Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens

The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors. NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals. To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed. The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences. Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.

The good, the bad and the ugly: The fall and resolution of Banco Espírito Santo

This case study – and accompanying teaching note – briefly describes the history of the Espírito Santo family, a banking dynasty who led one of Portugal’s leading economic and financial groups, along with its “crown jewel”, Banco Espírito Santo. It chronicles how the corporate governance issues at BES allowed the family to exploit the bank, its shareholders and its customers, so as to support its unprofitable non-financial businesses. This left the bank in a poor financial situation, which deteriorated beyond control, leaving regulators – whose actions are also analysed here – with no alternative, amidst a severe liquidity crisis, but to apply a resolution measure, pinning large losses on junior bondholders and shareholders before recapitalising the bank.

Leishmaniasis transmission: distribution and coarse-resolution ecology of two vectors and two parasites in Egypt

Introduction: In past decades, leishmaniasis burden has been low across Egypt; however, changing environment and land use has placed several parts of the country at risk. As a consequence, leishmaniasis has become a particularly difficult health problem, both for local inhabitants and for multinational military personnel. Methods: To evaluate coarse-resolution aspects of the ecology of leishmaniasis transmission, collection records for sandflies and Leishmania species were obtained from diverse sources. To characterize environmental variation across the country, we used multitemporal Land Surface Temperature (LST) and Normalized Difference Vegetation Index (NDVI) data from the Moderate Resolution Imaging Spectroradiometer (MODIS) for 2005-2011. Ecological niche models were generated using MaxEnt, and results were analyzed using background similarity tests to assess whether associations among vectors and parasites (i.e., niche similarity) can be detected across broad geographic regions. Results: We found niche similarity only between one vector species and its corresponding parasite species (i.e., Phlebotomus papatasi with Leishmania major), suggesting that geographic ranges of zoonotic cutaneous leishmaniasis and its potential vector may overlap, but under distinct environmental associations. Other associations (e.g., P. sergenti with L. major) were not supported. Mapping suitable areas for each species suggested that northeastern Egypt is particularly at risk because both parasites have potential to circulate. Conclusions: Ecological niche modeling approaches can be used as a first-pass assessment of vector-parasite interactions, offering useful insights into constraints on the geography of transmission patterns of leishmaniasis.

Biomimetic mineralization: encapsulation in calcium carbonate shells

Calcium carbonate biomineralization is a self-assembly process that has been studied to be applied in the biomedical field to encapsulate biomolecules. Advantages of engineering mineral capsules include improved drug loading efficiencies and protection against external environment. However, common production methods result in heterogeneous capsules and subject biomolecules to heat and vibration which cause irreversible damage. To overcome these issues, a microfluidic device was designed, manufactured and tested in terms of selectivity for water and oil to produce a W/O/W emulsion. During the development of this work there was one critical challenge: the selective functionalization in closed microfluidic channels. Wet chemical oxidation of PDMS with 1M NaOH, confirmed by FTIR, followed by adsorption of polyelectrolytes - PDADMAC/PSS - confirmed by UV-Vis and AFM results, render the surface of PDMS hydrophilic. UV-Vis spectroscopy also confirmed that this modification did not affect PDMS optical properties, making possible to monitor fluids and droplets. More important, with this approach PDMS remains hydrophilic over time. However, due to equipment constrains selectivity in microchannels was not achieved. Therefore, emulsion studies took place with conventional methods. Several systems were tried, with promising results achieved with CaCO3 in-situ precipitation, without the use of polymers or magnesium. This mineral stabilizes oil droplets in water, but not in air due to incomplete capsule formation.

Estudo da aplicação de adesivos de cura UV em conservação e restauro de vidro e vitral

Do presente trabalho resultou a submissão de um artigo, com o título UV Curing Adhesives for Conservation of Glass- Chemical and Mechanical Stability, para comunicação oral em conferência internacional intitulada Recent Advances in Glass and Ceramics Conservation, a ocorrer em Wroclaw, Polónia, de 25 a 29 de Maio de 2016, no âmbito do ICOM-CC Glass and Ceramics Working Group Interim Meeting.

