Nucleic acids and proteins, some of the building blocks of life, are not static structures but highly dynamic entities that need to interact with one another to meet cellular demands. The work presented in this thesis focuses on the application of highly sensitive fluorescence methods, both at ensemble and single-molecule level, to determine the dynamics and structure of specific biomolecular interactions with nanometer resolution and in temporal scales from nanoseconds to minutes, which includes most biologically relevant processes. The main aims of my PhD can be classified in three areas: i) exploring new fluorescent sensors with increased specificity for certain nucleic acid structures; ii) understanding how some of these nucleic acids sense the presence of small molecules in the cellular environment and trigger gene regulation by altering their structure; and iii) understanding how certain molecular machines, such as helicase proteins, are able to unwind the DNA double helix by using chemical energy in the form of ATP hydrolysis.

Notre travail de thèse porte sur la détection et la modélisation de configurations dynamiques dans des séquences d'images. Nous développons des approches statistiques locales sans apprentissage supervisé. Notre application principale est la microscopie de fluorescence, un outil fondamental de la biologie cellulaire moderne. Deux cas peuvent se présenter : 1. les objets étudiés n'interagissent pas, et les dynamiques individuelles peuvent être analysées indépendamment ; 2. les objets étudiés interagissent, et la dynamique à analyser est celle du groupe entier d'objets. En ce qui concerne les dynamiques individuelles, nous nous intéressons à des séquences d’images biologiques dans lesquelles des protéines évoluent au sein de la cellule. Plus précisément, nous étudions la fusion de vésicules à la frontière de la cellule, appelée membrane plasmique. Les vésicules sont des intermédiaires de transport qui véhiculent des molécules dans la cellule. À la fin du processus d’exocytose, la fusion des vésicules avec la membrane s’accompagne d’une diffusion desdites protéines. Les images sont acquises en microscopie de fluorescence par réflexion totale interne (TIRF). Afin de repérer les évènements de fusion, nous proposons une nouvelle méthode de détection de spots. Puis, nous modélisons les dynamiques des protéines et estimons les paramètres biophysiques associés dans les séquences TIRF. La dynamique de groupe, quant à elle, est notamment rencontrée dans les mouvements de tissus cellulaires, le développement embryonnaire ou dans d’autres domaines, comme les mouvements de foules dans des vidéos. Nous proposons une nouvelle méthode d’estimation du mouvement de groupe permettant de caractériser le mouvement à la fois de façon quantitative et qualitative. Elle est utilisée pour classifier le mouvement de groupe, retrouver les chemins principaux dans la scène et détecter des anomalies locales. Dans l'un ou l'autre cas d'étude, nous abordons les problématiques selon une démarche commune, essentiellement dirigée par les données et mettant en œuvre des tests statistiques. Par ailleurs, nous avons le souci de proposer des méthodes nécessitant le réglage d'un faible nombre de paramètres qui sont, de plus, peu sensibles ou calibrés avec des règles statistiques. Enfin, nous adoptons des approches locales, qui ont l’avantage d’être rapides, flexibles et peu sensibles aux variations de contexte, qu’elles soient spatiales (arrière-plan variable) ou temporelles (changement d’illumination globale comme le photoblanchiment en microscopie de fluorescence). / In this thesis, we investigate statistical methods to detect, estimate and characterize dynamical events in image sequences. Our main focus is on fluorescence microscopy images, which represent a fundamental tool for cell biology. There are two cases : 1. Studied objects do not interact, and individual dynamics can be independently analyzed ; 2. Studied objects interact, and group dynamics must be analyzed as a whole. In the case of individual dynamics, our primary focus is on biological image sequences showing proteins evolving in a cell, and more precisely at the cell frontier named plasma membrane. Proteins transported in the cell by vesicles, are observed in total internal reflection fluorescence microscopy (TIRFM), an observation technique well adapted to plasma membrane dynamics analysis. At the end of the exocytosis process, vesicles fuse to the plasma membrane and release proteins, which then diffuse. We first propose a new spot detection method aimed at localizing fusion events. Then, we model the protein dynamics and estimate the biophysical parameters in TIRFM image sequences for further biological analysis. We also address the processing of image sequences at lower magnifications, that is, depicting groups of cells, instead of an isolated cell. We propose a method to jointly estimate quantitative and qualitative motion measurements. It is used to classify the group motion, recover principal paths followed in the scene, and detect localized anomalies. Since they are free of appearance model, the developed methods are quite general and also applied to other applications including crowd motion analysis in videos. Whether it is for spot detection, protein dynamics estimation or group motion analysis, a common approach is ubiquitous, however. First, statistical arguments are used to automatically infer the method parameters. Secondly, we rely on local approaches, which have the advantage of being computationally efficient. Local modeling handles spatially varying image statistics much more easily and more accurately than global modeling. Local approaches also allow neglecting contextual variations such as spatially varying background contrast or, in fluorescence microscopy, temporal fading known as photobleaching.

