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Protein (mis)folding into highly ordered, fibrillar structures, amyloid fibrils, is a hallmark of several, mainly neurodegenerative, disorders. The mechanism of this supra-molecular self-assembly reaction, as well as its relationship to protein folding are not well understood. In particular, the molecular origin of the metastability of the soluble state of proteins with respect to the aggregated states has not been clearly established. In this dissertation, it is demonstrated, that highly accurate kinetic experiments, using a novel biosensing method, can yield fundamental insight into the dynamics of proteins in the region of the free energy landscape corresponding to protein aggregation. First, a section on Method development describes the extension and elaboration of the previously established kinetic assay relying on quartz crystal microbalance measurements for the study of amyloid fibril elongation (Chapter 3). This methodology is then applied in order to study in great detail the origin of the various contributions to the free energy barriers separating the soluble state of a protein from its aggregated state. In particular, the relative importance of residual structure, hydrophobicity (Chapter 4) and electrostatic interactions (Chapter 5) for the total free energy of activation are discussed. In the last part of this thesis (Chapter 6), it is demonstrated that this biosensing method can also be used to study the binding of small molecules to amyloid fibrils, a very useful feature in the framework of the quest for potential inhibitors of amyloid formation. In addition, it is shown that Thioflavin T, to-date the most frequently employed fluorescent label molecule for bulk solution kinetic studies, can in the presence of potential amyloid inhibitor candidates be highly unreliable as a means to quantify the effect of the inhibitor on amyloid formation kinetics. In summary, the work in this thesis contributes to both the fundamental and the applied aspects of the field of protein aggregation.

Os corantes ciânicos (CC) são compostos orgânicos que possuem uma estrutura facilmente variável, permitindo obter-se as características fotofísicas desejáveis. Devido a sua alta afinidade por estruturas biológicas, baixa citotoxicidade no escuro, alta solubilidade em meio aquoso e fotoatividade os CC são considerados compostos promissores para aplicações no tratamento do câncer por terapia fotodinâmica (TFD). CC possuem uma forte tendência de se agregar em meio aquoso, que modifica suas características fotofísicas, reduzindo os rendimentos quânticos de fluorescência e do estado tripleto, diminuindo assim sua eficiência em suas aplicações como sonda fluorescente e na TFD, todavia, a agregação aumenta a eficiência da conversão da sua energia de excitação em calor, que é importante para sua aplicação na terapia por hipertermia (HT). Sendo introduzido num organismo o CC se encontra no ambiente onde ele vai interagir com sais e estruturas nano-heterogêneas (membrana celular, ácidos nucléicos etc.), interações que podem influenciar na sua agregação. Nesse trabalho investigamos o fenômeno da agregação dos CC em suas interações com sistemas nano-heterogêneos naturais (DNA) e sintéticos (micelas) em função da sua própria estrutura, da estrutura destes sistemas e da composição da solução: as concentrações do corante e do sistema nano-heterogêneo e a força iônica. Entre os CC, escolhemos como modelos a Acridina Laranja (AL) e os corantes com dois cromóforos (BCD) que se diferem pelo ângulo formado entre seus cromóforos. Utilizamos técnicas espectroscópicas estacionárias e com resolução temporal de absorção óptica, fluorescência, espalhamento ressonante e dinâmico da luz e fotólise por pulso relâmpago. Descobrimos que em soluções aquosas homogêneas os sais induzem a agregação dos CC. No caso da AL, os sais suprimem sua fluorescência pelo aumento da agregação da AL e pela formação de um exciplexo entre a AL em seu estado excitado singleto e o ânion do sal. A interação dos CC com estruturas nano-organizadas é complexa. Observamos que na interação do CC com o DNA aparecem várias