Persistent asthma in adults: comparison of high resolution computed tomography of the lungs after one year of follow-up

OBJECTIVE: The aims of this study were to evaluate the role of high resolution computed tomography of the torax in detecting abnormalities in chronic asthmatic patients and to determine the behavior of these lesions after at least one year. METHOD: Fourteen persistent asthmatic patients with a mean forced expiratory volume in 1-second that was 63% of predicted and a mean forced expiratory volume in 1-second /forced vital capacity of 60% had two high resolution computed tomographys separated by an interval of at least one year. RESULTS: All 14 patients had abnormalities on both scans. The most common abnormality was bronchial wall thickening, which was present in all patients on both computed tomographys. Bronchiectasis was suggested on the first computed tomography in 5 of the 14 (36%) patients, but on follow-up, the bronchial dilatation had disappeared in 2 and diminished in a third. Only one patient had any emphysematous changes; a minimal persistent area of paraseptal emphysema was present on both scans. In 3 patients, a "mosaic" appearance was observed on the first scan, and this persisted on the follow-up computed tomography. Two patients had persistent areas of mucoid impaction. In a third patient, mucus plugging was detected only on the second computed tomography. CONCLUSIONS: We conclude that there are many abnormalities on the high resolution computed tomography of patients with persistent asthma. Changes suggestive of bronchiectasis, namely bronchial dilatation, frequently resolve spontaneously. Therefore, the diagnosis of bronchiectasis by high resolution computed tomography in asthmatic patients must be made with caution, since bronchial dilatation can be reversible or can represent false dilatation. Nonsmoking chronic asthmatic subjects in this study had no evidence of centrilobular or panacinar emphysema.

Introducing mapping standards in the quality assessment of buildings extracted from very high resolution satellite imagery

Many municipal activities require updated large-scale maps that include both topographic and thematic information. For this purpose, the efficient use of very high spatial resolution (VHR) satellite imagery suggests the development of approaches that enable a timely discrimination, counting and delineation of urban elements according to legal technical specifications and quality standards. Therefore, the nature of this data source and expanding range of applications calls for objective methods and quantitative metrics to assess the quality of the extracted information which go beyond traditional thematic accuracy alone. The present work concerns the development and testing of a new approach for using technical mapping standards in the quality assessment of buildings automatically extracted from VHR satellite imagery. Feature extraction software was employed to map buildings present in a pansharpened QuickBird image of Lisbon. Quality assessment was exhaustive and involved comparisons of extracted features against a reference data set, introducing cartographic constraints from scales 1:1000, 1:5000, and 1:10,000. The spatial data quality elements subject to evaluation were: thematic (attribute) accuracy, completeness, and geometric quality assessed based on planimetric deviation from the reference map. Tests were developed and metrics analyzed considering thresholds and standards for the large mapping scales most frequently used by municipalities. Results show that values for completeness varied with mapping scales and were only slightly superior for scale 1:10,000. Concerning the geometric quality, a large percentage of extracted features met the strict topographic standards of planimetric deviation for scale 1:10,000, while no buildings were compliant with the specification for scale 1:1000.

Sol-gel entrapped biosystems: enzyme(s) and whole cells

In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells. CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor β-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC). The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to “in-cell” Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.

Mechanisms underlying intracellular delivery

AuNPs are versatile systems used for different biomedical application including imaging, drug and gene delivery. These systems support the intracellular transport of active molecules, a step that is considered one of the crucial problems in drug delivery. Nevertheless, in order to design optimal multifunctional AuNPs for specific and efficient nanomedicine applications, the mechanism by which AuNPs interact with living cells must be fully understand. The main goal of this work consisted in the assessment of the cellular uptake mechanism of 14 nm spherical AuNPs by A549 cells, through fluorescent spectroscopy and microscopy, in combination with quantitative analysis by ICP-MS. TAMRA labeled AuNPs were characterized by UV-visible and fluorescent spectroscopy and the final hydrodynamic diameter of 22.5 ± 0.33 nm was obtained by DLS. Regarding the cellular uptake studies, the AuNPs presented a fast cellular uptake kinetics reaching a saturation point after 6 hours of incubation in A549 cells. Further investigation concerning the internalization mechanism of this AuNPs was evaluated using specific inhibitors for different endocytic pathways. Optimal inhibition was achieved using chlorpromazine, inhibitor of clathrin-mediated endocytosis, resulting in a 23.5 % inhibition of AuNPs after 1 hour of incubation. This preliminary result obtained by fluorescent spectroscopy suggests that these AuNPs were predominantly uptake by clathrin-mediated endocytosis, meaning that other endocytic pathways must be involved in the cellular uptake of this AuNPs. In what cell viability is concern, the prepared AuNPs and the endocytic inhibitors revealed no significant effect on the cell viability in A549 cell line.

