Lorsqu'une tumeur grossit, elle exerce une pression sur les tissus environnants et est comprimée en retour. Des expériences sur un modèle de tumeur in vitro, appelé sphéroïde, ont montré que cette pression influence largement le devenir du tissu cancéreux, notamment en freinant sa croissance, mais aussi en le rendant plus invasif. Pour mieux comprendre ce dernier effet, nous avons cherché à étudier le comportement migratoire des cellules à l'intérieur d'un sphéroïde sous pression. Observer l'intérieur d'un sphéroïde pose cependant un problème technique car les méthodes usuelles d'imagerie ne sont pas utilisables dans des tissus épais (> 100 μm). L'imagerie classique étant limitée en profondeur à cause de la diffusion de la lumière, nous nous sommes tournés vers une méthode qui utilise justement celle-ci : la diffusion dynamique de lumière ou DLS (Dynamic Light Scattering). Nous avons développé son application à la migration cellulaire, afin d'obtenir la distribution des déplacements relatifs des cellules au cours du temps. Cette mesure est faite sans utiliser de marqueurs spécifiques et est applicable à des sphéroïdes allant jusqu'à 400 μm de diamètre. Nous avons ainsi mis en évidence une organisation radiale du sphéroïde en termes de mobilité, avec des cellules rapides en surface et plus lentes au centre. Nous avons aussi montré qu'en appliquant une contrainte au sphéroïde, la vitesse moyenne diminue jusqu'à être réduite de moitié pour des pressions supérieures à 15kPa. Une autre équipe a mesuré par ailleurs une augmentation de la vitesse des cellules en surface suite à une compression, ce qui indique que l'organisation radiale se retrouve dans la réponse à la pression. Nous avons montré que cette sensibilité à la pression est une propriété qui émerge de l'organisation 3D du tissu, dans laquelle la matrice extracellulaire joue un rôle primordial. Enfin, pour explorer les possibilités qu'offre notre technique, nous l'avons appliquée à une autre question : comment la migration des macrophages est-elle affectée par les signaux provenant de cellules apoptotiques ? Les résultats ont montré que les cellules apoptotiques précoces augmentent la vitesse des macrophages tandis que les cellules apoptotiques tardives la réduisent. D'un cas à l'autre, la longueur de persistance du mouvement est conservée. / As a tumor grows, it exerts a mechanical pressure on its surrounding tissue and is compressed back as a reaction. Recent experiments on an in vitro tumor model, called spheroid, have shown that this pressure is crucial for the fate of the cancerous tissue. In particular, the pressure slows down its growth, but makes it more invasive. To further understand the latter effect, we decided to study the migration of cells inside spheroids under pressure. However, imaging the inside of a spheroid is technically challenging as usual microscopy methods do not work on thick tissues (> 100 μm). Standard imaging methods are limited in terms of depth penetration because of light scattering. For this reason, we decided to take advantage of this scattered light with a method called Dynamic Light Scattering (DLS). We developed its application to cell migration in order to measure the distribution of cells displacements over time. The measurement is label-free and works with spheroids as thick as 400 μm in diameter. By this means, we revealed a radial organization inside the spheroid in terms of mobility, with fast cells at the surface and slower cells in the core. We also showed that applying a pressure onto spheroids decreases the average cell speed by a factor up to two for pressure greater than 15 kPa. Another team reported an increase in the speed of cells located at the surface of a compressed spheroid, which implies that the radial organization is also true for the impact of pressure. We demonstrated that this sensitivity to an external pressure is a 3D emergent property, in which the extracellular matrix plays an essential role. Finally, we explored the potential of our technique by addressing another question: how do apoptotic cells signals affect the migration of macrophages? We found that early apoptotic cells increase the speed of macrophages whereas late apoptotic cells decrease it. In both cases, the persistence length of the motion is the same.

