Disabled-2 (Dab2) is an adapter protein that interacts with cell membranes and it is involved in several biological processes including endocytosis and platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane and helps co-localization with clathrin coats. Dab2, released from platelet alpha granules, inhibits platelet aggregation by binding to the °IIb? integrin receptor on the platelet surface through an Arg-Gly-Asp (RGD) motif located within the PTB domain. Alternatively, Dab2 binds sulfatides on the platelets surface, and this binding partition Dab2 in two pools (sulfatide and integrin receptor-bound states), but the biological consequences of lipid binding remain unclear. Dab2 binds sulfatides through two basic motifs located on its N-terminal region including the PTB domain (N-PTB). We have characterized the binding of Dab2 to micelles, which are widely used to mimic biological membranes. These micellar interactions were studied in the absence and presence of Dab2 lipid ligands, sulfatides and PIP2. By applying multiple biochemical, biophysical, and structural techniques, we found that whereas Dab2 N-PTB binding to PIP2 stabilized the protein but did not contribute to the penetration of the protein into micelles, sulfatides induced conformational changes and facilitated penetration of Dab2 N-PTB into micelles. This is in agreement with previous observation that sulfatides, but not PIP2, protect Dab2 N-PTB from thrombin cleavage. By studying the mechanism by which Dab2 targets membranes, we will have the opportunity to manipulate its function in different lipid-dependent biological processes. / Master of Science

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Spintronics is a new branch of electronics, which uses not only the charge of electron but also its spin for transfer and processing of information. For re- al applications it is necessary to understand, how the magnetic state of matter changes not only in time but also in space. This diploma thesis is therefore de- voted to the imaging of magnetic domains in ferromagnetic semiconductors by purely optical methods, which are based on the usage of the magneto-optical ef- fects. In first part of the thesis we concentrated on the optimalization of existing polarizing microscope, whose function is based solely on the polar Kerr effect. After the optimalization we try to use this setup to observe magnetic domains in ferromagnetic semiconductor GaMnAs by the means of the magnetic linear dichroism. 1

In these diploma thesis magnetically ordered materials are studied with the prospect of their application in spintronics. Specifically, we investigated metallic alloy FeRh, which undergoes a magnetic phase transition from antife- romagnetic phase to feromagnetic one around 100◦ C. This phenomenon can be readily used in memory devices. Laser spectroscopy is used as a nondestructive method without need of any electrical contacts. Magnetic properties of FeRh are studied by magnetooptical effects including quadratic magnetic linear dichroism. The measured polarization rotations are of the order of miliradians, therefore, the detection is realized by an optical bridge. At first, we concentrated on discrimina- ting of various magnetooptical effects from each other. The second part is focused on the phase transition induced by different means. Firstly, by heating the whole sample, secondly by illuminating the sample locally by continuous laser.

Studies of Lanthanide Complexes by a Combination of Spectroscopic Methods Monika Krupová (Department of Physical and Macromoecular Chemistry, Faculty of Science, Charles University in Prague) Since conventional structural analysis offers rather limited means for the chirality detection, a series of lanthanide tris-(β-diketonates) are investigated as effective receptors for a better chirality sensing in biomolecular substrates. These lanthanide complexes containing β-diketonate ligands are electrically neutral; they can further coordinate with various small organic molecules such as chiral alcohols, amino alcohols or amino acids in organic solvents and produce a strong chiral signal. Previously, a resonance in Raman scattering was observed in the studied systems due to the correspondence of europium electronic transition energy to the laser excitation wavelength, about a 100-fold signal enhancement if compared to non-resonant vibrational ROA was observed. This enabled shorter detection times as well as lower sample concentrations. In the current work, interaction of the Eu(FOD) complex with (R)- and (S)- enantiomer of 1-phenylethanol in n-hexane was studied using IR spectroscopy, Raman spectroscopy and Raman optical activity (ROA), UV-Vis spectroscopy and ultraviolet circular dichroism (UVCD). Only...