espécies em equilíbrio, tais como monômeros de CC livres e ligados ao DNA, agregados de CC ligados ao DNA e agregados de DNA ligados com os monômeros de CC. A ligação da AL ao DNA reduz a probabilidade do contato da AL com outras moléculas. Contudo, na presença do DNA os sais reduzem a agregação da AL devido à redução da constante de ligação da AL com o DNA. Na presença do dodecil sulfato de sódio (SDS), observamos que em baixas concentrações este estimula a agregação do CC. O aumento da concentração de SDS induz a desagregação do CC. Identificamos que os agregados dos CC com SDS apresentam uma dinâmica que pode perdurar por diversas horas. Durante esse período os agregados trocam suas formas H e J. Investigamos uma possível aplicação prática da agregação numa terapia de HT, identificando que a agregação protege o CC da fotodecomposição e aumenta a eficiência da geração de calor. Os resultados obtidos são importantes para avaliar o potencial de aplicação do CC como fotossensibilizadores em terapia fotodinâmica, fotohipertermia e sondas fluorescentes em diagnóstico por fluorescência. / Cyanine dyes (CD) are organic compounds that have an easily variable structure, thus allowing obtain desirable photophysical characteristics. Due to their high affinity to biological structures, low cytotoxicity in the dark, high solubility in aqueous medium and photoactivity the CD are promising materials for application as photosensitizers in cancer treatment by photodynamic therapy (PDT) and as fluorescence probes in fluorescence diagnostics (FD). CD have a strong tendency to aggregate in aqueous media, which modify their photophysical characteristics, reducing its fluorescence and triplet state quantum yields, thus decreasing their efficiency in applications in PDT and FD. At the same time, aggregation increases the probability of excitation energy conversion into heat, which is important for application in hyperthermia (HT) therapy. Being introduced into organism, CD will interact with salts and nano-heterogeneous structures (cell membrane, nucleic acids etc.). These interactions can affect its aggregation. In this work we have investigated the CD aggregation phenomenon at its interactions with natural (DNA) and synthetic (micelles) nano-heterogeneous systems in function of their own structure, structure of the nano-heterogeneous system and the solution characteristics like dye and nano-heterogeneous system concentrations and ionic strength. Among CD, we have chosen as models Acridine Orange (AO) and cyanine dyes with two chromophores (BCD) that differ by the angle between chromophores. Stationary and time-resolved optical absorption, fluorescence, resonant and dynamic light scattering spectroscopies and flash photolysis were used. We have found that in homogeneous aqueous solutions salts induce the CD aggregation. In the case of AO, the salts quench the AO fluorescence by increasing its aggregation and by forming an exciplex between the AO molecule in its singlet excited state and the salt anion. Interaction of CD with nano-organized systems is complex. We observed that at CD interaction with DNA there appear several species in equilibrium, such as CD monomers free and bound to DNA, CD aggregates bound to DNA and DNA aggregates bound to CD monomers. The aggregation of DNA molecules around AO monomers reduces the probability for AO contact with other molecules. In the presence of DNA salts reduce AO aggregation due to reduction of the AO binding constant to DNA. Sodium dodecyl sulfate (SDS) in low concentrations induces CD aggregation, while higher SDS concentrations stimulate CD disaggregation. The process of CD aggregation in the presence of SDS can continue for several hours. During this period, the form of aggregates may modify from H to J or from J to H depending on the dye structure. The irradiation of dye solutions with visible light increases the solution temperature. Aggregation protected CD from photodecomposition and increased heat generation. The results obtained may help in evaluation the potential of CD as photosensitizers in photodynamic therapy, photohyperthermia and fluorescent probes in fluorescence diagnostics.

