Esta tese apresenta resultados da caracterização estrutural de três sistemas de classes diferentes por meio, principalmente, da modelagem de dados de espalhamento de raios X a baixos ângulos (SAXS). No sistema surfactante-surfactante as micelas mistas são formadas por dodecil sulfato de sódio (SDS) e um dos surfactantes da série Tween (Tween 20, 40, 60 e 80). A modelagem adotada impôs vínculos moleculares uma vez que os dados de SAXS estavam em escala absoluta. Isso reduziu a ambiguidade nos valores dos parâmetros ajustáveis e permitiu verificar que os dados de SAXS são satisfatoriamente descritos considerando-se que as micelas são elipsoides de revolução core-shell, podendo ser prolatas ou oblatas dependendo do tipo de Tween empregado. Para o sistema proteína-surfactante, a metodologia experimental utilizada permitiu um estudo estrutural e termodinâmico dos complexos formados por meio do acompanhamento do processo de ligação de SDS às proteínas lisozima e alfa-lactalbumina. A técnica de calorimetria de titulação isotérmica (ITC) forneceu um panorama geral sobre a desnaturação proteica e norteou os experimentos seguintes de SAXS e dicroísmo circular (CD). Por meio da análise dos dados de CD concluiu-se que as proteínas perdem quase totalmente sua estrutura terciária, mas não a secundária (esse estado é conhecido como molten globule). Já a modelagem de SAXS em escala absoluta com imposição de vínculos permitiu concluir que os complexos proteína-surfactante podem ser entendidos micelas decoradas, isto é, a proteína está distribuída sobre a superfície de uma micela de SDS. Esse modelo, aliado à abordagem experimental empregada, permitiu a caracterização sistemática dos complexos durante a desnaturação proteica. Em relação ao sistema lipossomas-(bio)ativos, a análise dos dados de SAXS por meio do Método da Deconvolução Gaussiana usando bicamadas simétricas, para os sistemas Phospholipon 90H curcumina/vitamina D3 e dipalmitoilfosfatidilcolina de soja (DPPC) ácido láurico (LA), e assimétricas, para o caso fosfatidilcolina de ovo (EPC) sumatriptano (SMT), permitiu acompanhar mudanças na estrutura das mesmas ocasionadas pela presença dos (bio)ativos. Verificou-se em todos os casos que a espessura da bicamada se mantém praticamente constante. A flexibilidade membranar aumenta, seja em função da temperatura, para o sistema Phospholipon 90H curcumina/ vitamina D3, seja em função da concentração de (bio)ativos, como nos outros dois casos. Para estes, concluiu-se pela análise dos perfis de contraste de densidade eletrônica que os (bio)ativos interagem preferencialmente com as cabeças polares dos fosfolipídeos que constituem os lipossomas, possivelmente causando defeitos topológicos nessa região e ocasionando o aumento da flexibilidade membranar mencionada antes. LA, diferentemente de SMT, induz uma transição de lipossomas multilamelares para unilamelares, e esse fenômeno é grandemente influenciado pelo pH do meio. / This thesis presents a structural characterization of three systems of different classes through, mainly, the small angle X-ray scattering (SAXS) technique. In the surfactant-surfactant system the mixed micelles are composed by sodium dodecyl sulfate (SDS) and one of the Tween surfactants (Tween 20, 40, 60 and 80). The adopted modeling imposed molecular constraints since the SAXS data was in absolute scale. This procedure reduced the ambiguity in the values of the adjustable parameters and allowed to verify that SAXS data is satisfactorily described considering that the micelles are core-shell revolution ellipsoids which can be prolate or oblate depending on the type of Tween used in the micelle. For the protein-surfactant system, the applied experimental methodology allowed a structural and thermodynamic study of the complexes formed through monitoring the binding of SDS to the proteins lysozyme and alpha-lactalbumin. Isothermal titration calorimetry (ITC) technique provided an overview of proteic denaturation and guided the following experiments of SAXS and circular dichroism (CD). From CD data analysis it was concluded that the proteins lose almost totally their tertiary structure, but not the secondary one (this state is known as \"molten globule\"). On the other hand, SAXS data modeling in absolute scale with molecular constraints leaded to the conclusion that protein-surfactant complexes can be considered decorated micelles in which the protein is distributed over a SDS micelle surface. This model, combined to the adopted experimental procedure, allowed the systematic characterization of the complexes along the protein denaturation. In the liposome-(bio)actives system, the SAXS data analysis using the Gaussian Deconvolution Method assuming symmetric bilayers, for the systems Phospholipon 90H curcumin /vitamin D3 and soybean dipalmitoyl phosphatidylcholine (DPPC) lauric acid (LA), and asymmetric bilayers, in the case of egg phosphatidylcholine (EPC) sumatriptan (SMT) system, allowed to follow changes in the lipid bilayer structure induced by the presence of the (bio)actives. It has been found, in all cases, that the bilayer thickness remains approximately. Membrane flexibility increases, depending on the temperature, for the Phospholipon 90H curcumin /vitamin D3 system, or as a function of the (bio)actives concentrations, as in the other two cases. For those, it was concluded, by the analysis of electron density contrast profiles, that the (bio)actives preferentially interact with the polar heads of the phospholipids forming the liposomes, possibly causing topological defects in that region and leading the membrane flexibility increase. LA, unlike SMT, induces a transition from multilamellar to unilamellar liposomes, and this phenomenon is greatly influenced by the pH of the medium.

