The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus

Dissertation for the Master’s Degree in Structural and Functional Biochemistry

Avaliação de emissões de nanopartículas resultantes dos processos de soldadura TIG e SER.

Dissertação para obtenção do Grau de Mestre em Engenharia da Soldadura

Characterization of processed materials by electrical currents: development of equipment and applications

Dissertação para obtenção do Grau de Mestre em Engenharia Mecânica

Printable organic and inorganic materials for flexible electrochemical devices

Portuguese Science Foundation - project Electra PTDC/CTM/099124/2008 and the PhD grant SFRH/BD/45224. financial support: Professor E. Fortunato’s ERC 2008 Advanced Grant (INVISIBLE contract number 228144), “APPLE” FP7-NMP-2010-SME/262782-2 and “SMARTEC” FP7-ICT-2009.3.9/258203

Assessment of Notch1 ligands production - key protein targets in breast cancer

Cell-to-cell communication is required for many biological processes in development and adult life. One of the most common systems utilized by a wide range of eukaryotes is the Notch signalling pathway. Four Notch receptors and five ligands have been identified in mammals that interact via their extracellular domains leading to transcription activation. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer. Thus, any of the Notch1 ligands can be studied as possible therapeutic targets for breast cancer. To study Notch pathway proteins there is the need to obtain stable protein solutions. E. coli is the host of excellence for recombinant proteins for the ease of use, fast growth and high cell densities. However, the expression of mammalian proteins in such systems may overwhelm the bacterial cellular machinery, which does not possess the ability for post-translational modifications, or dedicated compartments for protein synthesis. Mammalian cells are therefore preferred, despite their technical and financial increased demands. We aim to determine the best expression and purification conditions for the different ligand protein constructs, to develop specific function-blocking antibodies using the Phage Display technology. Moreover, we propose to crystallize the Notch1 ligands alone and in complex with the phage display selected antibodies, unveiling molecular details. hJag2DE3 and hDll1DE6 proteins were purified from refolded inclusion bodies or mammalian cell culture supernatants, respectively, and purity was confirmed by SDS-PAGE (>95%). Protein produced in mammalian cells showed to be more stable, apparently with the physiological disulfide pattern, contrary to what was observed in the refolded protein. Several nano-scale crystallization experiments were set up in 96-well plates, but no positive result was obtained. We will continue to pursue for the best expression for the Notch ligand constructs in both expression systems.

Structural and functional characterization of ModA and TupA proteins belonging to the molybdate and tungstate transport system from desulfovibrio alaskensis G20

Fundação para a Ciência e Tecnologia - EXPL/BBB-BEP/0274/2012

Nanostructuring silicon probes via electrodeposition: characterization of electrode coatings for acute in vivo neural recordings

Understanding how the brain works will require tools capable of measuring neuron elec-trical activity at a network scale. However, considerable progress is still necessary to reliably increase the number of neurons that are recorded and identified simultaneously with existing mi-croelectrode arrays. This project aims to evaluate how different materials can modify the effi-ciency of signal transfer from the neural tissue to the electrode. Therefore, various coating materials (gold, PEDOT, tungsten oxide and carbon nano-tubes) are characterized in terms of their underlying electrochemical processes and recording ef-ficacy. Iridium electrodes (177-706 μm2) are coated using galvanostatic deposition under different charge densities. By performing electrochemical impedance spectroscopy in phosphate buffered saline it is determined that the impedance modulus at 1 kHz depends on the coating material and decreased up to a maximum of two orders of magnitude for PEDOT (from 1 MΩ to 25 kΩ). The electrodes are furthermore characterized by cyclic voltammetry showing that charge storage capacity is im-proved by one order of magnitude reaching a maximum of 84.1 mC/cm2 for the PEDOT: gold nanoparticles composite (38 times the capacity of the pristine). Neural recording of spontaneous activity within the cortex was performed in anesthetized rodents to evaluate electrode coating performance.

Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide

INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.

VO2 thin films and nanoparticles for energy-saving applications in architectural elements

Vanadium dioxide (VO2) is a promising material with large interest in construction industry and architecture, due to its thermochromic properties. This material may be used to create "smart" coatings that result in improvements in the buildings energy efficiency, by reducing heat exchanges and, consequently, the need for acclimatization. In this work, VO2 thin films and coatings were produced and tested in laboratory, to apply in architectural elements, such as glass, rooftop tiles and exterior paints. Thin films were produced by RF magnetron sputtering and VO2 nanoparticles were obtained through hydrothermal synthesis, aiming to create "smart" windows and tiles, respectively. These coatings have demonstrated the capability to modulate the transmittance of infrared radiation by around 20%. The VO2 nanoparticle coatings were successfully applied on ceramic tiles. The critical temperature was reduced to around 40ºC by tungsten doping. Ultimately, two identical house models were built, in order to test the VO2 coatings, in real atmospheric conditions during one of the hottest months of the year, in Portugal – August.