Este estudo reúne os principais resultados de fluorescência estática e resolvida no tempo sobre a interação da porfirina meso-tetrakis (4-metilpiridil), na forma de base livre (TMPyP) e complexada com Zn2+ (ZnTMPyP), com vesículas de fosfolipídio. Adicionalmente foram utilizadas as técnicas de potencial zeta e espalhamento de luz dinâmico (DLS, do inglês \"dynamic light scattering\"). As vesículas de fosfolipídio foram formadas por dois conjuntos de fosfolipídios: saturados e insaturados. O primeiro grupo é formado pela mistura dos fosfolipídios zwiteriônico 1,2-dipalmitoil-sn-glicero-3-fosfocolina (DPPC) e aniônico 1,2-dipalmitoil-sn-3-glicero-[fosfo-rac-(1- glicerol)] (DPPG), a diferentes razões molares. Os estudos utilizando tais sistemas foram realizados abaixo (25oC) e acima (50oC) da temperatura de transição de fase gel-líquido cristalino destes fosfolipídios (~ 41oC). O segundo grupo é formado pela mistura dos fosfolipídios zwiteriônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfocolina (POPC) e aniônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfo(1-rac-glicerol) (POPG). Como a transição de fase destes dois fosfolipídios ocorre a temperaturas negativas, todos os experimentos foram realizados a 25oC (vesículas no estado líquido cristalino). Todos os sistemas foram preparados através do método de extrusão para a obtenção de vesículas grandes unilamelares (LUV, do inglês \"large unilamellar vesicles\"). As análises dos dados de fluorescência indicaram que a atração eletrostática entre os substituíntes (positivamente carregados) das porfirinas TMPyP e ZnTMPyP e o grupo das cabeças polares (camada de Stern) das vesículas de fosfolipídio desempenha um papel fundamental na associação da porfirina. A distribuição da TMPyP entre o meio aquoso (tampão) e as vesículas de fosfolipídio foi evidenciada pela coexistência de um tempo de vida de fluorescência mais curto (~ 5 ns) e outro mais longo (~ 9-11 ns), respectivamente. Baseado nos valores das constantes pré-exponenciais, estudos adicionais mostram que a distribuição acima é afetada pela concentração de sal na solução. Os resultados de supressão de fluorescência com o supressor iodeto de potássio (KI) indicaram que ambas porfirinas estão localizadas, preferencialmente, na região da camada de Stern. Este resultado foi confirmado pelos estudos de potencial zeta e de DLS, os quais mostraram uma neutralização parcial das cargas negativas na superfície das vesículas devido à associação da porfirina. / This study presents time-resolved and steady-state fluorescence results on the interaction of the meso-tetrakis (4-methylpyridil) porphyrin, in free base form (TMPyP), and complexed with Zn2+ (ZnTMPyP), with phospholipid vesicles. Zeta potential and dynamic light scattering (DLS) techniques were also used. Phospholipid vesicles were formed by two phospholipid systems: saturated and unsaturated. The first group is a mixture of zwiterionic dipalmitoyl-sn-glycero-3-phosphocoline (DPPC) and anionic 1,2-dipalmitoyl-sn-3-glycero-[phospho-rac-(1-glycerol)] (DPPG) phospholipids, at different molar ratios. Measurements were performed bellow (25oC) and above (50oC) the main gel-liquid crystalline phase transition temperature (~ 41oC). The second group is constituted by a mixture of zwiterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1-rac-glycerol) (POPG) phospholipids, at different molar ratios. Since the gel-liquid crystalline phase transition of these phospholipids occurs at a very low temperature value, all experiments were performed at 25oC (liquid crystalline state vesicles). All phospholipid systems were prepared through the extrusion method in order to obtain large unilamellar vesicles (LUV). The fluorescence data analyses indicated that the electrostatic attraction between the porphyrin substituents (positively charged) and the polar head groups of the phospholipid vesicles (Stern layer) plays an important role on the porphyrin binding affinity. The distribution of TMPyP between the aqueous medium (buffer) and the phospholipid vesicles was characterized by the coexistence of a shorter (~ 5 ns) and a longer (~ 9-11 ns) fluorescence lifetimes, respectively. Based on the pre- exponential values, additional time-resolved experiments showed a redistribution of the porphyrin at increasing salt concentration. The quenching studies, using potassium iodide (KI) as quencher, indicated that both TMPyP and ZnTMPyP are preferentially located at the Stern layer region. This result is in agreement with the zeta potential and DLS findings, which demonstrated a partial neutralization of the negative charges at the vesicle surface due to the porphyrin association.