L'objet de cette étude est de mettre au point une méthode d'analyse et de modélisation des propriétés de complexation et d'acidité de la matière organique naturelle (MON) la plus rigoureuse possible, en représentant ses interactions par des paramètres fiables, et en s'affranchissant de la structure exacte des composés organiques la constituant. La MON a été définie à partir d'un modèle chimique constitué de quasi-particules reproduisant les interactions non-spécifiques de la MON vis-à-vis des cations, et les interactions spécifiques à un seul métal. Le logiciel PROSECE, spécifiquement développé à cet effet, permet de calculer la spéciation chimique et d'optimiser un grand nombre de paramètres de complexation à partir de la modélisation de données expérimentales. Comparé à d'autres traitements de données, PROSECE s'est montré nettement plus approprié, d'autant plus lorsqu'il est couplé à une technique d'ajouts logarithmiques. Les interactions MON-métaux traces ont été analysées par DPASV, sur la plus large fenêtre analytique possible. Un stand de mesure a été spécialement élaboré afin d'automatiser les titrations logarithmiques. Les interactions MON-proton ont été analysées par titration acido-basique. Une caractérisation simultanée des interactions entre une MON standard, l'acide fulvique Suwannee River, le cadmium, le plomb et le proton a été réalisée. Les propriétés d'acidité d'échantillons prélevés sur La Seine au cours d'une année ont été étudiées et reliées à différents facteurs biogéochimiques. Une concentration minimale de 0.04 meq en sites acides a été définie comme limite en dessous de laquelle l'optimisation des propriétés d'acidité d'une MON devient incorrecte. PROSECE a été également utilisé pour modéliser des expériences de quenching de fluorescence, rendant plus précise leur analyse. L'analyse des propriétés de MON marines a été initiée. Du fait de la complexité du milieu, la caractérisation des propriétés de complexation est plus difficile, nécessitant quelques adaptations analytiques.

