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This dissertation describes new methods for measuring surface forces using evanescent waves, and applications to non-DLVO forces. An evanescent wave, generated at a solid-liquid interface, is scattered by AFM tips or particles attached to AFM cantilevers. The scattering of this wave is used to determine absolute separation between surfaces and/or the refractive index as a function of separation in AFM measurements. This technique is known as evanescent wave atomic force microscopy (EW-AFM). The scattering of an evanescent wave by Si3N4 AFM tips is large and decays exponentially with separation from a refractive index boundary. Thus, scattering is a useful method for measuring the separation between a Si3N4 tip and sample. This method has been used to measure the absolute separation between a tip and sample in the presence of an irreversibly adsorbed polymer film. Measurement of the film thickness and time response of the polymer to applied loads has also been studied. These measurements are not possible using current AFM techniques. In addition to measurements in polymer systems, the simple scattering profile from Si3N4 tips was used to re-examine short range hydration forces between hydrophilic surfaces. Results presented in this thesis suggest this force does not depend on the hydrated radius of the ion between glass and silicon nitride. The scattering generated by a Si3N4 tip has also been used to measure the refractive index of bulk fluids and thin films between hydrophobic surfaces. Based on these results, I have shown that a long-range attraction between hydrophobic surfaces is accompanied by an increase in the refractive index between the tip and surface. From this I have concluded that the attractive force, measured in this study, is the result of an increase in the concentration of organic material between surfaces. Finally, I have shown that the scattering profile depends on the material and size of the scattering object. Scattering from silicon nitride tips is exponential with separation. In contrast, the scattering profile from silicon tips, which are similar in size and geometry, is not a simple exponential. The scattering profile of larger spherical particles attached to cantilevers is also not exponential. It is approximately the sum of two exponentials. The functional form of the scattering profile with separation is consistent with the transmission of evanescent light through flat planar films. This result would suggest that a re-examination of the separation-dependence of scattering in TIRM measurements is necessary. / Ph. D.

The composition and stability of polymer-magnetite complexes is essential for their use as a treatment for retinal detachment, for drug targeting and delivery, and for use as a MRI contrast agent. This work outlines a general methodology to design well-defined, stable polymer-magnetite complexes. Colloidal modeling was developed and validated to describe polymer brush extension from the magnetite core. This allowed for the observation of deviations from expected behavior as well as the precise control of polymer-particle complex size. Application of the modified Derjaguin-Verwey-Landau-Overbeek (DLVO) theory allowed the determination of the polymer loading and molecular weight necessary to sterically stabilize primary magnetite particles. Anchoring of polyethers to the magnetite nanoparticle surface was examined using three different types of anchor groups: carboxylic acid, ammonium, and zwitterionic phosphonate. As assessed by dynamic light scattering (DLS), the zwitterionic phosphonate group provided far more robust anchoring than either the carboxylic acid or ammonium anchor groups, which was attributed to an extremely strong interaction between the phosphonate anchor and the magnetite surface. Coverage of the magnetite surface by the anchor group was found to be a critical design variable for the stability of the zwitterionic phosphonate groups, and the use of a tri-zwitterionic phosphonate anchor provided stability in phosphate buffered saline (PBS) for a large range of polymer loadings. Incorporation of an amphiphlic poly(propylene oxide)-b-poly(ethyelene oxide) (PPO-b-PEO) diblock copolymer attached to the magnetite surface was examined through colloidal modeling and DLS. The relaxivity of the complexes was related to aggregation behavior observed through DLS. This indicated the presence of a hydrophobic interaction between the PPO layers of neighboring complexes. When this interaction was large enough, the complexes exhibited an increased relaxivity and cellular uptake. Thus, we have developed a methodology that allows for design of polymer-magnetite complexes with controlled sizes (within 8% of predicted values). Application of this methodology incorporated with modified DLVO theory aids in the design of colloidally stable complexes with minimum polymer loading. Finally, determination of an anchor group stable in the presence of phosphate salts at all magnetite loadings allows for the design of materials with minimum polymer loadings in biological systems. / Ph. D.