Small devices capable of self-propulsion have potential application in areas of nanoscience where autonomous locomotion and programmability are needed. The specific base-pairing interactions that arise from DNA hybridisation permit the programmed assembly of matter and also the creation of controllable dynamical systems. The aim of this thesis is to use the tools of DNA nanotechnology to design synthetic active matter at the micro and nano scales. In the first section, DNA was used as an active medium capable of transporting information faster than diffusion in the form of chemical waves. DNA waves were generated experimentally using a DNA autocatalytic reaction in a microfluidic channel. The propagation velocity of DNA chemical waves was slowed down by creating concentration gradients that changed the reaction kinetics in space. The second section details the synthesis of chemically-propelled particles and the use of DNA as a 'programmable glue' to mediate their interactions. Janus micromotors were fabricated by physical vapour deposition and a wet-chemical approach was demonstrated to synthesise asymmetrical catalytic Pt-Au nanoparticles that function as nanomotors. Dynamic light scattering measurements showed nanomotor activity that depends on H₂O₂ concentration, consistent with chemical propulsion. Gold nanoparticles/Origami hybrids were assembled in 2D lattices of different symmetries arranged by DNA linkers. The third section details the design process and synthesis of nanomotors using DNA as a structural scaffold. 3D DNA Origami rectangular prisms were functionalised site-specifically with bioconjugated catalysts, i.e. Pt nanoparticles and catalase. Enzymatic nanomotors were also conjugated to various cargoes and their motor activity was demonstrated by Fluorescence Correlation Spectroscopy. In the final section, control mechanisms for autonomous nanomotors are studied, which includes the conformational change of DNA aptamers in response to chemical signals, as well as a design for an adaptive dynamical system based on DNA/enzyme reaction networks.

This thesis focuses on the study of the interactions between polyelectrolytes and surfactants in aqueous solutions and at interfaces, as well as on the structural changes these molecules undergo due to that interaction. Small–angle neutron scattering, dynamic, and static light scattering were the main techniques used to investigate the interactions in bulk. The first type of polymer studied was a negatively charge glycoprotein (mucin); its interactions with ionic sodium alkyl sulfate surfactants and nonionic surfactants were determined. This system is of great relevance for several applications such as oral care and pharmaceutical products, since mucin is the main component of the mucus layer that protects the epithelial surfaces (e.g. oral tissues). Sodium dodecyl sulfate (SDS) on the other hand, has been used as foaming agent in tooth pastes for a very long time. In this work it is seen how SDS is very effective in dissolving the large aggregates mucin forms in solution, as well as in removing preadsorbed mucin layers from different surfaces. On the other hand, the nonionic surfactant n-dodecyl β-D-maltopyranoside (C12-mal), does not affect significantly the mucin aggregates in solution, neither does it remove mucin effectively from a negatively charge hydrophilic surface (silica). It can be suggested that nonionic surfactants (like the sugar–based C12-mal) could be used to obtain milder oral care products. The second type of systems consisted of positively charged polyelectrolytes and a negatively charged surfactant (SDS). These systems are relevant to a wide variety of applications ranging from mining and cleaning to gene delivery therapy. It was found that the interactions of these polyelectrolytes with SDS depend strongly on the polyelectrolyte structure, charge density and the solvent composition (pH, ionic strength, and so on). Large solvent isotopic effects were found in the interaction of polyethylene imine (PEI) and SDS, as well as on the interactions of this anionic surfactant and the sugar–based n-decyl β-D-glucopyranoside (C10G1). These surfactants mixtures formed similar structures in solutions to the ones formed by some of the polyelectrolytes studied, i.e. ellipsoidal micelles at low electrolyte concentration and stiff rods, at high electrolyte and SDS concentrations. / QC 20100901

The aim of this study was to investigate the molecular interactions of the anti-cancer drug Doxorubicin (DOX) with poly(methacrylic acid) grafted starch nanoparticles (PMAA-g-St). In order to fully understand the DOX/PMAA-g-St system, we conducted in-depth studies on DOX dimer dissociation and DOX/PMAA-g-St binding interactions using various techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence and absorption spectroscopy. Based on our experimental results, we developed a quantitative thermodynamic model with relevant parameters such as dissociation constant, Kd, as well as enthalpy of binding, ΔH, in order to explain DOX/PMAA-g-St interactions. In addition, we also studied the effect of environmental factors such as pH and NaCl on DOX self-association and DOX/PMAA-g-St complex formation. In conclusion, the combination of results obtained from various techniques as well as the multispecies equilibrium model, enables us to interpret quantitatively the data of drug loading onto and release from polymeric nanoparticles.

