La diffusion latérale des lipides et des protéines transmembranaires est essentielle pour les fonctions biologiques. Dans le contexte cellulaire, la fraction surfacique des protéines membranaires est élevée, atteignant environ de 50 à 70% selon le type de membrane. La diffusion se fait donc dans un milieu très encombré. Le but de ce travail est d'étudier in vitro l'effet de l'encombrement des protéines sur la diffusion des protéines et des lipides. Jusqu'à présent, les mesures de diffusion latérale ont généralement été réalisées à faible densité de protéines, et l'effet de l'encombrement des inclusions membranaires ou des protéines membranaires a été peu étudié expérimentalement. Nous avons utilisé une méthode de suivi de particules uniques (SPT) pour suivre les trajectoires de la pompe à protons Bactériorhodopsine (BR) et de lipides marqués avec des quantum dots au bas de vésicules unilamellaires géantes (GUVs) en fonction de la fraction de surface totale (Ф) de BR reconstituée dans la membrane constituée par ailleurs de 1,2-Dioleoyl-sn-glycéro-3-phosphocholine (DOPC). / Lateral diffusion of lipids and transmembrane proteins is essential for biological functions. In the cellular context, the surface fraction of membrane proteins is high, reaching approximately 50 to 70% depending on the membrane type. Therefore, diffusion occurs in a very crowded environment. The aim of this work is to study in vitro the effect of protein crowding on their own diffusion and on those of the surrounding lipids. So far, lateral diffusion measurements generally have been carried out at low protein density, and the effect of proteins crowding has not been much studied experimentally. We used a single particle tracking (SPT) method to track the trajectories of the Bacterorhodopsin (BR) proton pump and of lipids labeled with quantum dots at the bottom of giant unilamellar vesicles (GUVs) as a function of the total surface fraction (Ф) of BR reconstituted in 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane.

No description available.

Foamyviren (FV) gehören zur Familie der Retroviridae, werden aber aufgrund besonderer Eigenschaften in eine eigene Unterfamilie, die Spumaretrovirinae, eingeordnet. FV besitzen vor allem in vitro einen sehr breiten Tropismus, so dass bisher keine Zelllinie bekannt war, die nicht durch FV infiziert werden konnte. Obwohl diese Besonderheit darauf schließen lässt, dass ein sehr ubiquitäres Molekül auf der Wirtszelloberfläche für die FV-Bindung verwendet wird, ist der Rezeptor für die Virus-Aufnahme noch nicht bekannt. Dass FV einen pH-abhängigen Aufnahmemechanismus verwenden, lässt eine endozytotische Aufnahme vermuten. Dennoch sind die frühen Replikationsschritte, die zur Fusion der viralen und Wirtszellmembran führen, nur unzureichend charakterisiert. Deswegen wurden in der vorliegenden Arbeit funktionelle autofluoreszierende FV hergestellt, um die Bindung und Aufnahmemechanismen foamyviraler Partikel in Wirtszellen mit fluoreszenzmikroskopischen Analysen zu untersuchen. Mit diesen Methoden konnten erstmalig vier Zelllinien identifiziert werden, die nicht mit FV infizierbar sind, und damit mögliche Kandidaten für die Identifizierung des unbekannten FV Rezeptors darstellen. Des Weiteren wurden die fluoreszierenden FV erfolgreich eingesetzt, um die Fusionsereignisse zwischen viraler und zellulärer Membran in Echtzeit in lebenden Zellen zu untersuchen. Die durchgeführte „Single Virus Tracking“-Analyse zeigte, dass PFV (Prototype FV) Env-tragende Partikel sowohl an der Plasmamembran als auch in vermeintlichen Endosomen fusionieren können, wohingegen SFV (Simian FV) Env-tragende Partikel die Fusion wahrscheinlich nur in Endosomen auslösen können. Hinweis zur Nutzung der Filmdateien: Öffnen mit QuickTimePlayer bzw. ImageJ