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.

Movement of motor proteins along cytoskeletal filaments is fundamental for various cellular processes ranging from muscle contraction over cell division and flagellar movement to intracellular transport. Not surprisingly, the impairment of motility was shown to cause severe diseases. For example, a link between impaired intracellular transport and neurodegenerative diseases, such as Alzheimer’s, has been established. There, the movement of kinesin-1, a neuronal motor protein transporting vesicles along microtubules toward the axonal terminal, is thought to be strongly affected by roadblocks leading to malfunction and death of the nerve cell. Detailed information on how the motility of kinesin-1 deteriorates in the presence of roadblocks and whether the motor has a mechanism to circumvent such obstructions is scarce. In this thesis, kinesin-1 motility was studied in vitro in the presence of rigor kinesin-1 mutants, which served as permanent roadblocks, under controlled single-molecule conditions. The 25 nm wide microtubule track, consisting of 13 individual protofilaments, resembles a multi-lane environment for transport by processive kinesin-1 motors. The existence of multiple traffic-lanes, allows kinesin-1 to utilize different paths for cargo transport and potentially also for the circumvention of roadblocks. However, direct observation of motor encounters with roadblocks has been intricate in the past, mainly due to limitations in both, spatial and temporal resolution. These limitations, intrinsic to fluorescent probes commonly utilized to report on the motor positions, originate from a low rate of photon generation (low brightness) and a limited photostability (short observation time). Thus, studying kinesin-1 encounters with microtubule-associated roadblocks requires alternative labels, which explicitly avoid the shortcomings of fluorescence and consequently allow for a higher localization precision. Promising candidates for replacing fluorescent dyes are gold nanoparticles (AuNPs), which offer an enormous scattering cross-section due to plasmon resonance in the visible part of the optical spectrum. Problematic, however, is their incorporation into conventionally used (fluorescence) microscopes, because illumination and scattered light have the same wavelength and cannot be separated spectrally. Therefore, an approach based on total internal reflection (TIR) utilizing a novel parabolically shaped quartz prism for illumination was developed within this thesis. This approach provided homogenous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, single-molecule fluorescence as well as single-particle scattering were detectable with high signal-to-noise ratios. Importantly, AuNPs with a diameter of 40 nm provided sub-nanometer localization accuracies within millisecond integration times and reliably reported on the characteristic 8 nm stepping of individual kinesin-1 motors moving along microtubules. These results highlight the potential of AuNPs to replace fluorescent probes in future single-molecule experiments. The newly developed parabolic prism-type TIR microscope is expected to strongly facilitate such approaches in the future. To study how the motility of kinesin-1 is affected by permanent roadblocks on the microtubule lattice, first, conventional objective-type TIRF microscopy was applied to GFP-labeled motors. An increasing density of roadblocks caused the mean velocity, run length, and dwell time to decrease exponentially. This is explained by (i) the kinesin-1 motors showing extended pausing phases when confronted with a roadblock and (ii) the roadblocks causing a reduction in the free path of the motors. Furthermore, kinesin-1 was found to be highly sensitive to the crowdedness of microtubules as a roadblock decoration as low as 1 % sufficed to significantly reduce the landing rate. To study events, where kinesin-1 molecules continued their runs after having paused in front of a roadblock, AuNPs were loaded onto the tails of the motors. When observing the kinesin-1 motors with nanometer-precision, it was interestingly found that about 60 % of the runs continued by movements to the side, with the left and right direction being equally likely. This finding suggests that kinesin-1 is able to reach to a neighboring protofilament in order to ensure ongoing transportation. In the absence of roadblocks, individual kinesin-1 motors stepped sideward with a much lower, but non-vanishing probability (0.2 % per step). These findings suggest that processive motor proteins may possess an intrinsic side stepping mechanism, potentially optimized by evolution for their specific intracellular tasks.