Este trabalho tem como objetivos projetar, construir e caracterizar experimentalmente metamateriais quirais bi-planares para faixa de micro-ondas com o objetivo de investigar a rotação de campo como transdutor em aplicações de biossensoriamento. As estruturas metamateriais estudadas foram primeiramente modeladas e caracterizadas com o uso de aplicativos computacionais comerciais baseadas no método dos elementos finitos, onde os parâmetros geométricos das estruturas foram definidos para operarem na faixa de 8GHz a 12GHz. A partir dos projetos das estruturas simuladas, os metamateriais foram construídos e caracterizados experimentalmente. No estágio de fabricação, foi desenvolvida uma técnica inédita de confecção de metamateriais em placas de circuito impresso flexível. Esta técnica, baseada no processo tradicional de fabricação de placas de circuito impresso, utiliza uma combinação de filmes finos de alumínio e folhas plásticas adesivas flexíveis formando, assim, uma folha metalizada fina e flexível. Além de ser tão eficiente quanto o método convencional, a abordagem proposta utiliza materiais de baixo custo, podendo ser adaptada a outros substratos de qualquer material, formato e espessura. Também foram projetadas e construídas placas de metamateriais com geometrias similares via método tradicional (FR4 e cobre) com o objetivo de validar os procedimentos propostos neste trabalho. A caracterização experimental dos metamateriais quirais foi realizada, por meio de antenas cornetas e analisador de redes. As propriedades efetivas do meio (quiralidade, ângulo de azimute, elipticidade, e índice de refração) foram obtidas a partir dos parâmetros-S do material, por meio de um método de extração de parâmetros. Como esperado, os resultados experimentais tiveram boa concordância com os obtidos numericamente. Por fim, foi proposta uma aplicação de metamaterial quiral utilizando suas características de rotação de campo como transdutor para a medida de glicose em amostras aquosas. No experimento proposto, o ângulo de azimute de uma onda plana polarizada que atravessa o material foi obtido numericamente e sua rotação de campo foi diretamente correlacionada com a concentração de glicose. Os resultados indicaram uma elevada sensibilidade mesmo para baixas concentrações de glicose, demonstrando que a rotação de campo pode realmente ser utilizada para este propósito. / This work focus on the design, fabrication and experimental characterization of bi-planar chiral metamaterials in the microwave frequency range aiming at investigating field rotation as a possible transductor for biosensig applications. First, the metamaterial structures were simulated with commercial-based finite element methods, where the structures geometric parameters were defined to operate in the range of 8-12 GHz. Next, the metamaterials were fabricated and characterized experimentally. We have also developed a new technique for making metamaterials on flexible printed circuit boards. This proposed technique, based on traditional processes of printed circuit boards fabrication, utilizes combination of thin aluminum films and adhesive flexible plastic foils, thus forming a thin, flexible metallic foil. In addition to being as efficient as the conventional method, the proposed approach utilizes low cost materials with low insertion loss that can be adapted to substrates of any material, shape and thickness. We have also designed and fabricated metamaterials with similar geometries via the traditional circuit board method (FR4 and copper) in order to validate the procedures proposed in this work. The experimental characterization of the chiral metamaterials was carried out with antenna horns and a network analyzer. The effective medium parameters (chirality, azimuth angle, ellipticity, and refractive index) were then obtained from the Sparameters of the material via a parameter extraction method. The experimental results were in good agreement with those obtained numerically. Finally, we proposed a new chiral metamaterial application using its field rotation characteristics as a transductor for measuring glucose concentration in aqueous solutions. In the proposed experiment, the azimuth angle of a polarized plane wave propagating through the material was numerically calculeted and its polarization field rotation was directly correlated with the glucose concentration. The results indicated a very high sensitivity even for very low glucose concentrations, indicating that field rotation can indeed be used for this purpose.