Proton magnetic resonance spectroscopy of the frontal lobe in schizophrenics: a critical review of the methodology

Schizophrenic patients undergoing proton magnetic resonance spectroscopy show alterations in N-acetyl aspartate levels in several brain regions, indicating neuronal dysfunction. The present review focuses on the main proton magnetic resonance spectroscopy studies in the frontal lobe of schizophrenics. A MEDLINE search, from 1991 to March 2004, was carried out using the key-words spectroscopy and schizophrenia and proton and frontal. In addition, articles cited in the reference list of the studies obtained through MEDLINE were included. As a result, 27 articles were selected. The results were inconsistent, 19 papers reporting changes in the N-acetyl aspartate levels, while 8 reported no change. Methodological analysis led to the conclusion that the discrepancy may be due the following factors: (i) number of participants; (ii) variation in the clinical and demographic characteristics of the groups; (iii) little standardization of the acquisition parameters of spectroscopy. Overall, studies that fulfill strict methodological criteria show N-acetyl aspartate decrease in the frontal lobe of male schizophrenics.

Os médicos do trabalho e a nova organização da saúde e segurança do trabalho : quais as consequências na prática profissional? António de Sousa Uv

RESUMO - Em 1994/1995 o modelo legal de organização de serviços de Medicina do Trabalho instituído na década de 1960 foi substituído por uma nova superestrutura de serviços de Segurança, Higiene e Saúde dos Trabalhadores (SHST) nos locais de trabalho. O presente estudo pretende descrever e analisar em que medida o novo enquadramento jurídico de SHST, iniciado em 1994/1995 e correspondente genericamente à fase da Nova Saúde Ocupacional, foi acompanhado de alterações: (1) na percepção do grau de satisfação dos médicos do trabalho quanto ao seu papel e estatuto profissionais e (2) nas repercussões na sua prática profissional. O presente estudo (empírico, descritivo e comparativo) abrangeu um grupo de médicos do trabalho diplomados pela Escola Nacional de Saúde Pública (n = 153), de quem se recolheu, através de um questionário aplicado em 1993 e 2000, a opinião sobre as mudanças organizacionais da SHST. O papel e funções dos médicos do trabalho e as garantias de exercício profissional não se alteraram de forma importante, tendo a prática profissional da medicina do trabalho na modalidade «medicina do trabalho de empresa» (serviços internos e externos) diminuído, apesar de continuar a ser a forma de exercício predominante dos médicos do trabalho. O tempo dedicado à sua actividade situou-se num valor médio próximo das 20 horas semanais, sem alterações importantes entre 1993 e 2000. Concluiu-se que, no essencial, a publicação da nova legislação sobre organização de cuidados de MT/SHST/SO em 1994 e 1995 não reforçou significativamente as condições gerais de exercício da medicina do trabalho.

Chlorite Dismutase from perchlorate reducing bacteria. A potential biocatalyst to eliminate chlorinated species from polluted water resources and industrial wastes