Cette thèse porte sur l'utilisation du BLASSO, un problème d'optimisation convexe en dimension infinie généralisant le LASSO aux mesures, pour la super-résolution de sources ponctuelles. Nous montrons d'abord que la stabilité du support des solutions, pour N sources se regroupant, est contrôlée par un objet appelé pré-certificat aux 2N-1 dérivées nulles. Quand ce pré-certificat est non dégénéré, dans un régime de petit bruit dont la taille est contrôlée par la distance minimale séparant les sources, le BLASSO reconstruit exactement le support de la mesure initiale. Nous proposons ensuite l'algorithme Sliding Frank-Wolfe, une variante de l'algorithme de Frank-Wolfe avec déplacement continu des amplitudes et des positions, qui résout le BLASSO. Sous de faibles hypothèses, cet algorithme converge en un nombre fini d'itérations. Nous utilisons cet algorithme pour un problème 3D de microscopie par fluorescence en comparant trois modèles construits à partir des techniques PALM/STORM. / This thesis studies the noisy sparse spikes super-resolution problem for positive measures using the BLASSO, an infinite dimensional convex optimization problem generalizing the LASSO to measures. First, we show that the support stability of the BLASSO for N clustered spikes is governed by an object called the (2N-1)-vanishing derivatives pre-certificate. When it is non-degenerate, solving the BLASSO leads to exact support recovery of the initial measure, in a low noise regime whose size is controlled by the minimal separation distance of the spikes. In a second part, we propose the Sliding Frank-Wolfe algorithm, based on the Frank-Wolfe algorithm with an added step moving continuously the amplitudes and positions of the spikes, that solves the BLASSO. We show that, under mild assumptions, it converges in a finite number of iterations. We apply this algorithm to the 3D fluorescent microscopy problem by comparing three models based on the PALM/STORM technics.

Movement of motor proteins along cytoskeletal filaments is fundamental for various cellular processes ranging from muscle contraction over cell division and flagellar movement to intracellular transport. Not surprisingly, the impairment of motility was shown to cause severe diseases. For example, a link between impaired intracellular transport and neurodegenerative diseases, such as Alzheimer’s, has been established. There, the movement of kinesin-1, a neuronal motor protein transporting vesicles along microtubules toward the axonal terminal, is thought to be strongly affected by roadblocks leading to malfunction and death of the nerve cell. Detailed information on how the motility of kinesin-1 deteriorates in the presence of roadblocks and whether the motor has a mechanism to circumvent such obstructions is scarce. In this thesis, kinesin-1 motility was studied in vitro in the presence of rigor kinesin-1 mutants, which served as permanent roadblocks, under controlled single-molecule conditions. The 25 nm wide microtubule track, consisting of 13 individual protofilaments, resembles a multi-lane environment for transport by processive kinesin-1 motors. The existence of multiple traffic-lanes, allows kinesin-1 to utilize different paths for cargo transport and potentially also for the circumvention of roadblocks. However, direct observation of motor encounters with roadblocks has been intricate in the past, mainly due to limitations in both, spatial and temporal resolution. These limitations, intrinsic to fluorescent probes commonly utilized to report on the motor positions, originate from a low rate of photon generation (low brightness) and a limited photostability (short observation time). Thus, studying kinesin-1 encounters with microtubule-associated roadblocks requires alternative labels, which explicitly avoid the shortcomings of fluorescence and consequently allow for a higher localization precision. Promising candidates for replacing fluorescent dyes are gold nanoparticles (AuNPs), which offer an enormous scattering cross-section due to plasmon resonance in the visible part of the optical spectrum. Problematic, however, is their incorporation into conventionally used (fluorescence) microscopes, because illumination and scattered light have the same wavelength and cannot be separated spectrally. Therefore, an approach based on total internal reflection (TIR) utilizing a novel parabolically shaped quartz prism for illumination was developed within this thesis. This approach provided homogenous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, single-molecule fluorescence as well as single-particle scattering were detectable with high signal-to-noise ratios. Importantly, AuNPs with a diameter of 40 nm provided sub-nanometer localization accuracies within millisecond integration times and reliably reported on the characteristic 8 nm stepping of individual kinesin-1 motors moving along microtubules. These results highlight the potential of AuNPs to replace fluorescent probes in future single-molecule experiments. The newly developed parabolic prism-type TIR microscope is expected to strongly facilitate such approaches in the future. To study how the motility of kinesin-1 is affected by permanent roadblocks on the microtubule lattice, first, conventional objective-type TIRF microscopy was applied to GFP-labeled motors. An increasing density of roadblocks caused the mean velocity, run length, and dwell time to decrease exponentially. This is explained by (i) the kinesin-1 motors showing