<p>    </p> <p>Cerebral aneurysms are presented in 3-5% of the population and account for approximately 10% of all strokes. The clinical decision on treating unruptured aneurysms should not be taken lightly because a majority of the asymptomatic cerebral aneurysm will not rupture, while both endovascular and microsurgical treatments carry the risk of morbidity and mortality. Thus, there is a need for objective risk assessment to reliably predict the high-risk aneurysms to intervene. Recent studies have found that the blood flow hemodynamic metrics such as pressure and wall shear stress (WSS) are related to the growth and rupture of the aneurysms. 4D flow magnetic resonance imaging (MRI) measures time-resolved three-dimensional velocity fields in the aneurysms <em>in vivo</em>, allowing for the evaluation of hemodynamic parameters. This work presents the developments of flow-physics constrained data enhancement and augmentation methods for 4D flow MRI to assist the risk stratification of cerebral aneurysms. First, a phase unwrapping and denoising method is introduced to enhance the dynamic range and accuracy of 4D flow MRI velocity measurement by incorporating the divergence-free constraint of incompressible flow. Moreover, methods are developed to improve the estimation of hemodynamic parameters from 4D flow data including pressure and WSS. The pressure reconstruction method is also applied to the flow data acquired using particle imaging velocimetry (PIV) and particle tracking velocimetry (PTV) and shows superior performance as compared to the existing methods by solving the pressure Poisson equation. We also proposed a framework to estimate the uncertainty of the PIV/PTV based pressure estimation by propagating the velocity uncertainty. In addition, a multi-modality approach is introduced to enhances the resolution and accuracy of 4D flow data with sparse representation, which improves the reliability of the hemodynamic evaluation. Finally, we present a method to measure the left ventricular flow propagation velocity from cardiac imaging to help in assessing the diastolic function. </p>

L’identification de biomarqueurs des maladies mentales telles que l’autisme, la schizophrénie ou la maladie d’Alzheimer, est d’une importance capitale non seulement pour établir un diagnostic objectif, mais aussi pour suivre l’effet des traitements. La création et le maintien de fonctions neuronales sub-cellulaires, telle que la plasticité synaptique, sont fortement dépendants du transport intraneuronal, essentiel pour acheminer d’importants composants à des positions spécifiques. Un transport actif défaillant semble être partiellement responsable d’anomalies de la plasticité synaptique et de la morphologie neuronale présentes dans de nombreuses maladies neuropsychiatriques. Cette thèse décrit (i) la mise au point d’une méthode de quantification du transport intraneuronal reposant sur le suivi de nanoparticules de diamants fluorescents (fNDs); (ii) l’application de cette technique simple et faiblement invasive à l’analyse fonctionnelle de variants génétiques associés à des maladies neuropsychiatriques. Ce manuscrit comporte quatre chapitres. Le premier détaille l’architecture polygénique complexe des maladies mentales et démontre la pertinence d’étudier le transport intraneuronal. Les deuxième et troisième chapitres sont dédiés à la méthode et détaillent les stratégies d’internalisation des fNDs, les outils de quantification du transport intraneuronal et la validation de la technique. La forte brillance, la photo-stabilité parfaite et l’absence de toxicité cellulaire font des fNDs un outil de choix pour étudier la dynamique du transport intraneuronal sur une durée d’observation de plusieurs heures avec une haute résolution spatiotemporelle et une bonne puissance statistique. Enfin, dans le quatrième chapitre, nous appliquons cette nouvelle méthode d’analyse fonctionnelle pour étudier l’effet de variants génétiques associés à l’autisme et à la schizophrénie. Pour cela, nous utilisons des lignées de souris transgéniques ayant une faible surexpression des gènes MARK1 et SLC25A12, ainsi que des AAV-shRNA pour induire une haplo-insuffisance du gène AUTS2. Notre méthode de diagnostic moléculaire s’avère suffisamment sensible pour déceler des variations fines de la dynamique du transport intraneuronal, ouvrant la voie à de futurs développements en nanomédecine translationnelle. / The identification of molecular biomarkers of brain diseases as diverse as autism, schizophrenia and Alzheimer’s disease, is of crucial importance not only for an objective diagnosis but also to monitor response to treatments. The establishment and maintenance of sub-cellular neuronal functions, such as synaptic plasticity, are highly dependent on intracellular transport, which is essential to deliver important materials to specific locations. Abnormalities in such active transport are thought to be partly responsible for synaptic plasticity and neuronal morphology impairment found in many neuropsychiatric and neurodegenerative diseases. This thesis reports (i) the development of a quantification technic of intraneuronal transport based on fluorescent nanodiamonds (fNDs) tracking; (ii) the application of this simple and minimally invasive approach to the functional analysis of neuropsychiatric disease-related genetic variants.This manuscript falls into four chapters. The first one details the complex polygenic architecture of mental disorders and demonstrates the disease relevance of monitoring the intraneuronal transport. The second and the third chapters are dedicated to the nanodiamond-tracking assay and describe the fNDs internalisation strategies, the spatiotemporal quantitative readouts and the validation of the technic. The high brightness, the perfect photostability and the absence of cytotoxicity make fNDs a tool of choice to perform high throughput long-term bioimaging at high spatiotemporal resolution. Finally, in the fourth chapter, we apply this new functional analysis method to study the effect of genetic variants associated to autism and schizophrenia. We established transgenic mouse lines in which MARK1 and SLC25A12 genes were slightly overexpressed, and AAV-shRNA to induce AUTS2 gene haploinsufficiency. Our molecular diagnosis assay proves sufficiently sensitive to detect fine changes in intraneuronal transport dynamic, paving the way for future development in translational nanomedicine.