Abstract Spontaneous changes in protein systems, such as the binding of a ligand to an enzyme or receptor, are characterized by a decrease of free energy. Despite the recent developments in computing power and methodology, it remains challenging to accurately estimate free energy changes. Major issues are still concerned with the accuracy of the underlying model to describe the protein system and how well the calculation in fact emulates the behaviour of the system. This thesis is largely concerned with the quality of current free energy calculation methods as applied to protein-ligand systems. Several methodologies were employed to calculate Gibbs standard free energies of binding for a collection of protein-ligand complexes, for which experimental affinities were available. Calculations were performed using system description with different levels of accuracy and included a continuum approach, which considers the protein and the ligand at the atomic level but includes solvent as a polarizable continuum, and an all-atom approach that relies on molecular dynamics simulations. In most such applications, the effects of ionic strength are neglected. However, the severity of this approximation, in particular when calculating free energies of charged ligands, is not very clear. The issue of incorporating ionic strength in free energy calculations by means of explicit ions was investigated in greater detail and considerable attention was given to the affinities of charged peptides in the presence of explicit counter-ions. A second common approximation is concerned with the description of ligands that exhibit multiple protonation states. Because most of current methods do not model changes in the acid dissociation constants of titrating groups upon binding, protonation equilibria of such ligands are not taken into account in free energy calculations. The implications of this approximation when predicting affinities were analysed. Finally, when calculating free energies of binding, a correct description of the interactions between the protein and the ligand is of fundamental importance. However, active sites of enzymes, where strained conformations may hold a functional role, are not always accurately modelled by molecular mechanics force fields. The case of a strained planar proline in the active site of triosephosphate isomerase was investigated using an hybrid quantum mechanics/molecular mechanics method, which implies a higher level of accuracy.

Highly selective and sensitive phosphate sensors have been fabricated by constructing a solid membrane disk consisting of variable mixtures of aluminium powder (Al), aluminium phosphate (AlPO4) and powdered copper (Cu). Both binary and ternary electrode systems are produced depending on their composition. The ternary membranes exhibit greater selectivity over a wide range of concentrations. The ternary electrode with the composition 25% AlPO4, 25% Cu and 50% Al was selected as our preferred electrode. The newly fabricated ternary membrane phosphate selective electrodes exhibited linear potential response in the concentration range of 1.0 × 10−6 to 1.0 × 10−1 mol L−1. The electrodes also exhibit a fast response time of <60 s. Their detection limit is 1.0 × 10−6 mol L−1. The unique feature of the described electrodes is their ability to maintain a steady and reproducible response in the absence of an ionic strength control. The electrodes have a long lifetime and can be stored in air when not in use. The selectivity of the new phosphate selective electrodes with respect to other common ions is excellent. The results obtained provide further insight into the working principles of the newly fabricated phosphate selective electrodes. Dephosphorylation and phosphorylation reactions were monitored using the preferred phosphate selective electrode. The following reactions were studied and inferences drawn; (a) the reactions between *[{CoN4(OH)(OH2)}]2+ and *[OH(PO2O)]2- for 1:1, 2:1 and 3:1 *[{CoN4(OH)(OH2)}]2+ to *[OH(PO2O)]2- ratios.(b) the reactions between *[{CoN4(OH)(OH2)}]2+ and *[O2NC6H4PO2(O)(OH)]- for 1:1, 2:1 and 3:1 *[{CoN4(OH)(OH2)}]2+ to *[O2NC6H4PO2(O)(OH)]- ratios. (c) the reactions between *[{CoN4(OH)(OH2)}]2+ and *[(OH)2(PO2)2O]2- for 1:1, 2:1 and 3:1 [{CoN4(OH)(OH2)}]2+ to *[(OH)2(PO2)2O]2- ratios, and (d) the reactions between *[{CoN4(OH)(OH2)}]2+ and *[(OH)2(PO2)3O2]3- for the 1:1, 2:1 and 3:1 [{CoN4(OH)(OH2)}]2+ to *[(OH)2(PO2)3O2]3- ratios. Further insight into dephosphorylation and phosphorylation reactions is unravelled by the novel phosphate selective electrode monitoring. *For clarity of the complexes utilized, see chapter 4, table 4.1. KEY WORDS; Dephosphorylation, phosphorylation, ion selective electrodes, phosphate ion selective electrode, decontamination, electromotive force, potential difference, activity, concentration, selectivity coefficient, calibration, ionic strength, hydrolysis, inorganic phosphates, nitrophenylphosphate, pyrophosphate, tripolyphosphate, organophosphate esters. / Chemistry / D. Phil (Chemistry)