La plupart des bactéries vivent en communautés organisées, appelées biofilms, augmentant leur résistance aux traitements antibiotiques. Ainsi, la formation de biofilms sur les organes et matériels médicaux est considérée comme la cause de la majorité des infections bactériennes. Il est alors important de trouver des traitements pour empêcher ou perturber la formation de ces biofilms. Il a été proposé que les porines de P. Stuartii, Omp-Pst1 et Omp-Pst2, peuvent s’auto-assembler via un steric-zipper, étape responsable du développement initial du biofilm. Ainsi, notre objectif est de synthétiser des inhibiteurs d’interactions entre porines pour limiter ce contact intercellulaire. Nous avons développé des molécules peptidomimétiques basées sur la séquence LGNYR, active dans les deux porines. Pour cela, des réactions de chimie click sur phase solide ont été mises en oeuvre afin de synthétiser des analogues de cette séquence, comme la CuAAC pour introduire un motif triazole, dans une position variable au sein du peptide. Nous avons ainsi développé une méthode rapide et efficace afin de réaliser cette réaction par l’utilisation d’un catalyseur cuivre(I)-N-hétérocyclique carbène stable à l’air. Similairement, de nouvelles conditions ont aussi été mises au point en phase solide afin d’obtenir régiosélectivement des peptides comportant un motif isoxazole 3,4- ou 3,5-disubstitué, par la réaction entre un alcyne et un oxyde de nitrile. Ces cycloadditions 1,3-dipolaires nous ont ainsi permis d’obtenir une première librairie de peptidotriazoles et de peptidoisoxazoles. Il sera enfin possible d’étudier les biofilms grâce à la synthèse de sondes fluorescentes basées sur les inhibiteurs montrant de fortes affinités avec la cible, couplées à un fluorophore dérivé de coumarine. / In the environment, most of bacteria live as organized communities, known as biofilms, enhancing their resistance to antibiotic treatments. Thus the formation of biofilms on organ and indwelling medical devices is considered to cause the majority of bacterial infections in the human body. Thus, to enhance antibiotics efficacy, there is a high need to find treatments to prevent or disrupt biofilm formation. It has been proposed that P. stuartii porins, Omp-Pst1 and Omp-Pst2, can self-associate through a steric zipper, being responsible for the initial development of biofilms. Thus, our objective is to synthesize porin’s self-matching interactions (PSMI) inhibitors to counterfeit this intercellular contact. We developed peptidomimetic molecules based on the LGNYR sequence, that have shown to be active in both porins. Then we used click chemistry to synthesize on solid phase analogues of this sequence, as the solid phase Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to introduce a triazole moiety into the peptide chain at different positions. We thus developed a fast and efficient method to perform this reaction using a stable copper(I)-N-heterocyclic carbene catalyst. Similarly, new conditions were developed on solid phase to synthesize regioselectively peptides containing a 3,4- or 3,5-disubstituted isoxazole moiety, through the reaction between an alkyne and a nitrile oxide. These 1,3-dipolar cycloadditions allowed us to developed a first library of peptidotriazoles and peptidoisoxazoles. We also obtained fluorescent probes based on the inhibitors showing higher affinity for the target as tools to study biofilm formation.