No description available.

As the demand for water increases across the southwestern United States, the region's utilization of and dependence on water stored in groundwater aquifers has risen in kind. The Coconino Aquifer (C-aquifer) underlies much of the southwestern Colorado Plateau and is a primary source of groundwater in northeastern Arizona. One of the largest commercial users of water from the C-aquifer in Apache County, Arizona, is Springerville Generating Station, a coal-fired power plant owned and operated by Tucson Electric Power. The area surrounding the power plant, located between the cities of Springerville and St. Johns, Arizona (the Springerville-St. Johns area), is geologically complex: it contains the Cedar Mesa anticline, an underlying CO2 reservoir, extensive travertine deposits, and several faults, including the Coyote Wash fault. The Coyote Wash fault and Cedar Mesa anticline play a significant role in the relationships between the St. Johns CO2 gas field, groundwater flow, and the travertine deposits. Yet, the interaction between the structures and the effect they have on groundwater flow is poorly constrained. By mapping the subsurface geology utilizing borehole records and by creating a groundwater model of the area, this study determined that the Cedar Mesa anticline acts as a partial horizontal barrier to groundwater flow, whereas the Coyote Wash fault does not act as such a barrier. Particle tracking for the model indicates that despite the reduced water volume in the aquifer after decades of groundwater extraction, flow still occurs across the hinge of the Cedar Mesa anticline, accelerated by active pumping wells located west of the anticline axis. The model indicates that prior to the activation of the pumping wells, outflow from the C-aquifer would have occurred with greater frequency to Lyman Lake and along the extent of the Little Colorado River located downstream from the lake. The study also identified a zone of high hydraulic conductivity located between the Cedar Mesa anticline and the Coyote Wash fault that continues west of the Coyote Wash fault and may align with the Buttes anticline. This model of groundwater flow conditions improves the understanding of the complex subsurface geology and groundwater flow dynamics in the area.

The opportunistic environmental microbes, non-tuberculous Mycobacterium (NTM), pose an increasing risk of disease and death in both immunodeficient and immunocompetent individuals in the USA and across the world. NTM lung disease is particularly prevalent in Hawaii, although the modes of NTM acquisition and transport in Hawaii are not fully understood. This study evaluated 149 soil and 50 water samples across the Hawaiian Islands to determine geochemical factors controlling NTM. Non-metric multidimensional scaling (NMDS) and principal component analyses (PCA) of modern soils show variables such as Total Organic Carbon (TOC), pH, P, mafic silicate minerals, and Pb seem to control NTM presence and transition metals and oxides such as TiO2, Zr, and Nb seem to control the absence perhaps due to toxicity. Logistic regression modeling coupled with Kolmogorov-Smirnov testing supported that TOC and P could be used to explain the probability of NTM presence in modern soils. Kolmogorov-Smirnov, non-metric multidimensional scaling, and principal components analysis results suggest poor predictability of NTM presence in soils when evaluating mineralogy alone. The same statistical methods indicated that transition metals appeared to control NTM presence in stream water and major cations and anions seemed to control NTM absence. However, additional bacterial stream data is needed to strengthen this finding. Additionally, an Oahu source water assessment and protection groundwater model was refined by including stream discharge data, including losses to the aquifer. NTM inhabits many environmental niches, although little is understood regarding the transport of NTM from the environment to indoor plumbing. However, transport from surface water to water-supply aquifers is likely important. This study analyzes groundwater flow from stream losses as a mechanism of NTM transport to water supplies. An updated MODFLOW groundwater model was developed for the north-east Oahu, Waimea River drainage. Results show hundreds of meters of lateral and tens of meters of vertical transport of NTM in 1-3 months. Additionally, geochemical modeling with Geochemist’s Workbench showed Fe oxy/hydroxides oversaturated in 100% of streams. Fe oxy/hydroxide affixed to NTM would potentially satisfy NTM’s preference for attachment and allow for colloidal transport through the aquifer.