This thesis summarizes the results of studies on two aspects of nucleic acids. Chemically modified antisense oligonucleotides (AONs) have been evaluated with regards to their suitability for mRNA targeting in an antisense approach (Paper I – III). The chemically modified nucleotidic units 2'-O-Me-T, 2'-O-MOE-T, oxetane-T, LNA-T, azetidine-T, aza-ENA-T, carbocyclic-ENA-T and carbocyclic-LNA-T were incorporated into 15-mer AONs and targeted against a 15-mer RNA chosen from the coding region of SV-40 large T antigen. The comparative study showed that a single modified nucleotide in the AON with North-East locked sugar (oxetane-T and azetidine-T) lowered the affinity for the complementary RNA whereas North locked sugars (LNA-T, aza-ENA-T, carbocyclic-ENA-T, and carbocyclic-LNA-T) significantly improved the affinity. A comparative RNase H digestion study showed that modifications of the same type (North-East type or North type) in different sequences gave rise to similar cleavage patterns. Determination of the Michaelis-Menten parameters by kinetic experiments showed that the modified AONs recruit RNase H resulting in enhanced turnover numbers (kcat) although with weaker enzyme-substrate binding (1/Km) compared to the unmodified AON. The modified AONs were also evaluated with regards to resistance towards snake venom phosphodiesterase and human serum to estimate their stability toward exonucleases. The aza-ENA-T and carbocyclic-ENA-T modified AONs showed improved stability compared to all other modified AONs. In general, the modified AONs with North type nucleotides (except LNA-T) were found to be superior to the North-East type as they showed improved target affinity, comparable RNase H recruitment capability and improved exonuclease stability. The second aspect studied in this thesis is based on physicochemical studies of short RNA molecules utilizing NMR based pH titration and alkaline hydrolysis reactions (Paper IV – V). The NMR based (1H and 31P) pH titration studies revealed the effect of guaninyl ion formation, propagated electrostatically through a single stranded chain in a sequence dependent manner. The non-identical electronic character of the internucleotidic phosphodiesters was further verified by alkaline hydrolysis experiments. The internucleotidic phosphodiesters, which were influenced by guaninyl ion formation, were hydrolyzed at a faster rate than those sequences where such guaninyl ion formation was prevented by replacing G with N1-Me-G.

This thesis presents some new techniques that were developedfor continuous chemical analysis of a paper furnish. First, ageneral background is presented, covering topics from theorigins of papermaking to present day. A short introduction topapermaking chemistry and a variety of presently availableon-line chemical analysers and measurement strategies are alsodiscussed. A method is described for the continuous fractionation of apaper furnish containing coarse fibres (&gt;10µm) in orderto obtain a sample for analytical purposes (Paper I). Aconsistent sample, containing a representative fraction of thedissolved and colloidal substances (DCS) present in the bulkfurnish, was achieved by preventing cake formation on a filtersurface. A combination of turbulent flow above a membranefilter, while continuously withdrawing a relatively low samplevolume, were key factors in the prevention of filterfouling. For the continuous flow-extraction of DCS, a technique isdescribed whereby the extracting solvent was injected at a highvelocity into a continuous flow of analyte (Paper II).Comparison with conventional flow extraction showed anextraction enhancement of up to 9 times for colloidaltriglycerides. To achieve a continuous determination of chemicalsubstances, a real-time fully automated colorimetric titrationapparatus was developed (Paper III&amp;IV). This was achievedby using a series of micro-machined mixing channels for thecontinuous flow of analyte, with a sequence of detection unitsand titrant addition points along the flowpath (Paper III). Afuzzy logiccontroller was implemented to continuously adaptfor changes in the sample concentration, providing thepossibility of titrating over two orders of magnitude in sampleconcentration with minimal loss of accuracy (Paper IV). Also, a system is presented whereby the filtration apparatus(Paper I) is combined with the titration device (Paper III&amp;IV) in order to continuously determine total charge (orcolloidal charge) of a paper furnish in real-time (Paper V).This was achieved by utilising a back-titration approach andselected examples are presented showing the dynamicinteractions between wood fibres and polyelectrolyte adsorptionat various conditions of pH and polyelectrolyte molecularweight. Finally, some suggestions for a more comprehensive wet-endchemical monitoring platform are discussed and the role of thepresent work in evaluated in this context. <b>Keywords:</b>Chemical monitoring, continuous flowextraction, cross-flow filtration, dissolved and colloidalcomponents, fuzzy-logic control, on-line system, pitchanalysis, polyelectrolyte titration, process control, samplework-up, titrimetric analysis.

No description available.

