An NMR structural study of nickel-substituted rubredoxin

J Biol Inorg Chem (2010) 15:409–420 DOI 10.1007/s00775-009-0613-6

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

Biomol NMR Assign (2007) 1:81–83 DOI 10.1007/s12104-007-9022-3

Integrated study by NMR and X-ray Crystallography on the analysis of the molecular interactions in heme-binding proteins

Dissertação para obtenção do Grau de Doutor em Bioquímica, Especialidade Bioquímica Estrutural

Molecular determinants of ligand specificity in carbohydrate-binding modules: an NMR and X-ray crystallography integrated study

Dissertação para obtenção do Grau de Doutor em Bioquímica – Ramo Bioquímica Estrutural

The influence of CD 4+t cells, hiv disease stage and zidovudine on hiv isolation in Bahia, Brazil

HIV-l isolation was attempted on 72 individuais, including persons with knoum HIV infection and five without proven HIV infection but with indeterminate Western blot patterns, as well as on low-risk HIV seronegative persons. The ahility to detect HIV- 1 frorn culture supernatant by p24 antigen capture assay was evaluated by segregating patients by absolute CD4+ cell counts, clinicai stage of disease, p24 antigenemia and zidovudine use. The likelihood of a p24 positive HIV culture was highest among patients with CD4+ T-cell counts below 200/ul and patients with advanced clinical disease. Use of zidovudine did not affect the rate ofHIV positwity in cultures.

Application of NMR RDC’s, relaxation and self-diffusion for the study of dynamic processes of small molecules in solution

Spin-lattice Relaxation, self-Diffusion coefficients and Residual Dipolar Couplings (RDC’s) are the basis of well established Nuclear Magnetic Resonance techniques for the physicochemical study of small molecules (typically organic compounds and natural products with MW < 1000 Da), as they proved to be a powerful and complementary source of information about structural dynamic processes in solution. The work developed in this thesis consists in the application of the earlier-mentioned NMR techniques to explore, analyze and systematize patterns of the molecular dynamic behavior of selected small molecules in particular experimental conditions. Two systems were chosen to investigate molecular dynamic behavior by these techniques: the dynamics of ion-pair formation and ion interaction in ionic liquids (IL) and the dynamics of molecular reorientation when molecules are placed in oriented phases (alignment media). The application of NMR spin-lattice relaxation and self-diffusion measurements was applied to study the rotational and translational molecular dynamics of the IL: 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]. The study of the cation-anion dynamics in neat and IL-water mixtures was systematically investigated by a combination of multinuclear NMR relaxation techniques with diffusion data (using by H1, C13 and F19 NMR spectroscopy). Spin-lattice relaxation time (T1), self-diffusion coefficients and nuclear Overhauser effect experiments were combined to determine the conditions that favor the formation of long lived [BMIM][BF4] ion-pairs in water. For this purpose and using the self-diffusion coefficients of cation and anion as a probe, different IL-water compositions were screened (from neat IL to infinite dilution) to find the conditions where both cation and anion present equal diffusion coefficients (8% water fraction at 25 ºC). This condition as well as the neat IL and the infinite dilution were then further studied by 13C NMR relaxation in order to determine correlation times (c) for the molecular reorientational motion using a mathematical iterative procedure and experimental data obtained in a temperature range between 273 and 353 K. The behavior of self-diffusion and relaxation data obtained in our experiments point at the combining parameters of molar fraction 8 % and temperature 298 K as the most favorable condition for the formation of long lived ion-pairs. When molecules are subjected to soft anisotropic motion by being placed in some special media, Residual Dipolar Couplings (RDCs), can be measured, because of the partial alignment induced by this media. RDCs are emerging as a powerful routine tool employed in conformational analysis, as it complements and even outperforms the approaches based on the classical NMR NOE or J3 couplings. In this work, three different alignment media have been characterized and evaluated in terms of integrity using 2H and 1H 1D-NMR spectroscopy, namely the stretched and compressed gel PMMA, and the lyotropic liquid crystals CpCl/n-hexanol/brine and cromolyn/water. The influence that different media and degrees of alignment have on the dynamic properties of several molecules was explored. Different sized sugars were used and their self-diffusion was determined as well as conformation features using RDCs. The results obtained indicate that no influence is felt by the small molecules diffusion and conformational features studied within the alignment degree range studied, which was the 3, 5 and 6 % CpCl/n-hexanol/brine for diffusion, and 5 and 7.5 % CpCl/n-hexanol/brine for conformation. It was also possible to determine that the small molecules diffusion verified in the alignment media presented close values to the ones observed in water, reinforcing the idea of no conditioning of molecular properties in such media.