Biological systems carry inherent complexity, which pose difficulties observing behavioural properties, such as diffusion coefficients, kinetic constants and state switching occurrences. With constantly improving computing power and microscopy technologies, single molecule methods have become a viable alternative when probing the behaviour of proteins, enzymes, lipids and other molecules. Processed microscopy images and videos provide information such as particle intensities and trajectories, avoiding ensemble averaging and therefore allowing for a detailed breakdown of particle mobility and interactions. A single particle tracking (SPT) algorithm was developed which implements detection, localization and position linking on image stacks. Sub-pixel precise detection is done via either centroid determination, Gaussian fit, or radial symmetry centres, while tracking makes use of distance based global cost optimization. The detection algorithm is also used for single particle spectroscopy, where intensity information is used to determine the size of oligomers, as well as their interaction with other molecules through channel intensity cross-correlation. The algorithm underwent benchmarking with simulated videos and was applied to three different biological systems with comparison to other established methods of analysis. The first system studied was the diffusion of the fluorescent lipophilic dye DiD in a five-component mitochondria-like solid-supported lipid bilayer. Comparing line-scanning fluorescence correlation spectroscopy (FCS) and single particle tracking, the measured diffusion coefficients were found to be statistically different, with DFCS = 3 μm2s-1 and DSPT = 2 μm2s-1, indicating different operational ranges for the two methods. FCS outperforms SPT when the diffusion coefficient exceeds 1 μm2s-1, making it ideal for lipid diffusion in fluid membranes and proteins in solution with weak membrane interaction. SPT is best suited for mobile and immobile membrane inserted proteins, as well as lipid diffusion in viscous membranes. The second system studied was the interaction between the two proteins Bax and Bid when inserted in a membrane. Bax and Bid are both members of the Bcl-2 family of proteins, which plays a vital role in the apoptosis mechanism, by inducing mitochondrial outer membrane permeabilization. To study this system with single particle spectroscopy, fluorescently labelled Bax and truncated Bid (tBid) were imaged when interacting with a mitochondria-like supported lipid bilayer with confocal microscopy. Immobile and mobile particles were detected and distinguished based on the eccentricity of the observed fluorescence spot. The intensity of the particle signal was used to determine oligomer type (homo-oligomerization) while the interaction with the particles' counterpart (hetero-oligomerization) was determined by channel cross-correlation. This allowed the measurement of the 2D-KD values for mobile (0.6 μm-2) and immobile (0.08 μm-2) Bax/tBid complexes, showing that the degree of insertion of the proteins in the membrane greatly affect their affinity for each other. The third and final system studied was the motion of cellulases on cellulose fibers. Enzymatic hydrolysis of crystalline cellulose is a costly step in the generation of fermentable sugars for biofuel production. Due to the complex structure and many possible interaction states of the enzymes with cellulose, single particle tracking is a well-adapted technique to the gathering of information on the enzyme dynamics, which is essential for process optimization. The movement of cellulases on cellulose substrate was observed via labelled Thermobifidia fusca Cel5A, Cel6B and Cel9A on bacterial micro-crystalline cellulose substrate. The detected trajectories were analyzed using multiple diffusion models. A simple one-state diffusion model was insufficient to describe the observed radial displacement distributions and so a two-state model was introduced and confronted with the data using conventional least-squares fits , as well as a hidden Markov approach. The diffusion coefficients of the two states are found to be on the order of Dfast = 10-3 μm2s-1 and Dslow = 10-4 μm2s-1, with the slow state being more stable and therefore more likely to occur. Single particle tracking can give us better insight into complex interactions, such as synergistic binding of proteins existing in several different states and processive enzymatic behaviour, where ensemble averaging techniques can fall short. The uses of single molecule methods are plentiful and with the current rise of machine learning, higher levels of abstraction will provide us with more detailed insights into biological processes, driving promising developments in the medical field, as well as new technologies in many sectors of industry. / Thesis / Doctor of Science (PhD) / Proteins are the motors that drive most cellular processes, for example steering a cell’s life cycle, or decomposing sources of nutrients. Being able to observe the motion of individual proteins is key to understanding their behaviour. In this work a single particle tracking (SPT) program was developed to extract protein trajectories from fluorescence microscopy experiments. With this tool-set we investigated the following two systems. The first system of interest is the Bcl-2 protein family, which is vital during the pro- grammed cell death at the end of each cell’s life span. The failure of a controlled cell death can have dire consequences, such as necrosis and cancer. The Bcl-2 family proteins Bid and Bax are active on the outer membrane of the mitochondria, where they initiate the process of terminating the cell’s functions by forming pores. For our experiments we ar- tificially mimicked the outer membrane of the mitochondria, introduced Bid and Bax and observed their preferential groupings on the membrane surface. This provided indications of the mechanisms involved during binding and pore formation. The motivation behind the investigation of the second system is the improvement of biofuel generation from a renewable source: plant-based biomass. Cellulases are enzymes from bacteria or fungi that break down cellulose – one of the main building blocks of all plant cell walls – into fermentable sugars. In fluorescence microscopy experiments a purified cellulose substrate was used to monitor the motion of three types of cellulases. The insight which we gained into the cellulase behaviour may allow the optimization of the process of cellulose decomposition.