O entendimento dos princípios básicos do enovelamento protéico pode conduzir a muitas aplicações importantes. Embora não se conheçam todos os aspectos significativos envolvidos neste problema, experimentos e aproximações teóricas têm produzido avanços relevantes na sua compreensão. Um fato experimental importante tem sido a descoberta de que o logaritmo da taxa de enovelamento log kf se correlaciona linearmente com parâmetros estruturais globais, como a ordem de contato relativa c. Com o propósito de contribuir para o entendimento do processo de enovelamento, o objetivo primordial deste trabalho consiste em explicar o porquê de certas proteínas não seguirem o comportamento linear entre log kf e c, verificado para outras proteínas da mesma classe (usualmente proteínas pequenas e com termodinâmica descrita pela aproximação de dois estados). Para isso foi necessário identificar os parâmetros topológicos da estrutura nativa que constituíssem importantes determinantes da cinética do enovelamento de proteínas globulares. Também se estudou como as especificidades estéricas dos aminoácidos afetam o processo do enovelamento de proteínas, assim como influenciam na correlação entre a ordem de contato relativo e a taxa de enovelamento. Empregou-se neste estudo um modelo simplificado em rede cúbica, que foi tratado por meio de simulações Monte Carlo. Um conjunto de 52 estruturas maximamente compactas, correspondendo a cadeias de tamanho L = 27 monômeros, foi usado para representar estados nativos; estas estruturas foram escolhidas de forma a representar uma variedade significativa de padrões estruturais, independentemente de c. Através de uma análise detalhada da influência de parâmetros topológicos das configurações nativas na cinética do enovelamento, conclui-se que a taxa de enovelamento é fortemente dependente daquilo que denominamos aqui como “conteúdo de estruturas tipo-secundárias" da estrutura nativa. Adicionalmente, observou-se que aquela (taxa), independentemente do valor da ordem de contato relativo, é fortemente influenciada pelos padrões confíguracionais e suas combinações presentes na nativa. Por meio dessa premissa, foi então possível explicar de forma consistente os casos que não obedecem a pretensa relação linear entre log kf e c, levando a concluir que o logaritmo da taxa de enovelamento e a ordem de contato relativo são linearmente dependentes somente para aquelas configurações em que há uma certa quantidade equilibrada (que depende de c) de padrões estruturais, mesclando contatos efetivos de curto alcance (alto conteúdo de estruturas tipo-secundárias), com outros de longo alcance (baixo conteúdo de estruturas tipo-secundárias). Estruturas nativas que quebram este equilíbrio têm sua cinética de enovelamento afetada com respeito à reta de regressão linear ajustada para o conjunto de todas as configurações consideradas. Dessa forma, verificou-se que o mecanismo físico básico que relaciona o conteúdo de estruturas tipo-secundárias e a taxa de enovelamento, envolve o conceito de cooperatividade: se a estrutura nativa é rica em combinações de padrões estruturais ricos em contatos efetivos de curto alcance, o processo de enovelamento é mais rápido porque contatos locais são naturalmente estimulados por flutuações térmicas. / The understanding of basic principles of the protein folding problem can lead to many important applications. Although not all the involved significant aspects of this problem are known, experiments and theoretical approaches have produced important advances in its understanding. An important experimental fact has been the discovery that the logarithm of the folding rate log kf correlates linearly with global structural parameters, like the relative contact order c. In order to contribute for the understanding of folding process, the primordial goal of this work consists in to explain why certain proteins do not follow the linear behavior between log kf and c, as verified to other proteins from the same class (usually small two states proteins). For this, it was necessary to identify those topological parameters of the native structure that are important to the folding kinetic of globular protein. It was also studied how steric specificities of the aminoacids affect the protein folding process, as well how they influence the correlation between the relative contact order and the folding rate. It was employed in this study a simplified cubic lattice model, treated by Monte Carlo simulation. A set of 52 maximum compact structures, corresponding to chains of size L = 27 monomers, was used to represent the native states; these structures were chosen in such a way to represent a significant diversity of structural patterns, independently of c. Through a detailed analysis of the influence of topological parameters of the native configurations on the folding kinetic, it was concluded that the folding rate is strongly dependent of what we call here as “content of type-secondary" of the native. Additionally, it was observed that log kf is, independently of c, strongly influenced by the configurational patterns and its combinations in the native. Through this premise it was possible to consistently explain the cases that do not obey the pretense linear relation between log kf and c, leading to conclude that the logarithm of the folding rate and the relative contact order are linearly related only for those configurations in that there is a certain balanced amount of structural patterns (which depend on c) mixing short-range effective contacts (high contents of secondary-type structures) and long-range contacts (low contents of secondary-type structures). Structures that break this balance have its folding kinetic affected with respect to the linear fitting adjusted for the set of all the considered configurations. Of this form, it was verified that basic physical mechanism that relates the content of type-secondary structures and the folding rate involves the cooperativety concept: if the native structure presents combinations of structural standards rich in effective contacts of short-range, the folding process is faster because local contacts are naturally stimulated by thermal fluctuations.