extended pausing phases when confronted with a roadblock and (ii) the roadblocks causing a reduction in the free path of the motors. Furthermore, kinesin-1 was found to be highly sensitive to the crowdedness of microtubules as a roadblock decoration as low as 1 % sufficed to significantly reduce the landing rate. To study events, where kinesin-1 molecules continued their runs after having paused in front of a roadblock, AuNPs were loaded onto the tails of the motors. When observing the kinesin-1 motors with nanometer-precision, it was interestingly found that about 60 % of the runs continued by movements to the side, with the left and right direction being equally likely. This finding suggests that kinesin-1 is able to reach to a neighboring protofilament in order to ensure ongoing transportation. In the absence of roadblocks, individual kinesin-1 motors stepped sideward with a much lower, but non-vanishing probability (0.2 % per step). These findings suggest that processive motor proteins may possess an intrinsic side stepping mechanism, potentially optimized by evolution for their specific intracellular tasks.

Integration and miniaturisation in electronics has undoubtedly revolutionised the modern world. In biotechnology, emerging lab-on-a-chip (LOC) methodologies promise all-integrated laboratory processes, to perform complete biochemical or medical synthesis and analysis encapsulated on small microchips. The integration of electrical, optical and physical sensors, and control devices, with fluid handling, is creating a new class of functional chip-based systems. Scaled down onto a chip, reagent and sample consumption is reduced, point-of-care or in-the-field usage is enabled through portability, costs are reduced, automation increases the ease of use, and favourable scaling laws can be exploited, such as improved fluid control. The capacity to manipulate single cells on-chip has applications across the life sciences, in biotechnology, pharmacology, medical diagnostics and drug discovery. This thesis explores multiple applications of optical manipulation within microfluidic chips. Used in combination with microfluidic systems, optics adds powerful functionalities to emerging LOC technologies. These include particle management such as immobilising, sorting, concentrating, and transportation of cell-sized objects, along with sensing, spectroscopic interrogation, and cell treatment. The work in this thesis brings several key applications of optical techniques for manipulating and porating cell-sized microscopic particles to within microfluidic chips. The fields of optical trapping, optical tweezers and optical sorting are reviewed in the context of lab-on-a-chip application, and the physics of the laminar fluid flow exhibited at this size scale is detailed. Microfluidic chip fabrication methods are presented, including a robust method for the introduction of optical fibres for laser beam delivery, which is demonstrated in a dual-beam optical trap chip and in optical chromatography using photonic crystal fibre. The use of a total internal reflection microscope objective lens is utilised in a novel demonstration of propelling particles within fluid flow. The size and refractive index dependency is modelled and experimentally characterised, before presenting continuous passive optical sorting of microparticles based on these intrinsic optical properties, in a microfluidic chip. Finally, a microfluidic system is utilised in the delivery of mammalian cells to a focused femtosecond laser beam for continuous, high throughput photoporation. The optical injection efficiency of inserting a fluorescent dye is determined and the cell viability is evaluated. This could form the basis for ultra-high throughput, efficient transfection of cells, with the advantages of single cell treatment and unrivalled viability using this optical technique.

Pour certaines problématiques biomédicales, notamment en neurobiologie, l'imagerie des échantillons exige des résolutions spatiale et temporelle élevées, et seules des techniques de microscopie optique innovantes vont permettre d'imager la cellule dans des conditions physiologiques. Pour répondre à ces contraintes nous avons développé en intégralité un dispositif de microscopie de fluorescence en réflexion totale interne et résolue en temps. En effet, l'imagerie de fluorescence résolue en temps (FLIM) permet, en complément des informations de localisation et de topologie apportées par la microscopie conventionnelle, une analyse dynamique de processus moléculaires et métaboliques au sein des cellules. Associée à la technique de microscopie en réflexion totale interne, qui confère au système d'imagerie une résolution axiale sub-longueur d'onde tout en acquérant des images en champ large, la technique FLIM permet d'analyser l'activité membranaire des cellules en s'affranchissant de la fluorescence des autres entités de la cellule.<br />Ce travail s'est articulé autour de trois axes principaux : l'étude et le développement d'un dispositif d'excitation de la fluorescence basé sur un oscillateur laser solide picoseconde dont le spectre est élargi par effets non linéaires dans des fibres optiques microstructurées ; le développement et la caractérisation d'un dispositif de microscopie de fluorescence par onde évanescente (TIRF) couplé à une détection résolue en temps en plein champ ; et l'application de ce dispositif à l'étude d'un précurseur membranaire du peptide amyloïde impliqué dans la maladie d'Alzheimer.