Magnetospirillum (M.) sp. strain Lusitani, a perchlorate reducing bacteria (PRB), was previously isolated from a wastewater treatment plant and phylogenetic analysis was performed to classify the isolate. The DNA sequence of the genes responsible for perchlorate reduction and chlorite dismutation was determined and a model was designed based on the physiological roles of the proteins involved in the pcr-cld regulon. Chlorite dismutase (Cld) was purified from Magnetospirillum sp. strain Lusitani cells grown in anaerobiosis in the presence of perchlorate. The protein was purified up to electrophoretic grade using HPLC techniques as a 140 kDa homopentamer comprising five ~28 kDa monomers. Steady-state kinetic studies showed that the enzyme follows a Michaelis-Menten model with optimal pH and temperature of 6.0 and 5°C, respectively. The average values for the kinetic constants KM and Vmax were respectively 0.56 mM and 10.2 U, which correspond to a specific activity of 35470 U/mg and a turnover number of 16552 s-1. Cld from M. sp. strain Lusitani is inhibited by the product chloride, but not by dioxygen. Inhibition constants KiC= 460 mM and KiU= 480 mM indicated that sodium chloride is a weak mixed inhibitor of Cld, with a slightly stronger competitive character. The X-ray crystallography structure of M. sp. strain Lusitani Cld was solved at 3.0 Å resolution. In agreement with cofactor content biochemical analysis, the X-ray data showed that each Cld monomer harbors one heme b coordinated by a histidine residue (His188), hydrogen-bonded to a conserved glutamic acid residue (Glu238). The conserved neighboring arginine residue (Arg201) important for substrate positioning, was found in two different conformations in different monomers depending on the presence of the exogenous ligand thiocyanate. UV-Visible and CW-EPR spectroscopies were used to study the effect of redox agents, pH and exogenous ligands on the heme environment.

Estudo da influência de nanotubos de carbono funcionalizados nas propriedades de revestimentos de poliuretano

Dissertação de mestrado em Propriedades e Tecnologias de Polímeros

Mechanism of extracellular silver nanoparticles synthesis by Stereum hirsutum and Fusarium oxysporum

The increasing interest for greener and biological methods of synthesis has led to the development of non-toxic and comparatively more bioactive nanoparticles. Unlike physical and chemical methods of nanoparticle synthesis, microbial synthesis in general and mycosynthesis in particular is cost-effective and environment-friendly. However, different aspects, such as the rate of synthesis, monodispersity and downstream processing, need to be improved. Many fungal-based mechanisms have been proposed for the formation of silver nanoparticles (AgNPs), mainly those involving the presence of nitrate reductase, which has been detected in filtered fungus cell used for AgNPs production. There is a general acceptance that nitrate reductase is the main responsible for the reduction of Ag ions for the formation of AgNPs. However, this generally accepted mechanism for fungal AgNPs production is not totally understood. In order to elucidate the molecules participating in the mechanistic formation of metal nanoparticles, the current study is focused on the enzymes and other organic compounds involved in the biosynthesis of AgNPs. The use of each free fungal mycelium of both Stereum hirsutum and Fusarium oxysporum will be assessed. In order to identify defective mutants on the nitrate reductase structural gene niaD, fungal cultures of S.hirsutum and F.oxysporum will be selected by chlorate resistance. In addition, in order to verify if each compound identified as key-molecule influenced on the production of nanoparticles, an in vitro assay using different nitrogen sources will be developed. Lately, fungal extracellular enzymes will be measured and an in vitro assay will be done. Finally, The nanoparticle formation and its characterization will be evaluated by UV-visible spectroscopy, electron microscopy (TEM), X-ray diffraction analysis (XRD), Fourier transforms infrared spectroscopy (FTIR), and LC-MS/MS.

Structural, optical and magnetic properties of pulsed laser deposited Co-doped ZnO films

Zn1−xCoxO films with different Co concentrations (with x=0.00, 0.10, 0.15, and 0.30) were grown by pulsed laser deposition (PLD) technique. The structural and optical properties of the films were investigated by grazing incidence X-ray diffraction (GIXRD), Raman spectroscopy and photoluminescence (PL). The magnetic properties were measured by conventional magnetometry using a SQUID and simulated by ab-initio calculations using Korring–Khon–Rostoker (KKR) method combined with coherent potential approximation (CPA). The effect of Co-doping on the GIXRD and Raman peaks positions, shape and intensity is discussed. PL studies demonstrate that Co-doping induces a decrease of the bandgap energy and quenching of the UV emission. They also suggest the presence of Zn interstitials when x≥0.15. The 10% Co-doped ZnO film shows ferromagnetism at 390 K with a spontaneous magnetic moment ≈4×10−5 emu and coercive field ≈0.17 kOe. The origin of ferromagnetism is explained based on the calculations using KKR method.