured networks, in an intact and non-anesthetized vertebrate. Using this technique I have described for the first time, and in the time course of several days, the developmental dynamics of the functional properties of newborn neurons before and during their incorporation into the mature tectal circuit, the zebrafish most complex layered structure and highest visual center. Overall, these results suggest a developmental sequence of events during which newborn neurons capable of generating intrinsic activity dynamics first connect to their pre-synaptic sensory organ (the retina). At a second stage, the newborn neurons gradually incorporate into the tectal mature circuit showing sparse correlations with mature neurons. At a third stage, the spatial organization of the correlation between the newborn and the mature neurons is refined, becoming denser. I thus suggest that the newborn neurons first connect to a large population of sparsely located mature neurons and subsequently distant connections are pruned, permitting the newborn-labeled neuron to acquire a stable and robust functional signature (e.g. sharp receptive fields). In the recent years, treatments based on the transplantation of neural tissue have been developed to target neurodegenerative diseases such as Parkinson's disease. Because these therapies face the problem of poor survival and long-term functional incorporation, this study may provide better understanding of neuronal circuits formation and might pave the way to improve the efficiency of stem-cells-based treatments for human-brain reparation.

La "cryopolymérisation" permet d'obtenir des gels de polymère macroporeux ou "cryogels". Cette méthode a été utilisée pour la synthèse d'hydrogels thermosensibles à base de pNIPA. La température critique TC correspondant à la transition de volume a été déterminée par des mesures de taux de gonflement et par DSC. La macroporosité (distribution de la taille des pores et épaisseur des parois) et son évolution en fonction de T ont été étudiées par la microscopie biphotonique donnant des informations à l'échelle du µm à plusieurs dizaines de µm. La diffusion de rayons X (SAXS et WAXS) a été utilisée pour caractériser la structure multi-échelle (de quelques dixièmes à quelques dizaines de nm) du gel constituant les parois des macropores. Les courbes de diffusion ont été décrites analytiquement. L'évolution des dix paramètres contenus dans l'équation a été étudiée en fonction de T et discutée. Enfin, des expériences utilisant les phonons hyperfréquences générés par la technique des réseaux transitoires avec détection hétérodyne (HD-TG) ont été réalisées. Ces mesures ont permis de déterminer la vitesse de propagation de l'onde ultra-sonore (à 340 MHz), son atténuation, et la constante de diffusion thermique à différentes températures. / "Cryopolymerisation" yields macroporous gels named "cryogels". The method was used to synthesise thermosensitive pNIPA based hydrogels. The critical temperature TC corresponding to the volume phase transition was determined by swelling ratio measurements and DSC. The macroporosity (pore size distribution and wall thickness) and its change with temperature, was investigated by two-photon microscopy yielding information at the micrometer scale (a few tenths to tens of micrometers). X-ray scattering (SAXS and WAXS) was used to characterise the multi-scale structure of the gel forming the pore walls. The scattering curves were described analytically. The variation with temperature of the 10 parameters contained in the equation was investigated and discussed. Finally, heterodyne detected transient grating experiments were performed on a bulk pNIPA gel. These measurements allowed the determination of the speed of the ultrasonic wave (at 340 MHz), its attenuation and the thermal diffusion constant in the gel at different temperatures.

4 Abstract The present thesis focuses on testate amoebae (TA) and their relationship to their natural environment, as well as on relevant microscopic imaging methods. The bulk of the data has been published in original scientific papers and is compiled into three separate chapters (Pt I, Pt II and Pt III), each annotated by a brief introduction. (Pt I) The methods section is devoted to specialized microscopic techniques employed to broaden the scope of the ecological analyses. In particular, precise discrimination between live and dead individuals, biomass determination inside individual tests and a multi-modal visualization of the cytoplasm and organelles enhance the data. Laser scanning confocal microscopy and two-photon microscopy are the main imaging modalities employed to study TA morphology in detail. The data have implications for taxonomy and ecophysiology, including the use of TA as bioindicators of pollution. (Pt II) An actuoecological analysis focuses on the seasonal variability of TA species composition in a freshwater ecosystem, namely the Komo any ponds in Prague, during the course of the year. The species composition variation is correlated to simultaneously recorded limnological parameters such as temperature, pH, contamination by (heavy) metals (As, Cd, Mn, Ni, Fe, Pb), polycyclic aromatic...