O entendimento dos princípios básicos do enovelamento protéico pode conduzir a muitas aplicações importantes. Embora não se conheçam todos os aspectos significativos envolvidos neste problema, experimentos e aproximações teóricas têm produzido avanços relevantes na sua compreensão. Um fato experimental importante tem sido a descoberta de que o logaritmo da taxa de enovelamento log kf se correlaciona linearmente com parâmetros estruturais globais, como a ordem de contato relativa c. Com o propósito de contribuir para o entendimento do processo de enovelamento, o objetivo primordial deste trabalho consiste em explicar o porquê de certas proteínas não seguirem o comportamento linear entre log kf e c, verificado para outras proteínas da mesma classe (usualmente proteínas pequenas e com termodinâmica descrita pela aproximação de dois estados). Para isso foi necessário identificar os parâmetros topológicos da estrutura nativa que constituíssem importantes determinantes da cinética do enovelamento de proteínas globulares. Também se estudou como as especificidades estéricas dos aminoácidos afetam o processo do enovelamento de proteínas, assim como influenciam na correlação entre a ordem de contato relativo e a taxa de enovelamento. Empregou-se neste estudo um modelo simplificado em rede cúbica, que foi tratado por meio de simulações Monte Carlo. Um conjunto de 52 estruturas maximamente compactas, correspondendo a cadeias de tamanho L = 27 monômeros, foi usado para representar estados nativos; estas estruturas foram escolhidas de forma a representar uma variedade significativa de padrões estruturais, independentemente de c. Através de uma análise detalhada da influência de parâmetros topológicos das configurações nativas na cinética do enovelamento, conclui-se que a taxa de enovelamento é fortemente dependente daquilo que denominamos aqui como “conteúdo de estruturas tipo-secundárias” da estrutura nativa. Adicionalmente, observou-se que aquela (taxa), independentemente do valor da ordem de contato relativo, é fortemente influenciada pelos padrões confíguracionais e suas combinações presentes na nativa. Por meio dessa premissa, foi então possível explicar de forma consistente os casos que não obedecem a pretensa relação linear entre log kf e c, levando a concluir que o logaritmo da taxa de enovelamento e a ordem de contato relativo são linearmente dependentes somente para aquelas configurações em que há uma certa quantidade equilibrada (que depende de c) de padrões estruturais, mesclando contatos efetivos de curto alcance (alto conteúdo de estruturas tipo-secundárias), com outros de longo alcance (baixo conteúdo de estruturas tipo-secundárias). Estruturas nativas que quebram este equilíbrio têm sua cinética de enovelamento afetada com respeito à reta de regressão linear ajustada para o conjunto de todas as configurações consideradas. Dessa forma, verificou-se que o mecanismo físico básico que relaciona o conteúdo de estruturas tipo-secundárias e a taxa de enovelamento, envolve o conceito de cooperatividade: se a estrutura nativa é rica em combinações de padrões estruturais ricos em contatos efetivos de curto alcance, o processo de enovelamento é mais rápido porque contatos locais são naturalmente estimulados por flutuações térmicas. / The understanding of basic principles of the protein folding problem can lead to many important applications. Although not all the involved significant aspects of this problem are known, experiments and theoretical approaches have produced important advances in its understanding. An important experimental fact has been the discovery that the logarithm of the folding rate log kf correlates linearly with global structural parameters, like the relative contact order c. In order to contribute for the understanding of folding process, the primordial goal of this work consists in to explain why certain proteins do not follow the linear behavior between log kf and c, as verified to other proteins from the same class (usually small two states proteins). For this, it was necessary to identify those topological parameters of the native structure that are important to the folding kinetic of globular protein. It was also studied how steric specificities of the aminoacids affect the protein folding process, as well how they influence the correlation between the relative contact order and the folding rate. It was employed in this study a simplified cubic lattice model, treated by Monte Carlo simulation. A set of 52 maximum compact