Neste trabalho de Mestrado, foram feitos estudos do comportamento fotofísico da fisetina em diversos solventes através de medidas de absorção de luz UV-Visível, fluorescência estática e resolvida no tempo e da interação entre fisetina (3,3\',4\',7-tetrahidroxiflavona) e 7-hidroxiflavona com ciclodextrinas ( beta e gama) (CDs) através de experimentos de absorção de luz UV-Visível, sinal induzido de dicroísmo circular, fluorescência estática e resolvida no tempo, anisotropia de estado estacionário e RMN de 1H, focando a dependência destas medidas em função da temperatura e do pH. Os resultados experimentais foram comparados com estudos mecânico-quânticos baseados no modelo semi-empírico SAM1 (AMPAC), e nos funcionais B3LYP e MPW1PW91, da Teoria do Funcional de Densidade, empregando os conjuntos de funções de base 6-311G* e 3-21G**. Os estudos da fisetina em diferentes solventes próticos e apróticos mostraram que a fluorescência da sonda é fortemente dependente do solvente. Resultados experimentais indicam a formação de complexos de inclusão entre as CDs e os flavonóides acima. A fisetina com ?-CD forma complexo de estequiometria 1:1 nos meios neutro/ácido e básico, cujos valores de constante de complexação são 900 +- 100 +_ 240 +_ 90 (L/mol), respectivamente. Os dados teóricos evidenciaram que a inclusão da fisetina na beta-CD ocorre preferencialmente pelo anel fenila. O complexo com a gama-CD apresenta estequiometria de 1:1 em meio ácido/neutro e de 1:2 em meio básico, com constantes de complexação de 94 +- 30 e 130 +- 10 (L/mol), respectivamente. Os estudos com a 7-hidroxiflavona revelaram que somente ocorre a formação de complexos com a beta-CD de estequiometria 1:1 e não há dependência do pH. As constantes de complexação obtidas nos meios ácido/neutro e básico são similares, 1430 +-- 510 e 1220 +- 165 L/mol, respectivamente. / In this work, the photophysics of fisetin (3,3\',4\',7-tetrahydroxyflavone) in several solvents was studied through UV-vis absorption spectra, steady-state and time-resolved fluorescence measurements. The interaction between fisetin and 7-hydroxyflavone and cyclodextrins (b- e g-) (CDs) was also investigated by UV-vis absorption spectra, induced signal of circular dichroism, steady-state and time-resolved fluorescence, steady-state anisotropy, and 1H NMR, with dependence on pH and temperature. Some experimental data were compared with quantum-mechanics studies based on the SAM1 (AMPAC) semi-empirical model, as well as with the B3LYP and MPW1PW91 functional models from the Density Functional Theory using the 6-311G* and 3-21G* basis sets. The study of the photophysics of fisetin in protic and aprotic solvents showed a complex behavior and a strong dependence on the solvent. The occurrence of many equilibria between the possible structures of fisetin, e.g. the normal, a few deprotonated ones, and the tautomer due to the excited state intramolecular proton transfer can be responsible for the complex analyses of these experimental data. The spectroscopic measurements show that, at pH 4.0 and 6.5, the complex fisetin - b-CD is formed in a Fis:b-CD 1:1 stoichiometry and an equilibrium constant (K) of 900 ± 100 L/mol. In basic medium (pH 11.5), K decreases to 240 ± 90 L/mol. Molecular modeling points out that the inclusion complex is formed preferentially via entry of the fisetin phenyl group into b-CD. On the other hand, the fisetin - g-CD has a stoichiometry of 1:1 in acid/neutral solutions and of 1:2 in basic conditions. The K values are 94 ± 30 e 130 ± 10 (L/mol), respectively. The 7-hydroxyflavone can only form inclusion complexes with b-CD. The stoichiometry is 1:1 and there is no dependence on pH. Both equilibrium constants determined either in acid or basic medium is very similar to each other, 1430 ± 510, and 1220 ± 165 L/mol, respectively. For this reason, we suppose that the inclusion of this compound into b-CD is also through the phenyl ring of the flavonoid.