The Ph.D. thesis deals with the characterization of algal organic matter (AOM), which is difficult to remove in water treatment, and on the basis of AOM character, various methods for its elimination, e.g. coagulation, oxidation with subsequent coagulation and adsorption onto activated carbon are assesed. Special emphasis is placed on identifying the optimal conditions of the processes and on describing the mechanisms and interactions involved. In terms of anthropogenic micropollutants, the thesis deals with the highly topical issue of the occurrence of microplastics in water. It was found that the removal efficiency of the individual AOM components varies substantially depending on the elimination method used. The identified optimum conditions of individual methods and especially the mechanisms that apply to the removal of target substances varied widely. The non-proteinaceous fraction of AOM was removed with very low efficiency (max. 25%) by conventional coagulation even under optimized conditions (pH 6.6- 7.5 for aluminium sulfate as the coagulating agent and pH 7.5-9.0 for polyaluminium chloride) and it was given by the high content of low molecular weight (LMW) substances that are difficult to coagulate. The dominant coagulation mechanism was adsorption onto aluminium hydroxide precipitates....

Made available in DSpace on 2014-12-17T15:42:31Z (GMT). No. of bitstreams: 1 ClaudioLV.pdf: 1496908 bytes, checksum: 9da02c26d9351b21019488117fc27924 (MD5) Previous issue date: 2007-05-28 / Dispersions composed of polyelectrolyte complexes based on chitosan and poly(methacrylic acid), PMAA, were obtained by the dropping method and template polymerization. The effect of molecular weight of PMAA and ionic strength on the formation of chitosan/poly(methacrylic acid), CS/PMAA, complexes was evaluated using the dropping method. The increase in molecular weight of PMAA inhibited the formation of insoluble complexes, while the increase in ionic strength &#64257;rst favored the formation of the complex followed by inhibiting it at higher concentrations. The polyelectrolyte complexation was strongly dependent on macromolecular dimensions, both in terms of molecular weight and of coil expansion/contraction driven by polyelectrolyte effect. The resultant particles from dropping method and template polymerization were characterized as having regions with different charge densities: chitosan predominating in the core and poly(methacrylic acid) at the surface, the particles being negatively charged, as a consequence. Albumin was adsorbed on templatepolymerized CS/PMAA complexes (after crosslinking with glutardialdehyde) and pH was controlled in order to obtain two conditions: (i) adsorption of positively charged albumin, and (ii) adsorption of albumin at its isoelectric point. Adsorption isotherms and zeta potential measurements showed that albumin adsorption was controlled by hydrogen bonding/van der Waals interactions and that brushlike structures may enhance adsorption of albumin on these particles / Dispers?es formadas a partir de complexos polieletrol?ticos de quitosana e de poli(?cido metacr?lico), PMAA, foram obtidas tanto pelo m?todo de gotejamento, como pelo m?todo de polimeriza??o em molde. O efeito da massa molar do PMAA e da for?a i?nica na forma??o dos complexos de quitosana/poli(?cido metacr?lico), CS/PMAA, foi avaliado usando o m?todo de gotejamento. O aumento da massa molar do PMAA inibiu a forma??o dos complexos insol?veis, enquanto o aumento da for?a i?nica primeiramente favoreceu a forma??o dos complexos, depois a inibiu, em altas concentra??es de eletr?litos de baixa massa molar. A complexa??o dos polieletr?litos foi fortemente dependente das dimens?es macromoleculares, tanto em termos da massa molar quanto do efeito de expans?o/contra??o dos novelos, devido ao efeito polieletrol?tico. As part?culas resultantes tanto do m?todo de gotejamento, como da polimeriza??o em molde foram caracterizadas por apresentarem regi?es com diferentes densidades de carga: a quitosana predominantemente presente na regi?o central e o poli(?cido metacr?lico), na superf?cie, sendo, portanto, as part?culas carregadas negativamente. A albumina foi adsorvida nos complexos de CS/PMAA obtidos por polimeriza??o em molde (depois de sofrerem reticula??o covalente usando glutaralde?do) e o pH foi controlado a &#64257;m de se obter duas condi??es: (i) adsor??o de albumina carregada positivamente e (ii) adsor??o de albumina em seu ponto isoel?trico. As isotermas de adsor??o e as medidas de potencial zeta mostraram que a adsor??o da albumina foi controlada por liga??es de hidrog?nio/intera??es de van der Waals e que as estruturas em forma de escova puderam aumentar a adsor??o da albumina nessas part?culas

No description available.

