Modification of cell volume and proliferative capacity of Pseudokirchneriella subcapitata cells exposed to metal stress

The impact of metals (Cd, Cr, Cu and Zn) on growth, cell volume and cell division of the freshwateralga Pseudokirchneriella subcapitata exposed over a period of 72 h was investigated. The algal cells wereexposed to three nominal concentrations of each metal: low (closed to 72 h-EC10values), intermediate(closed to 72 h-EC50values) and high (upper than 72 h-EC90values). The exposure to low metal concen-trations resulted in a decrease of cell volume. On the contrary, for the highest metal concentrations anincrease of cell volume was observed; this effect was particularly notorious for Cd and less pronouncedfor Zn. Two behaviours were found when algal cells were exposed to intermediate concentrations ofmetals: Cu(II) and Cr(VI) induced a reduction of cell volume, while Cd(II) and Zn(II) provoked an oppositeeffect. The simultaneous nucleus staining and cell image analysis, allowed distinguishing three phases inP. subcapitata cell cycle: growth of mother cell; cell division, which includes two divisions of the nucleus;and, release of four autospores. The exposure of P. subcapitata cells to the highest metal concentrationsresulted in the arrest of cell growth before the first nucleus division [for Cr(VI) and Cu(II)] or after thesecond nucleus division but before the cytokinesis (release of autospores) when exposed to Cd(II). Thedifferent impact of metals on algal cell volume and cell-cycle progression, suggests that different toxic-ity mechanisms underlie the action of different metals studied. The simultaneous nucleus staining andcell image analysis, used in the present work, can be a useful tool in the analysis of the toxicity of thepollutants, in P. subcapitata, and help in the elucidation of their different modes of action.

Bionanoconjugados de Citocromo Tri- hémico e nanopartículas de Ouro para biorremediação de Cr(VI)

Dissertação para a obtenção do grau de Mestre em Biotecnologia

Heparinized nanohydroxyapatite/collagen granules for controlled release of vancomycin

The purpose of this study was to develop a bone substitute material capable of preventing or treating osteomyelitis through a sustainable release of vancomycin and simultaneously inducing bone regeneration. Porous heparinized nanohydroxyapatite (nanoHA)/collagen granules were characterized using scanning electron microscopy, micro-computed tomography and attenuated total reflectance Fourier transform infrared spectroscopy. After vancomycin adsorption onto the granules, its releasing profile was studied by UV molecular absorption spectroscopy. The heparinized granules presented a more sustainable release over time, in comparison with nonheparinized nanoHA and nanoHA/collagen granules. Vancomycin was released for 360 h and proved to be bioactive until 216 h. Staphylococcus aureus adhesion was higher on granules containing collagen, guiding the bacteria to the material with antibiotic, improving their eradication. Moreover, cytotoxicity of the released vancomycin was assessed using osteoblast cultures, and after 14 days of culture in the presence of vancomycin, cells were able to remain viable, increasing their metabolic activity and colonizing the granules, as observed by scanning electron microscopy and confocal laser scanning microscopy. These findings suggest that heparinized nanoHA/collagen granules are a promising material to improve the treatment of osteomyelitis, as they are capable of releasing vancomycin, eliminating the bacteria, and presented morphological and chemical characteristics to induce bone regeneration.

Comparação dos métodos ENF e 4-ENF para determinação da tenacidade ao corte de juntas adesivas

Ao longo destes últimos anos as ligações adesivas têm vindo a verificar um aumento progressivo em aplicações estruturais em detrimento das ligações mecânicas convencionais. Esta alteração de paradigma deve-se às vantagens que as juntas adesivas possuem relativamente aos outros métodos de ligação. A mecânica da fratura e os Modelos de Dano Coesivo (MDC) são critérios comuns para prever a resistência em juntas adesivas e usam como parâmetros fundamentais as taxas de libertação de energia. Pelo facto do ensaio 4-Point End Notched Flexure (4-ENF), aplicado em juntas adesivas, ainda estar pouco estudado é de grande relevância um estudo acerca da sua viabilidade para a determinação da taxa crítica de libertação de energia de deformação ao corte (GIIc). Esta dissertação tem como objetivo principal efetuar uma comparação entre os métodos End- Notched Flexure (ENF) e 4-ENF na determinação de GIIc em juntas adesivas. Para tal foram utilizados 3 adesivos: Araldite® AV138, Araldite® 2015 e SikaForce® 7752. O trabalho experimental passou pela conceção e fabrico de uma ferramenta para realização do ensaio 4-ENF, seguindo-se o fabrico e a preparação dos provetes para os ensaios. Pelo facto do ensaio 4-ENF ainda se encontrar pouco divulgado em juntas adesivas, e não se encontrar normalizado, uma parte importante do trabalho passou pela pesquisa e análise em trabalhos de investigação e artigos científicos. A análise dos resultados foi realizada por comparação direta dos valores de GIIc com os resultados obtidos no ensaio ENF, sendo realizada por série de adesivo, através da comparação das curvas P-δ e curvas-R. Como resultado verificou-se que o ensaio 4-ENF em ligações adesivas não é o mais versátil para a determinação do valor de GIIc, e que apenas um método de obtenção de GIIc é viável. Este método é baseado na medição do comprimento de fenda (a). Ficou evidenciado que o ensaio ENF, devido a ser um ensaio normalizado, por apresentar um setup mais simples e por apresentar uma maior disponibilidade de métodos para a determinação do valor de GIIc, é o mais recomendado. Conclui-se assim que o ensaio 4-ENF, embora sendo uma alternativa ao ensaio ENF, tem aplicação mais limitada.

Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria

Biochemistry, 2003, 42 (10), pp 3070–3080 DOI: 10.1021/bi026979d

O Códice Polifónico de Arouca. Estudo e Transcrição

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Ciências Musicais/Variante de Musicologia Histórica

Produção de carbonatos utilizando dióxido de carbono

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Double-walled PLLA/PLGA particles for the sustained release of Meloxicam: preparation, characterisation and contolled release studies

There were two main objectives in this thesis investigation, first, the production, characterisation, in vitro degradation and release studies of double walled microspheres for drug release control. The second one, and the most challenging, was the production of double walled nanospheres, also for drug control delivery. The spheres were produced using two polymers, the Poly(L-lactide)Acid, PLLA, and the Poly(L-lactide-co-glycolic)Acid, PLGA.Afterwards, a model drug, Meloxicam, which is an antiinflammatory drug, was encapsulated into the particles. Micro and nanospheres were produced by the solvent extraction/evaporation method, where perfect spherical particles were obtained. By varying the polymers PLLA/PLGA mass ratio, different core and shell composition, as well as several shell and core thickness were observed. In the particles with a PLLA/PLGA mass ratio 1:1, the shell is composed by PLLA and the core by PLGA. It was also verified that the Meloxicam has a tendency to be distributed in the PLGA layer. Micro and nanoparticles were characterised in morphology, size, polymer cristalinity properties and drug distribution. Particles degradation studies was performed, where the particles in a PVA solution of pH 7,4 where placed in an incubator, during approximately 40 days, at 120rpm, and 37ºC, simulating, as much as possible, the human body environment. From these studies, the conclusion was that particles containing a PLGA shell and a PLLA core degrade more rapidly, due to the fact that PLLA is more hydrophobic than the PLGA. Concerning the drug release controlled results, done also for 40 and 50 days, they showed that the microspheres containing a shell of PLLA release more slowly than when the shell is composed of PLGA. This result was predictable, since the drug is solubilised in the PLGA polymer and so, in that case, the PLLA shell works like a barrier between the drug and the outer medium. Another positive aspect presented by this study is the lower initial burst effect, obtained when using double walled particles, which is one of the advantages of the same. In a second part of this investigation, the production of the nanospheres was the main goal, since it was not yet accomplished by other authors or investigators. After several studies, referring to the speed, time and type of agitation, as well as, the concentration and volume of the first aqueous solution of poly-vinyl-alcohol (PVA) during the process of solvent extraction/evaporation it was possible to obtain double walled nanospheres.(...)

Da assessoria ao jornalismo : análise do processo de produção de press release na era digital em Portugal

A presente dissertação pretende fazer a análise do processo de produção do press release na assessoria em Portugal, a eficácia dessa ferramenta de comunicação junto dos jornalistas e, por inerência, a evolução da figura do assessor enquanto profissional reconhecido junto da comunidade jornalística. São também objetivos, compreender a relevância de um press release, perceber se gera efeito, analisar a possível forma de melhorar esta ferramenta e, ainda, perceber se esta ferramenta sofreu algum tipo de adaptação à era digital. A investigação inicia-se com a incursão pelos contextos e história das áreas profissionais em estudo, a assessoria de comunicação em agências e o jornalismo em Portugal, no quadro da crise económica e financeira de 2008 a 2013. O enfoque deste estudo será o procedimento e a eficácia de um press release, no período considerado. A pós-produção desta ferramenta implica o contacto entre dois interlocutores, os profissionais de assessoria e os profissionais do jornalismo. Finda esta investigação com análise baseada em seis entrevistas semiestruturadas, divididas em categorias profissionais e setores de atividade: jornalistas, assessores, assessores ex-jornalistas, nas áreas de saúde e consumo. Deste estudo resulta que o press release, privilegiando-se a sua estrutura e conteúdo, é, como foi, uma ferramenta fundamental muitas vezes, e nos dias de hoje, no auxílio às negociações one to one.