It is widely known that surfaces play an important role in numerous biological processes and technological applications. Thus, being able to modify surface properties provides an opportunity to control many phenomena occurring at interfaces. One way of controlling surface properties is to adsorb a polymer film onto the surface, for example through layer-by-layer (LbL) deposition of polyelectrolytes. This simple but versatile technique enables various polymers, proteins, colloidal particles etc. to be incorporated into the film, resulting in a multifunctional coating. Due to recent legislations and a consumer demand for more environmentally friendly products, we have chosen to use natural polymers (biopolymers) from renewable resources. The focus of this thesis has been on the adsorption of biopolymers and their layer-by-layer formation at solid-liquid interfaces; these processes have been studied by a wide range of techniques. The main method was the quartz crystal microbalance with dissipation monitoring (QCM-D), which measures the adsorbed mass, including trapped solvent and the viscoelastic properties of an adsorbed film. This technique was often complemented with an optical method, such as ellipsometry or dual polarization interferometry (DPI), which provided information about the “dry” polymer or protein adsorbed mass. From this combination, the solvent content and density of the layers was evaluated. In addition, the surface force apparatus (SFA), X-ray photoelectron spectroscopy (XPS), total internal reflection fluorescence (TIRF), and fluorescence resonance energy transfer (FRET) were utilized, providing further information about the film structure, chemical composition, and polymer inter-layer diffusion. Adsorption studies of the glycoprotein mucin, which has a key role in the mucousal function, showed that despite the net negative charge of mucin, it adsorbed on negatively charged substrates. The adsorbed layer was highly hydrated and the segment density on the substrate was low. We showed the importance of characterizing the mucin used, since differences in purity, such as the presence of albumin, gave rise to different adsorption behaviours in terms of both adsorbed amount and structure. The adsorbed mucin layer was to a large extent desorbed upon exposure to the anionic surfactant sodium dodecyl sulfate (SDS). In order to prevent desorption, we demonstrated that a protective layer of the cationic polysaccharide chitosan could be adsorbed onto the mucin layer and that the mucin-chitosan complexes resisted the desorption normally induced by association with SDS. Moreover, the association between chitosan and SDS was examined at the solid-liquid interface, in the bulk, and at the air-water interface. In all these environments chitosan-SDS complexes were formed and a net charge reversal of the complexes from positive to negative was observed when the concentration of SDS was increased. Furthermore, the LbL deposition method could be used to form a multilayer-like film by alternate adsorption of mucin and chitosan on silica substrates. The LbL technique was also applied to two proteins, lysozyme and β-casein with the aim of building a multilayer film consisting entirely of proteins. These proteins formed complexes at the solid-liquid interface, resulting in a proteinaceous layer, but the build-up was highly irregular with an increase in adsorbed amount per protein deposition cycle that was far less than a monolayer.Continuing with chitosan, known to have antibacterial properties we assembled multilayers with an anti-adhesive biopolymer, heparin, to evaluate the potential of this system as a coating for medical implants. Multilayers were assembled under various solution deposition conditions and the film structure and dynamics were studied in detail. The chitosan-heparin film was highly hydrated, in the range 60-80 wt-% depending on the deposition conditions. The adsorbed amount and thickness of the film increased exponential-like with the number of deposition steps, which was explained by inter-diffusion of chitosan molecules in the film during the build-up. In a novel approach, we used the distant dependent FRET technique to prove the inter-layer diffusion of fluorescent-labelled chitosan molecules within the film. The diffusion coefficient was insignificantly dependent on the deposition pH and ionic strength, and