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Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, 2015. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2016-05-06T15:01:10Z No. of bitstreams: 1 2015_KarolyneVileladeOliveira.pdf: 4124843 bytes, checksum: a896d190db2a100ab397386387efb480 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-05-23T18:18:59Z (GMT) No. of bitstreams: 1 2015_KarolyneVileladeOliveira.pdf: 4124843 bytes, checksum: a896d190db2a100ab397386387efb480 (MD5) / Made available in DSpace on 2016-05-23T18:18:59Z (GMT). No. of bitstreams: 1 2015_KarolyneVileladeOliveira.pdf: 4124843 bytes, checksum: a896d190db2a100ab397386387efb480 (MD5) / Neste trabalho foram preparados dois substratos com atividade SERS (surface-enhanced Raman scattering), baseados na dispersão de nanopartículas de prata em solução aquosa (AgNPs/Coloide) e em gel de agarose (AgNPs/Agar). Esses substratos foram caracterizados por MET e espectroscopia UV-vis. Ambos os substratos foram também investigados pelo efeito SERS, registrando-se 100 espectros em triplicata empregando o modo de mapeamento Raman numa região de 20x20 µm. Os diâmetros médios das AgNPs nas amostras de AgNPs/Coloide e AgNPs/Agar foram de 8 nm e 19 nm, respectivamente. Os espectros UV-vis desses materiais revelam que as AgNPs/Agar apresentam ressonâncias de plasmon de superfície na região de 600 nm. A adsorção da rodamina 6G (R6G) foi investigada para ambos os substratos, para concentrações entre 10-8 M e 10-5 M, por excitação SERS em 632,8 nm. As intensidades SERS da R6G nas AgNPs/Agar foram ca. 10 vezes mais fortes que aquelas apresentadas para a R6G adsorvida nas AgNPs/Coloide. As isotermas de adsorção obtidas para ambos os substratos foram bem ajustadas a isotermas de Langmuir com constantes de adsorção e energias livres de Gibbs de adsorção semelhantes, características de adsorção química. As AgNPs/Agar também foram investigadas como um substrato SERS-ativo para a adsorção de azul de metileno (MB), cristal violeta (CV) e benzotriazolato (BTA). Os resultados SERRS para o MB mostraram sinais de monômeros e dímeros de MB. Os resultados também mostraram que R6G e CV adsorvem na superfície das AgNPs/Agar formando pares iônicos com os íons cloreto adsorvidos usados para promover agregação, enquanto que o MB e BTA adsorvem quimicamente na superfície das AgNPs formando ligações Ag-N. Curvas de intensidade SERS vs. concentração do adsorbato em solução foram usadas para obter isotermas de adsorção. Em todos os sistemas investigados os dados experimentais apresentaram razoável ajuste para isotermas de Langmuir com constante de adsorção e energia livre de Gibbs de adsorção compatível com adsorção química (< -30 kJ.mol-1). Os resultados (SERS/SERRS) para R6G, MB e CV, em concentração de 10-8 M, adsorvidos em AgNPs/Agar mostraram que os sinais SERS/SERRS de moléculas únicas desses adsorbatos foram observados, mostrando que o material AgNPs/Agar obtido é um substrato adequado para a investigação SERS de adsorbatos em concentrações muito baixas. / Two different SERS (surface-enhanced Raman scattering) substrates have been prepared based on silver nanoparticles (AgNPs) dispersed in water solution (AgNPs/Colloid) and in agarose gel (AgNPs/Agar). The materials were characterized by TEM and UV-vis spectroscopy. Both substrates were also investigated by SERS by recording 100 spectra in triplicates, using the Raman mapping procedure in an area of 20x20 µm. The mean diameters of the AgNPs in these substrates were 8 nm and 19 nm, respectively. The UV-vis spectra of these materials show that the AgNPs/Agar present AgNPs aggregates with surface plasmon resonances in the region of 600 nm. The adsorption of Rhodamine 6G (R6G) at concentrations ranging from 10-8 M to 10-5 M on both substrates was investigated by SERS excitation at 632.8 nm. The SERS intensities of R6G on the AgNPs/Agar were ca. 10 times stronger than those displayed for R6G adsorbed on the AgNPs/Colloid. Adsorption isotherms obtained for both substrates were well fitted to Langmuir isotherms with similar adsorption constants and free Gibbs adsorption energy, characteristic of chemical adsorption. The AgNPs/Agar was further investigated as a SERS-active substrate for the adsorption of Methylene Blue (MB), Crystal Violet (CV) and benzotriazolate (BTA). The SERRS results for MB have shown signals from MB monomers and dimers. The results have also shown that R6G and CV adsorb on the AgNPs/Agar surface forming ion-pairs with the adsorbed chloride ions used to promote the AgNPs aggregation, while MB and BTA adsorb chemically on the AgNPs surface forming Ag-N bonds. Curves of SERS intensity vsadsorbate concentration in solution were used to obtain adsorption isotherms. In all investigated systems the experimental data presented reasonable fitting to Langmuir isotherm with adsorption constants and free Gibbs adsorption energy compatible with chemical adsorption (< -30 kJ.mol-1). The results (SERS/SERRS) for R6G, MB and CV at 10-8 M concentration adsorbed on AgNPs/Agar have shown that SERS/SERRS signals from single molecules of these adsorbates were observed, showing that the AgNPs/Agar material obtained is a suitable substrate for SERS investigation of adsorbates at very low concentrations.