structures, corresponding to chains of size L = 27 monomers, was used to represent the native states; these structures were chosen in such a way to represent a significant diversity of structural patterns, independently of c. Through a detailed analysis of the influence of topological parameters of the native configurations on the folding kinetic, it was concluded that the folding rate is strongly dependent of what we call here as “content of type-secondary” of the native. Additionally, it was observed that log kf is, independently of c, strongly influenced by the configurational patterns and its combinations in the native. Through this premise it was possible to consistently explain the cases that do not obey the pretense linear relation between log kf and c, leading to conclude that the logarithm of the folding rate and the relative contact order are linearly related only for those configurations in that there is a certain balanced amount of structural patterns (which depend on c) mixing short-range effective contacts (high contents of secondary-type structures) and long-range contacts (low contents of secondary-type structures). Structures that break this balance have its folding kinetic affected with respect to the linear fitting adjusted for the set of all the considered configurations. Of this form, it was verified that basic physical mechanism that relates the content of type-secondary structures and the folding rate involves the cooperativety concept: if the native structure presents combinations of structural standards rich in effective contacts of short-range, the folding process is faster because local contacts are naturally stimulated by thermal fluctuations.

"The effect of bacterial surface biopolymers on bacterial adhesion to surfaces was studied through experiments and modeling. Atomic Force Microscopy (AFM) provided the tool to measure the interaction forces between different bacterial cells and silicon nitride tips under different chemical conditions at a nanoscopic level. Two bacterial strains were considered: Pseudomonas putida KT2442 and Escherichia coli K-12 JM109. This study addressed the following issues: 1) the effect of solution ionic strength and solvent polarity on adhesion between Pseudomonas putida KT2442 and the silicon nitride AFM tip, 2) role of heterogeneity of bacterial surface biopolymers on bacterial adhesion, 3) role of lipopolysaccharides (LPS) on adhesion at three different scales: continuous, batch, and nanoscale, and 4) nature of interactions between E. coli JM109 and a model surface (silicon nitride tip). To address the first issue, formamide, water, and methanol were used to investigate the effect of polarity on surface characteristics of biopolymers on the bacterial surface while a range of salt concentrations between that of water to 1 M KCl were used to study the effect of ionic strength. The adhesion increased with decreasing polarity of the solvent, indicating that the polymers on the bacterial surface are hydrophilic in nature. The adhesion was slightly affected by ionic strength variations up to a concentration of 0.1 M KCl; this may have been due to the fact that the ionic concentration in the solution did not counterbalance the ionic concentration in the biopolymer brush on the bacterial surface. However, a dramatic increase in the adhesion magnitude was observed when the salt concentration increased above 0.1 M KCl. This transition in adhesion with ionic strength from a low to high value induced a transition in the elasticity of the bacterial surface biopolymers. The biopolymer brush layer did change from rigid to soft with increasing the ionic strength. The elasticity was quantified mainly by the use of the freely jointed chain (FJC) model. Our interest in investigating the role of heterogeneity on adhesion developed from the results of the first study. The bacterial surface polymers were thought to be different in their chemical and physical nature since they were found to span a range of segment lengths. Analyzing the adhesion forces for P. putida KT2442 showed that the bacterial surface is heterogeneous. The heterogeneity was evident on the same cell surface and between different cells from the same population. To resolve the third issue, approximately, 80% of the surface LPS of E. coli K-12 JM109 were removed by treating the cells with 100 mM ethylenediaminetetraacetic acid (EDTA). The effect of LPS removal on the adhesion of the cells to the silicon nitride tip was studied in water and phosphate