Membrane filtration processes are widely utilized across different industrial sectors for biological and environmental separations. Examples of the former are sterile filtration and protein fractionation via microfiltration (MF) and ultrafiltration (UF) while drinking water treatment, tertiary treatment of wastewater, water reuse and desalination via MF, UF, nanofiltration (NF) and reverse-osmosis (RO) are examples of the latter. A common misconception is that the performance of membrane separation is solely dependent on the membrane pore size, whereas a multitude of parameters including solution conditions, solute concentration, presence of specific ions, hydrodynamic conditions, membrane structure and surface properties can significantly influence the separation performance and the membrane’s fouling propensity. The conventional approach for studying filtration performance is to use a single lab- or pilot-scale module and perform numerous experiments in a sequential manner which is both time-consuming and requires large amounts of material. Alternatively, high-throughput (HT) techniques, defined as the miniaturized version of conventional unit operations which allow for multiple experiments to be run in parallel and require a small amount of sample, can be employed. There is a growing interest in the use of HT techniques to speed up the testing and optimization of membrane-based separations. In this work, different HT screening approaches are developed and utilized for the evaluation and optimization of filtration performance using flat-sheet and hollow-fiber (HF) membranes used in biological and environmental separations. The effects of various process factors were evaluated on the separation of different biomolecules by combining a HT filtration method using flat-sheet UF membranes and design-of-experiments methods. Additionally, a novel HT platform was introduced for multi-modal (constant transmembrane pressure vs. constant flux) testing of flat-sheet membranes used in bio-separations. Furthermore, the first-ever HT modules for parallel testing of HF membranes were developed for rapid fouling tests as well as extended filtration evaluation experiments. The usefulness of the modules was demonstrated by evaluating the filtration performance of different foulants under various operating conditions as well as running surface modification experiments. The techniques described herein can be employed for rapid determination of the optimal combination of conditions that result in the best filtration performance for different membrane separation applications and thus eliminate the need to perform numerous conventional lab-scale tests. Overall, more than 250 filtration tests and 350 hydraulic permeability measurements were performed and analyzed using the HT platforms developed in this thesis. / Thesis / Doctor of Philosophy (PhD) / Membrane filtration is widely used as a key separation process in different industries. For example, microfiltration (MF) and ultrafiltration (UF) are used for sterilization and purification of bio-products. Furthermore, MF, UF and reverse-osmosis (RO) are used for drinking water and wastewater treatment. A common misconception is that membrane filtration is a process solely based on the pore size of the membrane whereas numerous factors can significantly affect the performance. Conventionally, a large number of lab- or full-scale experiments are performed to find the optimum operating conditions for each filtration process. High-throughput (HT) techniques are powerful methods to accelerate the pace of process optimization—they allow for multiple experiments to be run in parallel and require smaller amounts of sample. This thesis focuses on the development of different HT techniques that require a minimal amount of sample for parallel testing and optimization of membrane filtration processes with applications in environmental and biological separations. The introduced techniques can reduce the amount of sample used in each test between 10-50 times and accelerate process development and optimization by running parallel tests.