The new field of protein-based nano-technology takes advantage of the complex interactions between proteins to form unique structures with properties that cannot be achieved with traditional components. Microtubules (MTs), self assembled proteinaceous hollow filaments, offer promise in the development of MT-based nano-systems. The compelling need for the controlled assembly of 3D MT arrays is the fundamental motivation for the first part of this research. We report on the morphology of MTs grown in a crowded environment in the form of high viscosity fluids containing agarose and a novel process that enables the assembly of MTs supported by gel-based 3D scaffolds. Our research on MTs and their interaction with other molecules lead us to discover extraordinary spherulitic structures that changed the course of the project. The novel subject situate us into a complicated dilemma that question the nature of MT asters reported in experiments carried out in cells. The second part of this research is focused in the crystallization ofTaxol, a MT stabilizing molecule used as anti-cancer drug. It was confirmed via fluorescent and differential interference contrast microscopy that Taxol crystals can be decorated with fluorescent proteins and fluorochromes without perturbing their morphology. We used theoretical calculations to further investigate Taxol-fluorescent agent interactions. Furthermore, the crystallization of Taxol was studied in pure water, aqueous solutions containing tubulin proteins and tubulin-containing agarose gels. We demonstrated that tubulin is able to heterogeneously nucleate Taxol spherulites. To explain the formation of tubulin-Taxol nuclei a new, secondary Taxol-binding site within the tubulin heterodimer is suggested. Results presented in this work are important for in vivo and in vitro microtubule studies due to the possibility of mistaking these Taxol spherulites for microtubule asters. Thus, we are confirming the need for careful interpretation of fluorescence microscopy observations of MT structures when large concentrations of Taxol are used as stabilizing agent in cells.

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This diploma thesis deals with microrheology measured via the fluorescence correlation spectroscopy. As microrheological probes, fluorescently marked nanoparticles of 5 sizes in the range of 10-100 nm, were used. The particles had been immersed in a variety of concentrated glycerol solutions and agarose gels of different concentrations, and the FCS measurement revealed a diffusion coefficient of individual particles in each environment. Based on the coefficient, the viscosity of the glycerol needed to stop the particles could be determined. Particles of 10 nm size were not stopped even by the 100 wt% glycerol. In the case of the agarose gels, a combination of higher agarose concentration and larger particles resulted in an increase in the diffusion coefficient to an unlikely high value. This was caused probably by an agarose autofluorescence and the value indicates stopping of the particles in the given agarose gel. Later, the data acquired by the FCS measurement were converted to MSD curves using MATLAB software. The thesis discusses the influence of the experimental parameters on the shape of the MSD curve. The results showed that the number of particles and autocorrelation function have the most significant effect.

This Diploma thesis deals with optimization of the hydrogel preparation with various additions of substances affecting adhesion, studying the adhesion of prepared hydrogel systems and modifying their adhesion properties. Prepared hydrogels were agarose, alginate and gellan polymer systems with additions of acrylic acid, acrylamide, calcium chloride and Tween 20. The experimental part of the diploma thesis focuses first on optimizing the preparation of individual hydrogels, from which, according to certain criteria, suitable representatives were selected to study adhesion and its controlled modification. Agarose and gellan hydrogels were selected as suitable representatives, which were then used in rheological measurements. Based on these measurements, the effect of the adhesive force and the adhesive work of individual materials was evaluated. It was found that the best adhesion properties of the materials used are unadjusted hydrogels, in the case of hydrogels with the addition of another substance, a negative effect on adhesion was observed, when the adhesion force was reduced and also the adhesion work was reduced.

