Ligand K-edge X-ray absorption spectroscopy and DFT calculations on [Fe3S4]0,+ clusters: delocalization, redox, and effect of the protein environment

Ligand K-edge XAS of an [Fe3S4]0 model complex is reported. The pre-edge can be resolved into contributions from the í2Ssulfide, í3Ssulfide, and Sthiolate ligands. The average ligand-metal bond covalencies obtained from these pre-edges are further distributed between Fe3+ and Fe2.5+ components using DFT calculations. The bridging ligand covalency in the [Fe2S2]+ subsite of the [Fe3S4]0 cluster is found to be significantly lower than its value in a reduced [Fe2S2] cluster (38% vs 61%, respectively). This lowered bridging ligand covalency reduces the superexchange coupling parameter J relative to its value in a reduced [Fe2S2]+ site (-146 cm-1 vs -360 cm-1, respectively). This decrease in J, along with estimates of the double exchange parameter B and vibronic coupling parameter ì2/k-, leads to an S ) 2 delocalized ground state in the [Fe3S4]0 cluster. The S K-edge XAS of the protein ferredoxin II (Fd II) from the D. gigas active site shows a decrease in covalency compared to the model complex, in the same oxidation state, which correlates with the number of H-bonding interactions to specific sulfur ligands present in the active site. The changes in ligand-metal bond covalencies upon redox compared with DFT calculations indicate that the redox reaction involves a two-electron change (one-electron ionization plus a spin change of a second electron) with significant electronic relaxation. The presence of the redox inactive Fe3+ center is found to decrease the barrier of the redox process in the [Fe3S4] cluster due to its strong antiferromagnetic coupling with the redox active Fe2S2 subsite.

Comparação dos Perfis Lipídicos de Plasma de uma População da Região Norte

Os ácidos gordos desempenham um papel fisiológico importante como componentes indispensáveis na estrutura celular, bem como fontes de energia. Nas últimas décadas, tem havido um aumento notável do interesse público nos ácidos gordos polinsaturados ómegas 3 e 6 e no seu impacto sobre a saúde humana, especialmente em doenças metabólicas e cardiovasculares. Estes ácidos gordos específicos podem prevenir e/ou tratar várias patologias metabólicas, atuando nomeadamente como compostos anti-inflamatórios. A menopausa é um fator de risco para doença cardiovascular, a diminuição de estrogénio, que ocorre neste estado fisiológico, provoca disfunção endotelial e stresse oxidativo. Consequentemente há uma redução dos níveis de ácidos gordos polinsaturados ómegas 3, o que contribui para o aparecimento de aterosclerose e doença cardiovascular. Neste contexto, o objetivo deste estudo foi avaliar e caracterizar o perfil lipídico de ácidos gordos de uma amostra de mulheres pós-menopausa e com este, estudar as associações entre o perfil lipídico determinado e parâmetros metabólicos de risco (parâmetros clínicos e bioquímicos). Inicialmente, os ácidos gordos foram extraídos da matriz plasmática através da derivatização destes e a sua composição percentual no plasma foi determinada com recurso a cromatografia gasosa com deteção de ionização de chama. De seguida, através do software IBM SPSS Statistics 21, foram estabelecidas associações entre os parâmetros clínicos e bioquímicos e o perfil lipídico determinado. A população em estudo foi divida em dois grupos consoante o período de entrada na menopausa (há menos de 7 anos e há 7 anos ou mais). Não há conhecimento de estudos semelhantes ao apresentado, que relacionem todo o perfil de ácidos gordos com parâmetros metabólicos de risco considerando o estado menopausal. Os resultados obtidos mostram que o perfil lipídico influencia vários marcadores metabólicos / endócrinos com relevância clínica que devem ser explorados em futuros ensaios clínicos. Para as mulheres na menopausa há menos de 7 anos foram estabelecidas as seguintes relações: i) entre os ácidos gordos saturados e insaturados cis e os níveis de ALP; ii) entre os ácidos gordos mono e polinsaturados cis e os níveis de GGT, IL10 e estradiol; iii) entre os ácidos gordos polinsaturados trans e o IMC e os níveis de IL6; iv) entre os ómegas 3 e os níveis de IL10 e ácido úrico; v) entre os ómegas 6 e os níveis de estradiol, ALP e GGT; vi) entre os ómegas 9 e os níveis de estradiol e GGT; vi) entre os ácidos gordos de curta cadeia e os níveis de colesterol total, LDL, triglicerídeos e IL10; vii) entre os ácidos gordos saturados de cadeia longa e o ΣÁcido láurico, mirístico, palmítico e esteárico e os níveis de triglicerídeos, ALP e GGT; viii) os níveis de IL10 podem ser simultaneamente associados com os ácidos gordos de curta cadeia e os ómegas 3. Para as mulheres na menopausa há 7 anos ou mais foram estabelecidas relações: i) entre os ómegas 3 e o IMC e os níveis de triglicerídeos; ii) entre os ácidos gordos monoinsaturados cis e os ómegas 9 com os níveis de ALT. Relações independentes do estado menopausal também foram estabelecidas, nomeadamente: i) entre os ácidos gordos polinsaturados cis e ómegas 6 e os níveis de ALT, triglicerídeos e AST; ii) entre os níveis de ácidos gordos monoinsaturados cis e ómegas 9 e os níveis de AST e triglicerídeos. O perfil lipídico de ácidos gordos pode ser considerado um biomarcador para a condição de saúde da mulher na menopausa.

Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

Application of ionic liquids and enzymes for the removal of proteinaceous layers from polychrome of works of art and evaluation of the cleaning effectiveness

Dissertação Apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Ciências da Conservação, especialização em Pintura

Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.

Streptococcus anginosus como agentes de infeção em Humanos: um estudo epidemiológico numa taxonomia em mudança

Dissertação para obtenção do grau de Mestre em Microbiologia Médica

Cylindrical spinning rotor gauge — A new approach for vacuum measurement

The spinning rotor gauge (SRG) is one of the most interesting vacuum gauges ever made, covering a pressure range of over seven orders of magnitude, with minimal gas interference (no pumping, ionization or heating of the measured gas), and a great stability of less than 1% drift per year. But despite its remarkable properties, apparently the SRG has not been further developed since the eighties, when it gained commercial interest. In this context, this dissertation aims at providing a starting point for a new line of investigation regarding this instrument, focused on the rotor itself. A brief study of different rotor geometries is provided, including a comparison between a cylindrical rotor and a spherical one. A cylindrical spinning rotor gauge (CSRG) is then proposed, based on the original SRG, but requiring a completely new lateral damping system. A prototype was built and tested against a non calibrated reference gauge.

Identificação e quantificação de adutos covalentes formados entre a valina Nterminal da hemoglobina e o fármaco nevirapina em doentes de HIV: Desenvolvimento e validação de um método analítico por espectrometria de massa de alta resolução

A nevirapina é o fármaco antirretroviral mais utilizado nos países em desenvolvimento para combater o vírus da imunodeficiência humana (HIV). Apesar dos seus efeitos benéficos, a nevirapina tem sido associada a casos de hipersensibilidade cutânea e hepatoxicidade graves ou mesmo fatais. Estudos recentes sugerem o envolvimento da bioactivação do seu metabolito de Fase I, 12-OHNevirapina, a espécies electrofílicas capazes de formar adutos covalentes com proteínas. A identificação dos adutos formados, por esta via de bioactivação, com a valina N-terminal da hemoglobina (Hb) isolada de doentes sob terapêutica com a nevirapina foi já efectuada. A técnica analítica utilizada envolveu a utilização do método de derivatização N-alquilo Edman que, por reacção com fenilisotiocianato (PITC), permite destacar o aduto formado com o resíduo N-terminal da Hb, sob a forma de uma hidantoina, que foi posteriormente analisado e quantificado por cromatografia líquida acoplado à espectrometria de massa de tandem por ionização de electrospray. O presente trabalho teve como objectivo inicial optimizar o procedimento experimental envolvido no procedimento de N-alquilo Edman, tendo-se inicialmente comparado a sensibilidades obtidas mediante a utilização de dois agentes derivatizante: o PITC e o isotiocianato de fluoresceína (FTIC). Este processo inicial permitiu estabelecer que a utilização do PTIC como agente de derivatização conduzia a melhores resultados, tendo-se prosseguido com o desenvolvimento e a validação do método analítico para a quantificação deste aduto por cromatografia líquida acoplado a espectrometria de massa de alta resolução (LC-HRMS). O método analítico desenvolvido apresentou recuperações entre 36 e 57 %, supressão de sinal de 19 % e um limite de quantificação (LOQ) de 1,4 ppb (média da exactidão = 98 %; CV = 5 %). A metodologia validada foi depois aplicada na quantificação de adutos de nevirapina em duas amostras de Hb isolada de doentes de HIV e os níveis de adutos obtidos foram de 95 e 92 pmol/g de Hb.