Glyphosate (N-(phosphonomethyl)glycine, PMG, H3L) is a widely used organophosphorous herbicide. It interacts with metal ions and mineral surfaces, which may affect its mobility, degradation and bioavailability in the environment. However, these interactions are far from fully understood. This thesis is a summary of five papers discussing the complexation of PMG with metal ions in aqueous solution and the adsorption of PMG and/or Cd(II) on different mineral surfaces. The complexation of PMG with the metals Cd(II) or Al(III) in aqueous solution was investigated with macroscopic and molecular scale techniques. Potentiometric titration data were combined with EXAFS, ATR-FTIR and NMR spectroscopic data to generate solution equilibrium models. In the PMG-Cd(II) system, only mononuclear complexes were formed, while both mono and binuclear complexes were observed in the PMG-Al(III) system. EXAFS, ATR-FTIR, and XPS measurements showed that PMG adsorbs to the surfaces of goethite (α-FeOOH), aged γ-alumina (γ-Al2O3) and manganite (γ-MnOOH) through one oxygen of its phosphonate group to singly-coordinated surface sites. Surface complexation models consistent with these spectroscopic results were fit to adsorption data using the 1pK reaction formalism. Electrostatic effects were accounted using either the Extended Constant Capacitance Model (ECCM) or the Basic Stern Model (BSM), and the charge of the surface complexes was distributed over the different planes. The formation of the surface complexes was described according to the following reactions: ≡MeOH(0.5-) + H3L &lt;=&gt; ≡MeHL(1.5-) + H2O + H+ ≡MeOH(0.5-) + H3L &lt;=&gt; ≡MeL(2.5-) + H2O + 2H+ The coadsorption of PMG and Cd(II) on the surfaces of goethite and manganite results in the formation of ternary mineral-PMG-Cd(II) surface complexes, as suggested from EXAFS results. Previous EXAFS measurements have also established the coordination geometries for the binary goethite-Cd(II) and manganite-Cd(II) surface complexes. In addition to the surface reactions in the binary mineral-Cd(II) and mineral-PMG systems, a single ternary complex with the stoichiometry ≡MeLCd(OH)(1.5-) was sufficient to explain coadsorption data: ≡MeOH(0.5-) + H3L + Cd2+ &lt;=&gt; ≡MeLCd(OH)(1.5-) + 3H+ It was concluded that the affinity of PMG for the three mineral systems decreases within the series: goethite &gt; aged γ-Al2O3 &gt; manganite. The formation of the ternary surface complex is more significant on goethite surfaces than on manganite surfaces.

The surface charges of cellulosic fibers contribute to several papermaking operations that influence the manufacture and final properties of paper. This thesis investigates the effect of the surface charges on wet-end chemistry, e.g. through the interaction of cationic polyelectrolytes with the fiber surface charges, and on the network strength of pulp suspensions. The polyelectrolyte titration method was used to investigate the interaction of the fiber charges with cationic polyelectrolytes. Techniques were developed to fluorescent label the adsorbing cationic polyelectrolyte in order to visualize the adsorption behavior. Fluorescent confocal laser scanning microscopy (CLSM) was used to determine the extent to which the cationic polyelectrolyte adsorbs into the porous fiber wall. It was shown that the polyelectrolyte charge density limits the adsorption to the surface under electrolyte-free conditions. Adsorption into the fiber wall only occurs for two conditions: 1) if the molecular mass is sufficiently low or 2) the electrolyte concentration is high enough to screen the charges along the polyelectrolyte backbone but not the interactions between the polyelectrolyte and the fiber charges. Aside from the polyelectrolyte properties, the fiber charge density contributes to the adsorption behavior of cationic polyelectrolytes. The fiber charge profile was altered by bulk and surface carboxymethylation. The electrolyte concentration at which a deviation from 1:1 stoichiometry occurs was shown to be dependent on the amount of surface charges, such that the deviation in stoichiometry occurs at a higher electrolyte concentration for pulps having a higher surface charge. A hypothesis was developed to test the conditions at which the deviation in adsorption stoichiometry occurs, which was defined as the critical electrolyte concentration (CEC). It was found that the CEC corresponded to the electrolyte concentration at which the distance between the fiber charges was on the order of the Debye length. Electron spectroscopy for chemical analysis (ESCA) was used as an independent calibration procedure to validate for which a 1:1 stoichiometry occurs. The analysis with ESCA agreed well with the polyelectrolyte titration method for measurement of fiber surface charges. When measured under appropriate conditions, i.e. electrolyte concentration and molecular properties, the fiber surface charge can accurately be measured by the polyelectrolyte titration method. The charge profiles of various pulp types and treatments were also examined. Having been established as a valid technique, the polyelectrolyte titration method was again used to measure the surface charge while conductometric titration was used to measure the total charge content. The amount of bulk and surface charges vary depending on the pulping method and type of wood, although the ratio between the bulk and surface charge (i.e. the charge ratio) is similar for chemical pulps. The mechanical pulp has a higher charge ratio because it contains more fines material than chemical pulp. Bleaching of the chemical pulp decreases the amount of bulk and surface charges, although the charge ratio remains essentially constant. However, methods such as beating or carboxymethyl cellulose (CMC) grafting are available to increase the charge ratio. The effect of the charge profile on fiber-fiber interactions was studied on both a microscopic and macroscopic level. Colloidal probe microscopy (CPM) was used to investigate the microscopic interactions between two cellulose surfaces. Cellulose surfaces, prepared by spin-coating a dissolving pulp onto silica, were used to model the fiber surface, which is too rough for surface force measurements. The charge density of the model surface was increased by CMC grafting. Results showed that increasing the surface charge density created large electrosteric repulsions, due to CMC the chains protruding out from the surface. These interactions on the microscopic scale affect the fiber network strength, which was measured with a parallel plate rheometer. When the repulsion is increased between the fibers, caused by the increase in the surface charge, fiber flocs break apart more easily due to a reduced friction between the fiber surfaces. The forces acting on the fiber network can also be mechanical in origin. The fiber length and flexibility were altered in order to study the influence of mechanical surface linking and elastic fiber bending on the fiber network strength. Using the storage modulus (G’0) as a measure of fiber network strength, longer fibers were found to create a stronger network due to an increased amount of fiber contacts. Flexible fibers have a lower network strength than stiff fibers because the fibers come to rest in a less strained position such that the the influence of elastic fiber bending on the fiber network strength is predominant. / QC 20100831