This thesis describes the results of experimental work on polyelectrolyte gels and their interaction with oppositely charged surfactants, and presents two new algorithms applicable to the simulation of colloid and polymer systems. The model systems investigated were crosslinked poly(acrylate) (PA) and poly(styrene sulphonate) (PSS), and the surfactant was dodecyl trimethylammonium bromide (DoTAB). Pure gel materials were studied using dynamic light scattering. It was shown that the diffusion coefficient (D) increases with increasing degree of swelling and the concentration dependence is larger than predicted by scaling arguments. For gels at swelling equilibrium D increases with increasing degree of crosslinking. In subsequent studies on gel particles in DoTAB solution, Raman spectra were recorded at different positions in the gel. For both types of gels two distinct regions could be observed. For PA the surfactant is localised in the outer phase without any surfactant in the core, while for PSS the surfactant was distributed such that it had the same concentration relative to the polymer throughout the gel. In a second experiment, the kinetics for the deswelling of microscopic PSS particles in DoTAB solution was studied. It was found that the final volume varied linearly with the DoTAB concentration, and the rate of volume decrease could be fitted to a single exponential indicating stagnant layer diffusion to be the rate limiting process for the deswelling of the PSS particles. In the second part, I first describe an algorithm showing an efficient way to detect percolation in simulations, with periodic boundary conditions, using recursion. Spherical boundary conditions is an alternative to periodic boundary conditions for systems with long-range interactions. In the last part, the possibility to use the surface of a hypersphere in four dimensions for simulations of polymer systems is investigated, and algorithms for Monte Carlo and Brownian dynamics simulations are described.

Polyaniline was chemically synthesized at three different temperatures of 25, 0, and -25oC, by oxidative polymerization with ammonium peroxidisulfate at equimolar of aniline to oxidant ratio and 1M HCl. The resulted polyaniline was in a powder form which was characterized by several techniques such as: electrical conductivity, elemental analysis, thermal analysis, wide-angle X-Ray diffraction, and scanning electron microscope. The solution properties of the reduced polymer were studied by viscometry, static and dynamic light scattering. It was found that as the polymerization temperature decreased, the molecular weight, crystallinity, and thermal stability of polyaniline increased, while the electrical conductivity was independent of the polymerization temperature. Moreover, the morphology of the polymer was changed from granular to tubular with reducing polymerization temperature. Viscometry and static light scattering showed that polyaniline has a flexible random coil conformation when dissolved in N-methyl-2-pyrrolidinone which proved to be a good solvent for this polymer. Dynamic light scattering indicated that the polymer solution is a polyelectrolyte with high hydrodynamic radius at low polymer concentrations. All mechanical features except Young&#039 / s modulus of polyaniline-filled low density polyethylene composites became poorer as polyaniline content increased. Moreover, a sudden increase in the electrical conductivity with increasing polyaniline contents was also observed. The conductivity of the tubular composites of multi wall nanotubes (MWNTs)-filled polyaniline increased with increasing MWNTs loading, and became weakly temperature dependent. The morphological analysis indicated that the MWNTs were well dispersed and isolated, and the tubes became crowded proportionally to MWNTs weight percent used in the composites.

Dynamic light scattering (DLS) is a method to size submicron particles by measuring their thermal motion (diffusion) in suspensions and emulsions. However, the validity of the Stokes-Einstein equation that relates the diffusion coefficient and the particle size is limited to spherical particles and very low concentrations. Within this thesis, DLS is used for the characterization of suspensions of pyrogenic silica which consists of fractal-like aggregates composed of sintered spherical primary particles. These structural features clearly complicate the understanding of DLS experiments and have been a severe obstacle to employing DLS as routine standard tool for the characterization of pyrogenic silica. The main objective of this thesis is therefore to evaluate the application of DLS in product development and quality assurance of pyrogenic silica industry, what essentially means to identify those structural properties of fractal aggregates which are measurable with DLS and to quantify the method’s sensitivity to changes in these properties. The investigations presented here are split up into four parts, simulations that establish a relation between structural and hydrodynamic properties, experiments validating the simulation results, the characterization of concentrated suspensions and the application-oriented analysis of DLS data for specific industrially relevant measurement tasks. / Die Dynamische Lichtstreuung (DLS) ist eine Messmethode zur Größenbestimmung submikroner Partikel. Dabei wird primär die stochastische Bewegung der Teilchen (Diffusion) in Suspensionen und Emulsionen bewertet. Die Stokes-Einstein Gleichung, die das Verhältnis zwischen gemessenem Diffusionskoeffizienten und Partikelgröße wiedergibt, ist jedoch nur für kugelförmige Teilchen, die in sehr niedriger Konzentration vorliegen, gültig. In der vorliegenden Arbeit wird die dynamische Lichtstreuung zur Charakterisierung von Suspensionen pyrogener Kieselsäure eingesetzt. Diese besteht aus fraktalen Aggregaten, die wiederum aus versinterten aber meist kugelförmigen Primärpartikeln zusammengesetzt sind. Diese strukturellen Eigenschaften erschweren die Anwendbarkeit der DLS bzw. die Interpretation der Messergebnisse und verhinderten bisher den Einsatz der DLS als Routinemethode zur Charakterisierung pyrogener Kieselsäuren. Das Hauptziel dieser Arbeit ist daher eine Bewertung der Möglichkeiten der DLS für die Produktentwicklung und Qualitätssicherung in der Herstellung pyrogener Kieselsäuren. Das bedeutet im Besonderen, dass sowohl die messbaren granulometrischen Eigenschaften als auch die Sensitivität der Methode bei Eigenschaftsänderungen ermittelt werden müssen. Die hier durchgeführten Arbeiten sind in vier Teile gegliedert: Simulationen, die eine Beziehung zwischen strukturellen und hydrodynamischen Eigenschaften herstellen, Experimente zur Validierung der Simulationsergebnisse, die Charakterisierung konzentrierter Suspensionen und die anwendungsorientierte Auswertung von DLS-Daten für spezifische industrierelevante Messaufgaben.