hence on the film structure. With the use of a pH sensitive dye buried under seven chitosan-heparin bilayers, we showed that the dye remained highly sensitive to the charge of the outermost layer. From complementary QCM-D data, we suggested that an increase in the energy dissipation does not necessarily indicate that the layer structure becomes less rigid. / Det är välkänt att ytor spelar en viktig roll i många biologiska processer och tekniska tillämpningar. Att kunna modifiera en ytas egenskaper ger därför en möjlighet att kunna kontrollera många fenomen som sker på ytor. Ett sätt att kontrollera ytegenskaperna är genom att adsorbera en polymerfilm på ytan, till exempel genom att växelvis adsorbera olika polyelektrolyter (LbL-teknik). Denna enkla men mångsidiga teknik möjliggör att många olika material kan införlivas i filmen, vilket resulterar i en multifunktionell beläggning. På grund av dagens lagstiftning och konsumenters ökade efterfrågan på miljövänliga material beslutade vi oss för att använda biologiska polymerer (biopolymerer) i detta projekt. Fokus i den här avhandlingen har varit på adsorption av biopolymerer och deras LbL-formation på gränsytan vätska-fast fas, där adsorptionsförloppet och det adsorberade skiktet bestående av biopolymerer studerats med en mängd olika tekniker. Huvudtekniken var kvartskristallmikrovåg med energidissipations-registrering (QCM-D), som mäter massan inklusive inkorporerat vatten, samt de viskoelastiska egenskaperna hos ett adsorberat skikt. Som komplement till denna teknik användes ofta optiska metoder, till exempel ellipsometri och ”dubbel polarisationsinterferometri (DPI)”, två tekniker som endast mäter massan av de adsorberade biopolymererna. Genom denna kombination av metoder kunde massan av inkorporerat vatten i filmen och filmens densitet bestämmas. Dessutom användes ytkraftsapparaten (SFA), röntgenfotoelektronspektrometri (XPS), och fluorescens-spektroskopiteknikerna TIRF och FRET i några undersökningar för att erhålla information om skiktens struktur, kemiska sammansättning och polymerernas diffusion inom skiktet.Adsorptionsstudier av glycoproteinet mucin, som har en central roll i funktionen av slemhinnan, avslöjade att trots att mucinet har en negativ nettoladdning adsorberade det ändå på negativt laddade substrat. Det adsorberade lagret var väldigt hydratiserat och hade en låg andel mucin i direkt kontakt med ytan. Vi påvisade vikten av att noga undersöka mucinet som användes, eftersom olika renhet, till exempel i form av förekomsten av albumin gav upphov till olika adsorptionsbeteende gällande både adsorberad mängd och struktur. En stor andel av det adsorberade mucinlagret desorberade när det exponerades för den anjoniska tensiden natriumdodecylsulfat, SDS. Vi visade att ett skyddande lager av den katjoniska polysackariden chitosan kunde adsorberas på mucinet och att mucin-chitosan-komplexen inte desorberade när SDS tillsattes. Därtill studerades växelverkan mellan chitosan och SDS på gränsytan vätska-fast fas, i bulken och på luft-vattengränsytan. Komplex av chitosan-SDS bildades i samtliga miljöer och en nettoladdningsomsvängning från positiv till negativ observerades när koncentrationen av SDS ökades.Vidare kunde LbL-tekniken nyttjas för att skapa ett multilagerlikt skikt genom att alternerande adsorbera mucin och chitosan på kiseldioxidsubstrat. Denna teknik användes även med två proteiner, lysozym och β-kasein, med målet att skapa ett multilager bestående av endast proteiner. Dessa proteiner bildade komplex på gränsytan vätska-fast fas i form av ett blandat proteinlager, men uppbyggnaden var väldigt oregelbunden med en ökning i adsorberad mängd per proteindeponeringscykel som var avsevärt mindre än ett monolager.Inom området för biomaterial utgör de antibakteriella och antihäftande egenskaperna hos chitosan respektive heparin en lovande blandning för beläggningar av medicinska implantat. Baserat på detta konstruerade vi multilagerfilmer av chitosan och heparin med olika deponeringslösningar och undersökte dynamiken och filmens struktur i detalj. Chitosan-heparin-filmen var starkt hydratiserad, bestående av cirka 60-80 vikt-% vatten beroende på deponeringsbetingelserna. Den adsorberade mängden och tjockleken på filmen ökade nästan exponentiellt med antal deponeringar, vilket förklarades med chitosanets förmåga att diffundera genom filmen under uppbyggnaden. Med ett nytt angreppssätt använde vi FRET för att bevisa diffusionen av fluorescerande färgmärkt chitosan i filmen under uppbyggnaden. Diffusionskoefficienten var i princip oberoende av pH och jonstyrka under deponeringen och följaktligen av filmens struktur. Genom att använda ett pH-känsligt färgämne begravt under sju biskikt av chitosan-heparin visade vi att färgämnet i hög grad påverkades av laddningen på det yttersta lagret. Från QCM-D-data lade vi fram teorin om att en ökning av energidissipationen för ett lager inte nödvändigtvis indikerar att lagrets struktur har blivit mindre styvt. / QC 20100729