Lipoprotein (a): Structure, Pathophysiology and Clinical Implications

The chemical structure of lipoprotein (a) is similar to that of LDL, from which it differs due to the presence of apolipoprotein (a) bound to apo B100 via one disulfide bridge. Lipoprotein (a) is synthesized in the liver and its plasma concentration, which can be determined by use of monoclonal antibody-based methods, ranges from < 1 mg to > 1,000 mg/dL. Lipoprotein (a) levels over 20-30 mg/dL are associated with a two-fold risk of developing coronary artery disease. Usually, black subjects have higher lipoprotein (a) levels that, differently from Caucasians and Orientals, are not related to coronary artery disease. However, the risk of black subjects must be considered. Sex and age have little influence on lipoprotein (a) levels. Lipoprotein (a) homology with plasminogen might lead to interference with the fibrinolytic cascade, accounting for an atherogenic mechanism of that lipoprotein. Nevertheless, direct deposition of lipoprotein (a) on arterial wall is also a possible mechanism, lipoprotein (a) being more prone to oxidation than LDL. Most prospective studies have confirmed lipoprotein (a) as a predisposing factor to atherosclerosis. Statin treatment does not lower lipoprotein (a) levels, differently from niacin and ezetimibe, which tend to reduce lipoprotein (a), although confirmation of ezetimibe effects is pending. The reduction in lipoprotein (a) concentrations has not been demonstrated to reduce the risk for coronary artery disease. Whenever higher lipoprotein (a) concentrations are found, and in the absence of more effective and well-tolerated drugs, a more strict and vigorous control of the other coronary artery disease risk factors should be sought.

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

Pharmacokinetic profile of tert-butylaminoethanethiol

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethanethiol (TBAESH) was performed after administration of a single dose (35 mg/kg) either orally or intravenously. Plasma or blood samples were treated with dithiothreitol, perchloric acid and, after filtration, submitted to further purification with anionic resin. In the final step the drug was retained on a cationic resin column, eluted with NaCl lM and detected according to the method of Ellman (1958). The results suggested a pharmacokinetic behavior related to a one open compartment model with the following values for the total drug: area under the intravenous curve (AUC i.v.): 443(+ ou -) 24.0; AUC oral: 85.5(+ ou -) 14.5 ug min.ml(elevado a -1); elimination rate constant: 0.069(+ ou -) 0.0055 min(elevado a -1), biological half-life: 10.0(+ ou -) 0.80 min; distribution volume 1.15(+ ou -) 0.15 ml/g; biodisponibility: 0.19(+ ou -) 0.02. From a pharmacokinetic standpoint, TBAESH seems to have no advantage over the analogous disulfide compound.

Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL.

Unlike other tumor necrosis factor family members, the cytotoxic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L contains an unpaired cysteine residue (Cys(230)) in its receptor-binding domain. Here we show that the biological activity of both soluble recombinant TRAIL and cell-associated, full-length TRAIL is critically dependent on the presence of Cys(230). Mutation of Cys(230) to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200-fold, and the stability of its trimeric structure was reduced. In recombinant TRAIL, Cys(230) was found engaged either in interchain disulfide bridge formation, resulting in poorly active TRAIL, or in the chelation of one zinc atom per TRAIL trimer in the active, pro-apoptotic form of TRAIL.

Preparació de puntes de tungstè per a microscòpia d'efecte túnel (STM)

Es presenten els resultats experimentals obtinguts durant l’estudi sistemàtic realitzat de la preparació electroquímica de puntes de tungstè per al Microscopi d’Efecte Túnel (STM), fent servir dos electròlits: KOH i NaOH. L’estudi sobre la morfologia, longitud de la punta i radi de curvatura de la punta en funció del voltatge aplicat i les concentracions de l’electròlit es descriu al capítol 3. La caracterització de les puntes es va dur a terme, per una part, mitjançant un microscòpic electrònic de rastreig (SEM) i per l’altre banda, amb el ús de les puntes obtingudes al STM. En resumen, els resultats mostren que ambdós electròlits permeten obtenir puntes que es poden fer servir amb èxit per l’obtenció d’imatges amb l’STM. Les millors puntes són aquelles que s’obtenen dins de rangs de concentracions d’electròlit baixes, entre valor de 10 a 15% en pes pel NaOH i entre 10 i 20% pel KOH i rangs de voltatge entre 3 a 7 V pel NaOH i 4 a 8 V pel KOH. S’observa que es requereixen temps d’atac electroquímic menors fent servir com a electròlit NaOH. S’estudia, en el capítol 4, el tractament que requereix la punta per tal d’eliminar les impureses de la seva superfície. Es realitzen diferents proves amb tres mètodes de neteja: (1) tractament químic, (2) bombardeig iònic i (3) tractament tèrmic de recuit. En el capítol 5 del projecte s’analitzen les imatges d’una mostra d’or, Au(110), d’estructura coneguda, amb el microscopi d’efecte túnel STM) del laboratori fent servir les puntes obtingudes sota les condicions considerades òptimes. El resultat confirma el bon comportament de les puntes obtingudes sota les condicions descrites en els capítols anteriors i establert una pauta a seguir per obtenir puntes d’una manera senzilla i reproduïble.