A aprendizagem e o ensino do método cartesiano no plano com o CD-ROM da escola virtual

Esta investigação visa estudar a forma como os materiais educativos em ambientes interactivos e dinâmicos, no CD-ROM da Escola Virtual, desenvolvem a competência dos alunos na aprendizagem da geometria e em particular, no tema relativo ao método cartesiano no plano. A metodologia de investigação insere-se no paradigma interpretativo e segue uma abordagem qualitativa. Foi seleccionada uma turma do 11.º ano de escolaridade de um curso profissional, de uma Escola Secundária do distrito de Lisboa. A recolha de dados decorreu ao longo de dois meses, e recorreu a técnicas diversas como observação das aulas, questionários, registo vídeo, entrevistas e seu registo áudio. Os resultados obtidos neste estudo permitem concluir que a utilização do CDROM da Escola Virtual foi muito vantajosa no desenvolvimento do método cartesiano do plano e que a interactividade e animação gráfica presente contribuíram para uma boa compreensão dos conteúdos. Contudo, verificou-se que as potencialidades, dinâmicas e interactivas desta ferramenta nem sempre são suficientes e eficazes para a compreensão e apropriação de conceitos, por parte dos alunos. Quando os alunos não conseguem acompanhar as explicações no CD-ROM, o professor tem um papel preponderante, no sentido de os orientar, na procura de informação, ou de os ajudar na superação das dificuldades. Também permitiu concluir, que a utilização do CD-ROM influenciou de modo positivo a motivação dos alunos e favoreceu o trabalho colaborativo. A aprendizagem dos conceitos matemáticos processou-se a partir das interacções entre os grupos e entre os elementos de cada grupo, e através das acções mediadas pelos artefactos, designadamente o CD-ROM da Escola Virtual e seus recursos.

Fitorremediação de águas residuais contaminadas com Cd e Ni por três genótipos de Miscanthus

Fitorremediação, o uso de vegetação para a descontaminação de diferentes matrizes, é uma estratégia de baixo custo e tem sido apontada como uma metodologia passível de atenuar e estabilizar águas residuais. Neste contexto, este estudo tem como principal objectivo avaliar a capacidade fitorremediadora de três genótipos de Miscanthus (duas espécies - Miscanthus sinensis e Miscanthus floridulus, e um híbrido – Miscanthus x giganteus) a águas residuais contaminadas com cádmio e níquel (2,0 mg Ni/L e 0,2 mg Cd/L). Os resultados demonstraram que a irrigação com águas residuais contaminadas com Cd e Ni não afecta a produtividade dos três genótipos de Miscanthus em estudo e que o M. x giganteus e o M. floridulus registaram produtividades mais elevadas que o M. sinensis. Contudo, o M. x giganteus, demonstrou tendencialmente uma redução da sua produção com a contaminação de Cd. A biomassa obtida dos ensaios irrigados com águas residuais contaminadas apresentaram maior teor de Cd e Ni, do que a biomassa recolhida dos ensaios Controlo. O sistema Miscanthus-solo registou valores de remoção de Cd de 100%, e de Ni de 96-100%, no caso do M. x giganteus e do M. sinensis. O M. floridulus, contudo, demonstrou menos potencial de fitorremediação no que diz respeito à remoção de Ni das águas residuais: apenas 58% de Ni foi removido pelo sistema “biofiltrador”. Ainda assim, verificou-se a acumulação de Cd e Ni no perfil superior do solo devido à irrigação com as águas residuais contaminadas, o que demonstra a necessidade de controlo desta técnica de modo a prevenir possíveis efeitos negativos no ambiente.

Development and application of NMR techniques to study water-in-CO2 microemulsions

Self-assembly is a phenomenon that occurs frequently throughout the universe. In this work, two self-assembling systems were studied: the formation of reverse micelles in isooctane and in supercritical CO2 (scCO2), and the formation of gels in organic solvents. The goal was the physicochemical study of these systems and the development of an NMR methodology to study them. In this work, AOT was used as a model molecule both to comprehensively study a widely researched system water/AOT/isooctane at different water concentrations and to assess its aggregation in supercritical carbon dioxide at different pressures. In order to do so an NMR methodology was devised, in which it was possible to accurately determine hydrodynamic radius of the micelle (in agreement with DLS measurements) using diffusion ordered spectroscopy (DOSY), the micellar stability and its dynamics. This was mostly assessed by 1H NMR relaxation studies, which allowed to determine correlation times and size of correlating water molecules, which are in agreement with the size of the shell that interacts with the micellar layer. The encapsulation of differently-sized carbohydrates was also studied and allowed to understand the dynamics and stability of the aggregates in such conditions. A W/CO2 microemulsion was prepared using AOT and water in scCO2, with ethanol as cosurfactant. The behaviour of the components of the system at different pressures was assessed and it is likely that above 130 bar reverse microemulsions were achieved. The homogeneity of the system was also determined by NMR. The formation of the gel network by two small molecular organogelators in toluene-d8 was studied by DOSY. A methodology using One-shot DOSY to perform the spectra was designed and applied with success. This yielded an understanding about the role of the solvent and gelator in the aggregation process, as an estimation of the time of gelation.