Seit mehr als einem Jahrhundert ist ein wachsendes wissenschaftliches Interesse an Tropfen und den Vorgängen bei deren Aufprall auf die verschiedensten Substrate zu verzeichnen, wohl auch durch die Fotografien von Worthington (1908) ausgelöst. Inzwischen wurden viele Erkenntnisse durch große Fortschritte bei der experimentellen Untersuchung (z.B. mittels Hochgeschwindigkeitsaufnahmen) und durch theoretische und computergestützte Untersuchung (z.B. durch skalenfreie und numerische Modellierung) gewonnen. Trotzdem bleibt durch die Vielfältigkeit und Komplexität der Phänomene während des Tropfenaufpralls sowie wegen der ständig erweiterten Anwendungsbereiche dieses Forschungsgebiet hochaktuell. Insbesondere sehr kleine und gleichzeitig sehr schnelle Tropfen (Tropfendurchmesser 10µm bis 100µm, Tropfengeschwindigkeit 10m/s bis 100m/s) kommen in vielen modernen Anwendungen vor (z.B. Verbrennungsmotoren, Tintenstrahldrucker, Reinigung von Oberflächen). In diesem wichtigen, aber für Untersuchungen schwer zugänglichen Parameterbereich gibt es immer noch offene Fragen. Die vorliegende Arbeit beschäftigt sich daher mit diesen schnellen Mikrotropfen in Bezug auf ihre Herstellung und den Aufprallvorgang auf ein festes, trockenes oder benetztes Substrat. Zunächst wird eine Methode zur Erzeugung eines Hochgeschwindigkeitssprays realisiert, welche auf dem durch Ultraschall gesteuerten Plateau-Rayleigh-Zerfall eines Flüssigkeitsstrahls beruht. Sie ermöglicht es, sowohl Tropfengröße als auch –geschwindigkeit präzise und mit hoher Reproduzierbarkeit über den gesamten oben angegebenen Parameterbereich einzustellen. Durch gezielte Manipulation eines Einzeltropfens durch elektrische Felder wird anschließend der Tropfenaufprall auf Substrate unterschiedlicher Benetzbarkeit mit sehr hoher zeitlicher Auflösung (ca. 100 Mio. Bilder pro Sekunde) bei gleichzeitig hoher räumlicher Auflösung (< 1µm) untersucht. Es zeigt sich, dass bekannte Modelle für langsamere und größere Tropfen im Millimeterbereich auch für schnelle Mikrotropfen Gültigkeit behalten. Somit ist bei gleichen dimensionslosen Kennzahlen (z.B. Reynolds-Zahl, Weber-Zahl, Ohnesorge-Zahl) eine skalenfreie Beschreibung des Tropfenaufpralls möglich. Schließlich wird die Methode zur Tropfenerzeugung auf einen für Anwendungen in der Reinigung relevanten Fall übertragen. Hierbei geht es um den Tropfenaufprall auf ein von einem Flüssigkeitsfilm überströmten Substrat. Es werden die während des Auftreffvorgangs auftretenden Geschwindigkeiten in der sich bildenden radialen Strömung in Abhängigkeit von verschiedenen Prozessparametern bestimmt. Aus den Ergebnissen lassen sich Aussagen über die zu erwartende Reinigungswirkung durch derartige Tropfen und den Einfluss der Prozessparameter treffen.