A proteína P21 do Trypanosoma cruzi participa no processo de infecção da célula hospedeira, desse modo é de grande importância: elucidar as vias de sinalização induzidas pela proteína, bem como caracterizar a nível molecular e estrutural a P21. O que vai auxiliar no entendimento da função biológica da P21 e sua participação no processo de infecção. A P21 recombinante é expressa Escherichia coli em sua maioria na fração insolúvel. Visando aumento de proteína na fração solúvel, foi realizada a clonagem do gene da P21 em vetor pSMT3, bem como testes de expressão subsequentes em diferentes cepas de expressão de E. coli, em vetor pET-28 e pSMT3 e variadas condições de expressão e lise celular. Desse modo obtiveram-se as condições que permitissem uma maior concentração da P21 na fração solúvel. A expressão foi realizada no vetor pET-28, cepa BL21, a 37°C com meio 2xYT a 0.5 mM de IPTG, utilizando a técnica de sonicação como lise. A P21 foi purificada em cromatografia de afinidade e posteriormente em coluna de exclusão molecular. Foram realizados estudos de modelagem por homologia levaram a elaborar a hipótese de que a P21 tem a função de inibidor de serinoprotease do tipo kunitz. Posteriormente essa hipótese foi confirmada com ensaios de avaliação da atividade inibitória da P21 frente à tripsina, quimiotripsina e elastase neutrofílica, o qual a P21 mostrou capaz de inibir a elastase neutrofílica. Estudos com espalhamento dinâmico da luz (DLS) revelaram que as amostras testadas de P21 contém diferentes concentrações de agregados de alto peso molecular em todos os pHs avaliados. Outras medidas foram realizadas para avaliar o estado de agregação da P21 de acordo com a temperatura e verificou-se que entre 32-52 °C, a proteína apresenta menor raio hidrodinâmico, indicando menor agregação nesse intervalo. Estudos de dicroísmo circular revelaram que a P21 apresenta por volta de 20% de α hélice, 32% de folha-β, 22% de volta e 23 % estrutura randômica. De acordo com a curva de desnaturação referente ao espectro de CD obtido, a P21 se mostra desnaturada a partir de 64°C. / The Trypanosoma cruzi protein P21 participates in the host cell infection process, but its specific function is poorly described. Thus it is important to elucidate the signaling pathways induced by the protein and characterize the P21 at the structural and molecular levels, as a contribution towards the understanding of the P21 biological function and its role in the infection process. The Escherichia coli recombinant P21 is expressed mostly in the insoluble fraction. Aiming to increase protein in the soluble fraction, we performed the cloning of the P21 gene in pSMT3 vector and subsequent expression tests in different expression strains of E. coli in pET-28 and pSMT3 vectors and varied expression conditions and cell lysis. Thus we obtained conditions that allow a greater concentration of the P21 in the soluble fraction. The expression vector was performed using vector pET-28, in Bl21 strain at 37°C in 2xYT culture medium with 0.5 mM IPTG, using the sonication technique in cell lysis. The recombinant P21 was purified by Ni affinity chromatography and subsequently a molecular exclusion column. We performed homology modeling studies which led to assume that P21 can be a serinprotease inhibitor of Kunitz type. Furthermore, this hypothesis was confirmed in the experiments testing the P21 inhibitory activity against the trypsin, chymotrypsin and elastase. We found the P21 exclusively inhibit neutrophil elastase. Studies using dynamic light scattering (DLS) revealed that the samples containing P21 contained large molecular weigth aggregates in different concentrations at all evaluated pHs. Others measurements were performed to assess the P21 aggregation state according to the temperature and we found that between 32-52 °C the protein had a smaller hydrodynamic radius, indicating less aggregation in this range. Circular dichroism (CD) studies revealed that P21 has about 20% α-helix, 32% β, -sheet, 22% turn and 23% of random structure. According to the denaturation curve for the CD spectrum obtained, the P21 was denatured from 64°C.

Tritrpticina (TRP3) é um peptídeo antimicrobiano com 13 resíduos de amino ácidos com três Ws sequenciais. Com o objetivo de contribuir para a compreensão de seu mecanismo de ação, realizaram-se estudos funcionais e conformacionais da TRP3 e de dois análogos onde um (WLW) ou dois (LWL) W foram substituídos por L. Os peptídeos foram igualmente ativos contra bactérias Gram positivas e negativas. Sua atividade hemolítica requereu concentrações maiores, diminuindo na ordem TRP3>WLW>LWL. Os peptídeos permeabilizaram membranas modelo de E. coli ou contendo fosfolipídios carregados negativamente. Espectros de CD sugeriram que os peptídeos adquirem diferentes conformações ao se ligarem a bicamadas e micelas. Estudos de fluorescência mostraram que a ligação a membranas decresce na ordem: TRP3>WLW>LWL e que os peptídeos se localizam próximos à interface membrana-água. Espectros de RPE de marcadores de spin lipídicos indicaram que a ligação dos peptídeos altera a organização dos lipídios, aumentando o empacotamento molecular / Tritrpticin (TRP3) is a 13-residue antimicrobial peptide that contains three sequential Ws. With the aim of contributing to the understanding of its mechanism of action, functional and conformational studies were performed with TRP3 and two of its analogues where one (WLW) or two (LWL) of the W were replaced by L. The peptides were equally active against both Gram positive and Gram negative bacteria. Higher concentrations were required for hemolytic activity which varied in the order: TRP3>WLW>LWL. The peptides permeabilized membranes model membranes mimicking E. coli\'s lipid composition or containing different negatively charged phospholipids. CD spectra suggested the peptides acquired different conformations upon binding to bilayers or micelles. Fluorescence studies showed that membrane binding decreases in the order: TRP3>WLW>LWL and that the peptides are located close to the water-membrane interface. EPR spectra of lipid spin labels indicated that peptide binding alter lipid organization, increasing molecular packing