Teores de As, Pb, Cd e Hg e fertilidade de solos da região do Vale do Alto Guaporé, sudoeste do estado de Mato Grosso

O diagnóstico da fertilidade e teores de elementos-traço (ETs) em solos é importante, pois estes dados são escassos na literatura para áreas de transição Pantanal-Cerrado-Floresta Amazônica. Esse trabalho avaliou diversos parâmetros relacionados à fertilidade, teores biodisponíveis de Fe, Mn, Zn, Cu e B e semitotais de As, Cd, Hg e Pb de solos do Vale do Alto Guaporé, região sudoeste do estado de Mato Grosso. Foram coletadas amostras de solos (0-0,20 e 0,20-0,40 m de profundidade) em áreas de vegetação nativa (VN), pastagem (AP), cultura anual (CA) e garimpo de ouro (G). As amostras foram analisadas conforme métodos de rotina para avaliação da fertilidade do solo e ETs pelo método SW-3051A e os resultados médios comparados com os valores de referência de qualidade (VRQ) para solos estipulados pela Companhia de Tecnologia de Saneamento Ambiental (CETESB). Teores mais elevados de As e Hg foram verificados em VN e G com médias, respectivamente, iguais a 43,9 e 101,13 para o As; e 0,12 e 0,14 mg kg-1 para o Hg. Exceto Pb, vários locais de amostragem apresentaram teores dos ETs superiores ao VRQ: 46% em VN; 60% em G; 28% em CA; e 44% em AP, para o As; 20,8; 50; 55; e 22% em VN, G, CA e AP, respectivamente, para o Cd; 75; 65 e 67% das áreas de VN, G e CA e AP, respectivamente, para o Hg. A saturação por bases foi alta (60-80%) em 51,5% das amostras, enquanto o P foi baixo em todas áreas. Valores de referência de qualidade de solo para o As e Hg devem ser estipulados para solos dessa região, tendo em vista que os teores observados em áreas nativas foram superiores ao VRQ.

Metabolic profiling and classification of propolis samples from Southern Brazil: an NMR-based platform coupled with machine learning

The chemical composition of propolis is affected by environmental factors and harvest season, making it difficult to standardize its extracts for medicinal usage. By detecting a typical chemical profile associated with propolis from a specific production region or season, certain types of propolis may be used to obtain a specific pharmacological activity. In this study, propolis from three agroecological regions (plain, plateau, and highlands) from southern Brazil, collected over the four seasons of 2010, were investigated through a novel NMR-based metabolomics data analysis workflow. Chemometrics and machine learning algorithms (PLS-DA and RF), including methods to estimate variable importance in classification, were used in this study. The machine learning and feature selection methods permitted construction of models for propolis sample classification with high accuracy (>75%, reaching 90% in the best case), better discriminating samples regarding their collection seasons comparatively to the harvest regions. PLS-DA and RF allowed the identification of biomarkers for sample discrimination, expanding the set of discriminating features and adding relevant information for the identification of the class-determining metabolites. The NMR-based metabolomics analytical platform, coupled to bioinformatic tools, allowed characterization and classification of Brazilian propolis samples regarding the metabolite signature of important compounds, i.e., chemical fingerprint, harvest seasons, and production regions.

New self-assembled supramolecular hydrogels based on dehydropeptides

Supramolecular hydrogels rely on small molecules that self-assemble in water as a result of the cooperative effect of several relatively weak intermolecular interactions. Peptide-based low molecular weight hydrogelators have attracted enormous interest owing to the simplicity of small molecules combined with the versatility and biocompatibility of peptides. In this work, naproxen, a well known non-steroidal anti-inflammatory drug, was N-conjugated with various dehydrodipeptides to give aromatic peptide amphiphiles that resist proteolysis. Molecular dynamics simulations were used to obtain insight into the underlying molecular mechanism of self-assembly and to rationalize the design of this type of hydrogelators. The results obtained were in excellent agreement with the experimental observations. Only dehydrodipeptides having at least one aromatic amino acid gave hydrogels. The characterization of the hydrogels was carried out using transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy and also rheological assays.

Toxicity induced by a metal mixture (Cd, Pb and Zn) in the yeast Pichia kudriavzevii: The role of oxidative stress

The present work aims to contribute for the elucidation of the role of oxidative stress in the toxicity associated with the exposure of Pichia kudriavzevii to multi-metals (Cd, Pb and Zn). Cells of the non-conventional yeast P. kudriavzevii exposed for 6 h to the action of multi-metals accumulated intracellular reactive oxygen species (ROS), evaluated through the oxidation of the probe 2,7-dichlorodihydrofluorescein diacetate. A progressive loss of membrane integrity (monitored using propidium iodide) was observed in multi-metal-treated cells. The triggering of intracellular ROS accumulation preceded the loss of membrane integrity. These results suggest that the disruption of membrane integrity can be attributed to the oxidative stress. The exposure of yeast cells to single metal showed that, under the concentrations tested, Pb was the metal responsible for the induction of the oxidative stress. Yeast cells coexposed to an antioxidant (ascorbic acid) and multi-metals did not accumulate intracellular ROS, but loss proliferation capacity. Together, the data obtained indicated that intracellular ROS accumulation contributed to metal toxicity, namely for the disruption of membrane integrity of the yeast P. kudriavzevii. It was proposed that Pb toxicity (the metal responsible for the toxic symptoms under the conditions tested) result from the combination of an ionic mechanism and the intracellular ROS accumulation.