AtMAP65-4 est une protéine associée aux microtubules appartenant à la famille des AtMAP65s qui compte 9 membres identifiés chez Arabidopsis thaliana. Ces protéines appartiennent à une famille conservée au cours de l'évolution, les MAP65s. Ainsi, des protéines homologues sont présentes chez de mammifères (PRC1), chez la levure (Ase1p) ou chez la drosophile (FEO). Jusqu'ici l'étude des propriétés moléculaires et fonctionnelles des AtMAP65s s'est portée essentiellement sur l'étude d'AtMAP65-1 et AtMAP65-5. La principale caractéristique de ces protéines est d'induire la formation de faisceaux de microtubules in vitro. La distribution des AtMAP65s in vivo est très régulée, celle-ci sont localisées avec des réseaux des microtubules bien définis. Ainsi, leur rôle supposé est de mettre en place ces réseaux puis de participer à leur maintient. La localisation d'AtMAP65-4 apparait comme très intéressante car elle est strictement associée avec les microtubules du fuseau mitotique. D'après les résultats obtenus au cours de ce travail, nous avons suggéré que la fonction in vivo d'AtMAP65-4 est de participer à la mise en place et au maintient des microtubules en faisceaux dans les fibres kinétochoriennes lors de la division cellulaire. Lors d'une étude in vitro nous avons montré qu'AtMAP65-4 modifie les paramètres dynamiques de polymérisation des microtubules. Outre sa capacité à former des faisceaux, AtMAP65-4 permet une croissance régulière des microtubules au sein des faisceaux qu'elle induit. Le mécanisme d'action de la MAP à l'échelle moléculaire a été analysé à travers une étude bioinformatique où nous avons modélisé l'activité d'AtMAP65-4. Les données obtenues montrent qu'AtMAP65-4 peut bloquer les évènements de dépolymérisation des microtubules. Par ailleurs, l'activité d'AtMAP65-4 pourrait être régulée in vivo par des modifications post traductionnelles. En effet, nous avons montré et étudié l'effet de la phosphorylation d'AtMAP65-4 par les kinases Auroras. Cette phosphorylation pourrait être impliquée dans la régulation de l'activité d'AtMAP65-4 au cours de la mitose. / AtMAP65-4 is a microtubule-associated protein belonging to the AtMAP65s family that comprises 9 members identified in Arabidopsis thaliana. These proteins belong to a family conserved during evolution, MAP65s. Thus, homologous proteins are present in mammals (PRC1), in yeast (Ase1p) or Drosophila (FEO). So far the study of molecular properties and functional AtMAP65s has focused mainly on AtMAP65-1 and AtMAP65-5. The main feature of these proteins is to induce the formation of microtubule bundles in vitro. In vivo, these AtMAP65s are localized with subsets of microtubule bundles as they are suggested to play a role in establishing and maintaining these networks. From the results we obtained on AtMAP65-4 properties during this work such as the in vivo localization, biochemical properties and functional effetc on the MT polymerization, we suggested that the in vivo function of AtMAP65-4 is involved in setting up and maintaining microtubule bundles within kinetochore fibers during cell division. In vitro studies allowed us to show that AtMAP65-4 changes the dynamic parameters of microtubule. In addition to its ability to form bundles, AtMAP65-4 allows steady growth of microtubules in bundles it induces. The mechanism of action of the MAP at the molecular level was analyzed through a bioinformatics study where we modeled the activity of AtMAP65-4 and concluded that it could block the depolymerization events. Moreover, the activity of AtMAP65-4 could be regulated in vivo by post-translational modifications. Indeed, we have shown that AtMAP65-4 is phosphorylated by Aurora kinases in vitro. The effect of this phosphorylation during mitosis is under investigation.