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.

The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co-factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non-covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase.

<p>This thesis presents some new techniques that were developedfor continuous chemical analysis of a paper furnish. First, ageneral background is presented, covering topics from theorigins of papermaking to present day. A short introduction topapermaking chemistry and a variety of presently availableon-line chemical analysers and measurement strategies are alsodiscussed.</p><p>A method is described for the continuous fractionation of apaper furnish containing coarse fibres (>10µm) in orderto obtain a sample for analytical purposes (Paper I). Aconsistent sample, containing a representative fraction of thedissolved and colloidal substances (DCS) present in the bulkfurnish, was achieved by preventing cake formation on a filtersurface. A combination of turbulent flow above a membranefilter, while continuously withdrawing a relatively low samplevolume, were key factors in the prevention of filterfouling.</p><p>For the continuous flow-extraction of DCS, a technique isdescribed whereby the extracting solvent was injected at a highvelocity into a continuous flow of analyte (Paper II).Comparison with conventional flow extraction showed anextraction enhancement of up to 9 times for colloidaltriglycerides.</p><p>To achieve a continuous determination of chemicalsubstances, a real-time fully automated colorimetric titrationapparatus was developed (Paper III&IV). This was achievedby using a series of micro-machined mixing channels for thecontinuous flow of analyte, with a sequence of detection unitsand titrant addition points along the flowpath (Paper III). Afuzzy logiccontroller was implemented to continuously adaptfor changes in the sample concentration, providing thepossibility of titrating over two orders of magnitude in sampleconcentration with minimal loss of accuracy (Paper IV).</p><p>Also, a system is presented whereby the filtration apparatus(Paper I) is combined with the titration device (Paper III&IV) in order to continuously determine total charge (orcolloidal charge) of a paper furnish in real-time (Paper V).This was achieved by utilising a back-titration approach andselected examples are presented showing the dynamicinteractions between wood fibres and polyelectrolyte adsorptionat various conditions of pH and polyelectrolyte molecularweight.</p><p>Finally, some suggestions for a more comprehensive wet-endchemical monitoring platform are discussed and the role of thepresent work in evaluated in this context.</p><p><b>Keywords:</b>Chemical monitoring, continuous flowextraction, cross-flow filtration, dissolved and colloidalcomponents, fuzzy-logic control, on-line system, pitchanalysis, polyelectrolyte titration, process control, samplework-up, titrimetric analysis.</p>