The purpose of this experiment is to study as a model the gelation phase transition of agarose solutions with transverse relaxation (T2) quantitative magnetic resonance imaging (qMRI). The focus is on the reduction of T2 of agarose solution upon gelation. The sol-to-gel phase transition of agarose may provide a useful and controllable experimental model of tissue formation. Furthermore, it may provide the basis for exact mathematical models useful for understanding the much reduced transverse relaxation times (T2) observed in solid tissues relative to simple liquids. In this context, the purpose of this work was to monitor dynamically with T2 quantitative MRI the liquid-to-gel phase transition of pure agarose as a function of gel concentration. Samples of agarose at various concentrations were allowed to cool down while scanning dynamically with T2 qMRI, 32 x 10milliseconds (ms) echoes, CarrPurcell-Meiboom-Gill (CPMG), 3Tesla.T2 versus; (temperature).curves of each agarose solution show a distinct phase transition region characterized by a sharp T2 reduction. Four agarose solutions were sequentially prepared by dissolving agarose powder in distilled water at concentrations of 1%, 2%, 3%, and 4% by weight/volume. Immediately after preparation and boiling at 98°C, each liquid agarose solution was poured into a plastic container and scanned dynamically at 3.0T as it cooled down with a whole body MRI scanner (Achieva, Philips Medical Systems, Cleveland, OH). A single axial slice multi spin echo CPMG pulse sequence with the following parameters was used: 32 echoes, 10ms echo spacing, 1.5s repetition time (TR), 160 x 160 matrix size, and 2 SENSE factor. The time per dynamic scan was 1minute. The DICOM images were further processed with an adaptive T2 qMRI algorithm programed in Mathcad (Parametric Technology Corporation, Needham, MA) whereby the number of echoes used in the semi-logarithmic linear regression varies automatically from pixel to pixel depending on noise level. The T2 values of agarose gels have been measured during the entire gelation phase transition process at four different concentrations. The T2 versus time (temperature) curves of all the four concentrations shows a rapid drop at about 24 minutes (T~40°C) at which time the gelation phase transition begins. At all temperatures, T2 decreases as a function of increasing agarose concentration. The data shows similar behaviors for all concentrations with a phase transition characterized by a drastic drop in T2 occurring while the temperature drops by approximately 8°C. These results may be useful for testing theoretical models of the Nuclear Magnetic Resonance (NMR) T2 relaxation properties during tissue formation. Quantitative magnetic resonance imaging (qMRI) differs sharply from conventional directly acquired MRI in that objective measures [such as the trio of the basis MR properties: longitudinal relaxation (T1), T2 and Proton Density (PD)] are used for analysis as well as further post-processing rather than relative signal intensities. Q-MRI portrays the spatial distribution of absolute biophysical parameter measurements on a pixel-by-pixel basis; Kevin J. Chang et al 2005

La cellulose est l’une des bio-ressources les plus abondantes sur terre ; elle forme des microfibrilles où alternent régions cristallines, de taille nanométrique, et régions amorphes. L’utilisation des nanocristallites de cellulose, appelés nanowhiskers, comme renforts dans des composites à matrice polymère thermoplastique a fait l’objet de nombreuses publications. En revanche, l’utilisation de nanowhiskers comme éléments structurants d’hydrogels n’a pas été vraiment explorée, en dépit d’un intérêt potentiel pour la formulation d’hydrogels verts innovants. L’objectif de la thèse était d’étudier les relations entre état structural à différentes échelles et propriétés rhéologiques de systèmes hydrocolloïdaux constitués d’hydrogels d’agarose chargés de nanowhiskers de cellulose issus de tunicier, un animal marin. Les travaux expérimentaux menés au cours de cette thèse ont montré que l’effet renfort apporté par les nanowhiskers à la matrice agarose pouvait être marqué, et qu’il pouvait être modulé en faisant varier la densité de charges électriques à la surface des nanowhiskers. Aux fractions volumiques étudiées, inférieures à 0,2%, les résultats ont montré que les nanowhiskers ne percolaient pas et l’effet renfort a été attribué à des modifications topologiques du réseau d’agarose, et à un transfert des contraintes efficace entre la matrice et les charges, qui interagissent via des liaisons hydrogène. Les résultats obtenus au cours de cette thèse permettent d’améliorer la connaissance des mécanismes qui gouvernent les propriétés renfort apportées par l’ajout de nanocharges cellulosiques à une matrice hydrogel, et donnent des pistes réalistes pour une formulation maîtrisée d’hydrogels verts innovants ayant de bonnes propriétés mécaniques. / Cellulose is the most abundant organic compound on Earth; it is composed of microfibrils, containing nanocrystalline regions, and amorphous regions. Cellulose nanocrystallites, called nanowhiskers, have been studied as reinforcement agents in polymer composites with thermoplastic matrix in numerous publications. However, the use of nanowhiskers to reinforce hydrogels has not really been explored up to now, despite potential interest in the formulation of green innovative hydrogels. The aim of this PhD thesis was to study the relationship between the structural state, on different length scales, and the rheological properties of hydrocolloid systems consisting of agarose hydrogels filled by cellulose nanowhiskers from tunicate, amarine animal. The experimental work performed in this thesis has shown that the reinforcing effect provided by the nanowhiskers could be marked, and could also be modulated by varying the density of electrical charges on the surface of nanowhiskers. Within the volume fraction range studied, that is less than 0.2%, the results have shown that the nanowhiskers could not percolate, and reinforcement effect was attributed to topological modifications of the agarose network, and to an efficient stress transfer between the matrix and the fillers, which interact via hydrogen bonds.The results obtained in this thesis improve the understanding of the mechanisms that govern there inforcement effect provided by the addition of cellulose nanofillers in a hydrogel matrix; they could also help to design innovative green hydrogels having good mechanical properties.