Advances in microbial diagnosis: using mass spectrometry for proteomics and genomics applications

Since the last two decades mass spectrometry (MS) has been applied to analyse the chemical cellular components of microorganisms, providing rapid and discriminatory proteomic profiles for their species identification and, in some cases, subtyping. The application of MS for the microbial diagnosis is currently well-established. The remarkable reproducibility and objectivity of this method is based on the measurement of constantly expressed and highly abundant proteins, mainly important conservative ribosomal proteins, which are used as markers to generate a cellular fingerprint. Mass spectrometry based on matrix-assisted laser desorption ionization-time of flight (MALDI- TOF) technique has been an important tool for the microbial diagnostic. However, some technical limitation concerning both MALDI-TOF and its used protocols for sample preparation have fostered the research of new mass spectrometry systems (e.g. LC MS/MS). LC MS/MS is able to generate online mass spectra of specific ions with further online sequencing of these ions, which include both specific proteins and DNA fragments. In this work a set of data for yeasts and filamentous fungi diagnostic obtained through an international collaboration project involving partners from Argentina, Brazil, Chile and Portugal will be presented and discussed.

Development of magnetoelectric CoFe2O4/poly(vinylidene fluoride) microspheres

Magnetoelectric microspheres based on piezoelectric poly(vinylidene fluoride) (PVDF) and magnetrostrictive CoFe2O4 (CFO), a novel morphology for polymer-based ME material, have been developed by an electrospray process. The CFO nanoparticles content in the (3-7 μm diameter) microspheres reaches values up to 27 wt.%, despite their concentration in the starting solution reaching values up to 70 wt.%. Additionally, the inclusion of magnetostrictive nanoparticles into the polymer spheres has no relevant effect on the piezoelectric β-phase content (≈60%), crystallinity (40%) and the onset degradation temperature (460º-465ºC) of the polymer matrix. The multiferroic microspeheres show a maximum piezoelectric reponse |d33|≈30 pC.N-1, leading to a magnetoelectric response of Δ|d33|≈5 pC.N-1 obtained when a 220 mT DC magnetic field was applied. It is also shown that the interface between CFO nanoparticles and PVDF (from 0 to 55%) has a strong influence on the ME response of the microspheres. The simplicity and the scalability of the processing method suggest a large application potential of this novel magnetoelectric geometry in areas such as tissue engineering, sensors and actuators.

Electrochemical vs antibacterial characterization of ZrCN-Ag coatings

Nowadays, antibacterial properties are becoming a viable feature to be introduced in biomaterials due to the possibility of modifying the materials' surface used in medical devices in a micro/nano metric scale. As a result, it is mandatory to understand the mechanisms of the antimicrobial agents currently used and their possible failures. In this work, the antibacterial activity of ZrCNAg films is studied, taking into consideration the ability of silver nanoparticles to be dissolved when embedded into a ceramic matrix. The study focuses on the silver release evaluated by glow discharge optical emission spectroscopy and the effect of the fluid composition on this release. The results revealed a very low silver release of the films, leading to non-antibacterial activity of such materials. The silver release was found to be dependent on the electrolyte composition. NaCl (8.9 g L? 1) showed the lowest spontaneously silver ionization, while introducing the sulfates in Hanks' balanced salt solution (HBSS) such ionization is increased; finally, the proteins incorporated to the (HBSS) showed a reduction of the silver release, which also explains the low ionization in the culture medium (tryptic soy broth) that contains high quantities of proteins.

Mesenchymal stem cells culture on poly(vinylidene fluoride) piezoelectric microspheres

Dissertação de mestrado em Biofísica e Bionanossistemas

Desenvolvimento de métodos analíticos para controlo de qualidade de resinas naturais

Dissertação de mestrado em Técnicas de Caracterização e Análise Química

Antibacterial properties of multifunctional coatings Ag-ZrCN for biomedical devices: a novel approach

PhD in Sciences Specialty in Physics

Enzymatic hydrophobic modification of jute fibers via grafting to reinforce composites

Horseradish peroxidase (HRP)/H2O2 system catalyzes the free-radical polymerization of aromatic compounds such as lignins and gallate esters. In this work, dodecyl gallate (DG) was grafted onto the surfaces of lignin-rich jute fabrics by HRP-mediated oxidative polymerization with an aim to enhance the hydrophobicity of the fibers. The DG-grafted jute fibers and reaction products of their model compounds were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results clearly indicated the grafting of DG to the jute fiber by HRP. Furthermore, the hydrophobicity of jute fabrics was determined by measuring the wetting time and static contact angle. Compared to the control sample, the wetting time and static contact angle of the grated fabrics changed from ~1 s to 1 h and from ~0° to 123.68°, respectively. This clearly proved that the hydrophobicity of jute fabrics improved considerably. Conditions of the HRP-catalyzed DG-grafting reactions were optimized in terms of the DG content of modified jute fabrics. Moreover, the results of breaking strength and elongation of DG-grafted jute/ polypropylene (PP) composites demonstrated improved reinforcement of the composite due to enzymatic hydrophobic modification of jute fibers.