The light scattering methods were used for characterization of properties and formation of networks of different dimension, based on N-vinylcaprolactam (VCL). Formation of PVCL microgels in presence of poly(vinyl alcohol) as stabilizer was studied. Size of resulting microgels strongly depends on the temperature and heating rate: interparticle aggregation was observed during slow heating and intraparticle collapse during fast heating. Angular dependence of measured diffusion coefficient on the angle of observation was studied for the microgel in the swollen, shrunken and aggregated states. Thermo-sensitive microgels based on N-vinylcaprolactam and acetoacetoxyethyl methacrylate were prepared under surfactant free conditions. The presence of internal part with low thermo-sensitivity and highly thermo-sensitive outer part of the particle (the core-shell structure of microgel) was deduced from static and dynamic light scattering experiments. Results obtained from combined SLS and DLS show the change of conformation from "swollen" soft sphere to compact shrunken "hard sphere". Thermo-sensitive microgel based on N-vinylcaprolactam and N-vinylpyrrolidone was used for investigation of the internal modes in microgel dispersion in the wide range of qRg values. Two internal motions and translation diffusion were observed in the asymptotic range. Angular dependence of the normalized diffusion coefficient showed power law behavior in this range. The experimentally determined value of exponent n = 0.96 was in good agreement with the value predicted for ZIMM limit for polymer chains with hydrodynamic interaction. The reduced first cumulant Ã*(q) reached a constant value in the range of large qRg values. Appearance of plateau value indicates ZIMM limit of hydrodynamic interaction but experimental value was much lower than the theoretically predicted plateau value for linear chains in good solution. 3,3?-(ethane-1,1-diyl)bis(1-vinyl-2-pyrrolidone) was used as a cross-linker of VCL in solution by radical polymerization. The network formation was investigated by dynamic light scattering. It was shown, that monitoring of the light scattered intensity in all cases is quite sensitive to detect the gelation threshold even in the presence of very low amount of cross-linker. The power law of time correlation function at the gel point is a sufficient but not a necessary condition for critical gelation. The exponent calculated from power law depends on cross-linker concentration and can be attributed to the degree of branching. Critical exponents obtained at the gel point by DLS and rheology for hydrogel system based on VCL and hydroxyethyl methacrylate were compared. The theoretically predicted equality of exponents from these methods was found as not valid at least for this studied system.

While much is known about the structure and interactions of the β-carboxysomal proteins, interactions of the proposed vertex protein CcmL with the other components have not yet been directly demonstrated. A fluorescence resonance energy transfer based method combined with other complementary spectrophotometry techniques as well as x-ray crystallography and transmission electron microscopy was used in a Thermosynechococcus elongatus BP-1 model to study these interactions. CcmL was found to interact with the various CcmK shell proteins with a clear binding preference for CcmK2; the previously proposed interaction of CcmL with CcmM was shown to not occur in vitro, and a possible CcmL-CcmL interaction was observed tentatively. In addition, analysis of a novel x-ray crystal structure of Nostoc sp. PCC7120 CcmL in a decameric form suggests a possibility of a CcmL-CcmL back-to-back interaction. This study gives the first direct experimental evidence for the biological role of CcmL as the carboxysomal vertex protein.