buffered silane (PBS). The adhesion results from the AFM experiments were compared to batch retention experiments with glass as the substratum and column attachment experiments with columns packed with quartz sand. LPS controlled bacterial adhesion to the different surfaces in the study at three scales: batch, continuous, and nano-scale. Finally, the nature of interactions between E. coli JM109 and a model surface (silicon nitride tip) were investigated in solvents of varying polarity (formamide, water, and methanol). The Young’s modulus of elasticity for the bacterial surface was estimated by fitting of the Hertzian model to the force-indentation curves. Young’s modulus values increased as the solvent polarity decreased, indicating a stiffer bacterial surface in lower polarity solvents. The average adhesion force in each solvent was negatively correlated with the dielectric constant of the solvent, suggesting hydrophilic biopolymers. Specific and non-specific interaction forces between the AFM tip and the biopolymers were further characterized by applying a Poisson statistical analysis to the discrete adhesion data. The specific and non-specific interaction forces were the highest in methanol (-4 and -1.48 nN respectively). These values are in accordance with the high adhesion magnitude values measured with AFM in methanol. The results of my different studies emphasized the important role of AFM in studying biological interactions to different surfaces and in characterizing bacterial surface biopolymers."

In this thesis the interactions in cellulose systems havebeen studied with emphasis on surface forces and adsorptionphenomenon. The study was limited to surfaces and polymers ofinterest for papermaking. During papermaking both therunability of the paper machine as well as the properties ofthe final paper will depend on the molecular interactionsbetween the components present in the suspension. The objectiveof this work was to gain a better understanding of theseinteraction forces and how they are affected by solutionconditions like pH and electrolyte concentration. The maintechniques employed for this purpose were direct surface forcemeasurements using both the interferometric surface forceapparatus (SFA) and the atomic force microscopy (AFM)colloidal-probe technique. In addition, both electronspectroscopy for chemical analysis (ESCA) and AFM imaging wereused for surface characterisation and to study adsorptionbehaviour. Since a smooth and transparent surface is needed forsurface force studies using SFA, Langmuir-Blodgett films ofcellulose were employed. The interactions between cellulose surfacesand xylansurfaces were studied. Cellulose is the main constituent of thewood fibre. In close association with cellulose in the cellwall are the hemicelluloses, xylan being a commonhemicellulose. Steric forces were found to dominate theinteractions between these substances in aqueous solutions.Still factors like charge density, solution pH and electrolyteconcentration were important. The adhesion between cellulosesurfaces in air depended on the contact area. The effect of acationic polyelectrolyte on the forces between cellulosesurfaces and between cellulose and mineral surfaces wasinvestigated to obtain a better understanding of the effects ofcationic retention and strength additives. In the presence of acationic polyelectrolyte the forces at large distances weredominated by double-layer repulsion. Bridging attraction was insome cases observed, but the presence and magnitude of thisforce was dependent on the properties of the substratesurface. Lignin on the fibre surface affects brightness, swelling andstrength of paper. In addition, dissolved lignin interfereswith added polymers during papermaking. For this reason theadsorption of lignin on cellulose fibre surfaces was explored.The adsorption of lignin was influenced by the concentration oflignin in solution and by the electrolyte composition of thesolution. The retention of lignin was affected by the presenceof a cationic polyelectrolyte and particularly by how thepolyelectrolyte was added. The AFM imaging revealed thestructure of the adsorbed lignin, which varied with the way ofadding the polyelectrolyte, and gave an idea about theadsorption mechanism. The effect of lignin on strengthproperties of paper sheets was also evaluated. Keywords: cellulose, hemicellulose, xylan, lignin, surfaceforces, adsorption, polyelectrolyte, SFA, AFM, ESCA,Langmuir-Blodgett films, chitosan, steric forces,bridging,adhesion