Made available in DSpace on 2016-06-02T19:56:33Z (GMT). No. of bitstreams: 1 2277.pdf: 933463 bytes, checksum: 01507ff9166cf8406e37c047022142e1 (MD5) Previous issue date: 2009-02-27 / Universidade Federal de Sao Carlos / The objective of this work was stabilize and immobilize &#946;-galactosidase from activated agarose and chitosan supports. Initially, it was evaluated the buffer type, ionic strength and bivalent ions Mn2+ and Mg2+ on the hydrolytic activity of the enzyme using as substrates lactose and o-NPG (o-nitrophenyl galactopyranoside). Then the enzyme was covalently immobilized on glyoxyl-agarose, epoxy-chitosan-alginate and chitosan activated with glutaraldehyde, encapsulation in agarose and chitosan and ionic adsorption on MANAE-agarose. After immobilization, different strategies were used to stabilize the derivative such as crosslinking with glutaraldehyde and polyaldehyde dextran for the enzyme immobilized by ionic adsorption and reduction with sodium borohydride (NaBH4) for the enzyme covalently immobilized. For the derivative with maximum catalytic activity obtained, we estimated the kinetic and biochemical parameters as well as thermal stability at different temperatures and storage, operational stability, effect of inhibition by galactose and lactose yield. In the lactose hydrolysis, the best conditions were evaluated at 45°C in 100 mM potassium phosphate buffer pH 7.0 and addition of 2 mM MgCl2 and 0.1 mM MnCl2 (6786.5 U/mL of crude extract) and in the hydrolysis of synthetic substrate, o-NPG, the conditions for maximum catalytic activity was buffer sodium phosphate pH 7.0 50 mM with 2 mM MgCl2 at 25°C (4466.1 U/mL of crude extract). The enzyme immobilization on glyoxyl-agarose at pH 10.05 inactivated the enzyme. At pH 7.0, the enzyme was not immobilized. Similar behavior was observed for the enzyme immobilized on epoxy-chitosan-alginate. The ionic adsorption of the enzyme on MANAE-agarose allowed obtaining derivatives with high catalytic activity and immobilization yield around 100%. Neverthelless the crosslinking of the immobilized enzyme using polyaldehyde dextran and glutaraldehyde reduced drastically the hydrolytic activity of the derivatives by distortion of the enzyme structure. Besides the thermal stability of these derivatives at 10°C showed a similar behavior to the free enzyme. In order to obtain a derivative with high thermal stability on catalytic activity, the covalent immobilization on coagulated chitosan by different solutions and temperature. The derivative that provided higher catalytic activity was coagulated in a solution of 0.5 M KOH at 50°C and activated with glutaraldehyde 0.8% (v/v), with immobilization yield and recovered activity of 100%. An assay of looding of the support showed that 247,0 mg protein/ g gel was the maximum load. However diffusional limitation was verified even at 25 mg/g of gel. The immobilization process did not change the biochemical properties of the enzyme (optimum temperature and pH). In the storage stability at 10 ° C, the derivative covalently immobilized lost only 20% of initial activity after 90 days. The enzyme immobilized on chitosan was 3-5 fold more stable than the soluble enzyme at 20 and 40°C. The operational stability in 4 cycles showed a loss of 17% of hydrolytic activity after 4 cycles. Another important fact was the smallest effect of inhibition by galactose compared to soluble enzyme, even at high concentrations (5 g/L). In the hydrolysis of lactose the conversion was 70 % using insoluble and immobilized enzymes. We can conclude that the &#946;-galactosidase immobilized on chitosan activated with glutaraldehyde showed good properties, because it allows the development of continuous processes, facility of downstream process (product purification), avoiding contamination of the product by the biocatalyst. This advantage is very important specially for food industry. / O objetivo deste trabalho foi imobilizar e estabilizar &#946;-galactosidase de Kluyveromyces fragilis utilizando diferentes estratégias de imobilização em suportes orgânicos quitosana e agarose, com diferentes protocolos de ativação. Inicialmente, foi avaliado o tipo de tampão, força iônica e a suplementação com íons bivalentes Mn2+ e Mg2+ sobre a atividade hidrolítica da enzima empregando lactose e o-NPG (o-nitrofenil galactopiranosídeo) como substratos. A enzima foi imobilizada covalentemente em glioxil-agarose, epóxi-quitosana-alginato e quitosana ativada com glutaraldeído, por encapsulação em agarose e quitosana e por adsorção iônica em MANAE-agarose. Após a imobilização, diferentes estratégias foram adotadas para a estabilização do derivado como o entrecruzamento com glutaraldeído e polialdeído dextrana para a enzima imobilizada por adsorção iônica e a redução com borohidreto de sódio (NaBH4) para a enzima imobilizada covalentemente. Para o melhor derivado de &#946;-galactosidase foram estimados pH e temperatura de máxima atividade catalítica; estudados estabilidade térmica e operacional; efeito de inibição pela galactose e conversão de lactose. Na hidrólise da lactose, as melhores condições avaliadas foram a 45°C em tampão fosfato de potássio 100 mM pH 7,0 e adição de 2 mM MgCl2 e 0,1 mM MnCl2 (6786,5 U/mL de extrato) e na hidrólise do substrato sintético, o-NPG, a máxima atividade foi obtida em tampão fosfato de sódio 50 mM pH 7,0 com 2 mM MgCl2 a 25°C (4466,1 U/mL de extrato). A imobilização da enzima em gel glioxil-agarose não forneceu um derivado ativo, pois no pH de imobilização (10,05) a enzima sofreu inativação. Comportamento similar foi verificado para a enzima imobilizada em epóxi-quitosana-alginato. A adsorção iônica da enzima em MANAE-agarose forneceu derivados com elevada atividade catalítica e rendimento de imobilização da ordem de 100%. Porém, o entrecruzamento com polialdeído dextrana e glutaraldeído pós-imobilização reduziu drasticamente a atividade hidrolítica dos derivados provavelmente por distorção da estrutura ativa da enzima. Mesmo com o entrecruzamento, a estabilidade térmica destes derivados a 10°C apresentou um comportamento similar à enzima livre. Com o propósito de obter um derivado mais estável termicamente e com elevada atividade catalítica, foram avaliadas diferentes estratégias de imobilização covalente em quitosana coagulada por diferentes soluções e temperatura. O derivado que forneceu maior atividade catalítica foi obtido imobilizando a enzima em quitosana coagulada em solução 0,5 M de KOH a 50°C e ativado com glutaraldeído 0,8% (v/v), com atividade recuperada e rendimento de imobilização de 100%. A máxima concentração de enzima imobilizada neste suporte foi de 247,0 mg de proteína/g de gel. Após o carregamento de 25 mg/g de gel, limitação difusional foi verificada. A imobilização não alterou o pH e temperatura de máxima atividade hidrolítica da &#946;-galactosidase. A enzima imobilizada em quitosana-glutaraldeído perdeu apenas 20% da atividade inicial após 90 dias incubada no tampão fosfato de potássio 20 mM pH 7,0 com íons bivalentes Mn2+ e Mg2+a 10°C, foi 3-5 vezes mais estável que a enzima solúvel nas temperaturas de 20 e 40°C, pH7,0. A estabilidade operacional (40ºC e pH7,0) realizada em 4 ciclos mostrou uma perda de 17% da atividade hidrolítica inicial ao final do quarto ciclo. Outro fato relevante foi o menor efeito de inibição da enzima imobilizada pela galactose em comparação à enzima solúvel, mesmo em altas concentrações (5 g/L). Na hidrólise de lactose a 40ºC e pH 7,0 foi verificada uma conversão de 70% da lactose para ambas as enzimas, solúvel e imobilizada. De acordo com os resultados obtidos, pôde-se verificar que a imobilização de &#946;-galactosidase em quitosana ativada com glutaraldeído foi vantajosa, pois permite o desenvolvimento de processos contínuos de produção, simplifica a etapa de purificação do produto final e especificamente para fins alimentícios, reduz ou evita a contaminação do produto pelo biocatalisador.