Analysis of metabolic flux distributions in relation to the extracellular environment in Avian cells

Continuous cell lines that proliferate in chemically defined and simple media have been highly regarded as suitable alternatives for vaccine production. One such cell line is the AG1.CR.pIX avian cell line developed by PROBIOGEN. This cell line can be cultivated in a fully scalable suspension culture and adapted to grow in chemically defined, calf serum free, medium [1]–[5]. The medium composition and cultivation strategy are important factors for reaching high virus titers. In this project, a series of computational methods was used to simulate the cell’s response to different environments. The study is based on the metabolic model of the central metabolism proposed in [1]. In a first step, Metabolic Flux Analysis (MFA) was used along with measured uptake and secretion fluxes to estimate intracellular flux values. The network and data were found to be consistent. In a second step, Flux Balance Analysis (FBA) was performed to access the cell’s biological objective. The objective that resulted in the best predicted results fit to the experimental data was the minimization of oxidative phosphorylation. Employing this objective, in the next step Flux Variability Analysis (FVA) was used to characterize the flux solution space. Furthermore, various scenarios, where a reaction deletion (elimination of the compound from the media) was simulated, were performed and the flux solution space for each scenario was calculated. Growth restrictions caused by essential and non-essential amino acids were accurately predicted. Fluxes related to the essential amino acids uptake and catabolism, the lipid synthesis and ATP production via TCA were found to be essential to exponential growth. Finally, the data gathered during the previous steps were analyzed using principal component analysis (PCA), in order to assess potential changes in the physiological state of the cell. Three metabolic states were found, which correspond to zero, partial and maximum biomass growth rate. Elimination of non-essential amino acids or pyruvate from the media showed no impact on the cell’s assumed normal metabolic state.

Diagnosis of latent Mycobacterium tuberculosis infection: tuberculin test versus interferon-gamma release

Abstract: INTRODUCTION: The treatment of individuals with active tuberculosis (TB) and the identification and treatment of latent tuberculosis infection (LTBI) contacts are the two most important strategies for the control of TB. The objective of this study was compare the performance of tuberculin skin testing (TST) with QuantiFERON-TB Gold In TUBE(r) in the diagnosis of LTBI in contacts of patients with active TB. METHODS: Cross-sectional analytical study with 60 contacts of patients with active pulmonary TB. A blood sample of each contact was taken for interferon-gamma release assay (IGRA) and subsequently performed the TST. A receiver operating characteristic curve was generated to assess the cutoff points and the sensitivity, predictive values, and accuracy were calculated. The agreement between IGRA and TST results was evaluated by Kappa coefficient. RESULTS: Here, 67.9% sensitivity, 84.4% specificity, 79.1% PPV, 75% NPV, and 76.7% accuracy were observed for the 5mm cutoff point. The prevalence of LTBI determined by TST and IGRA was 40% and 46.7%, respectively. CONCLUSIONS: Both QuantiFERON-TB Gold In TUBE(r) and TST showed good performance in LTBI diagnosis. The creation of specific diagnostic methods is necessary for the diagnosis of LTBI with higher sensitivity and specificity, preferably with low cost and not require a return visit for reading because with early treatment of latent forms can prevent active TB.

Blocking tumor exosome release using nanovectorization systems

Exosomes are small membrane vesicles secreted by most cell types, either normal or malignant and are found in most body fluids such as saliva, plasma and breast milk. In the past decade, the interest in these vesicles has been growing more and more since it was found that besides their beneficial functions such as the removal of cellular debris and unnecessary proteins during cell maturation process, they can also interact with other cells and transfer information between them, thus helping diseases like cancer to progress. The present work intended to use gold nanoparticles as vehicles for gene silencing in an attempt to reduce the tumor-derived exosome secretion, regulated by Rab27a protein, and also aimed to compare the exosome secretion between two breast cell lines, MCF7 and MDA. Changes in RAB27A gene expression were measured by Real-time Quantitative PCR and it was revealed a decreased in RAB27A gene expression, as expected. Exosomes were isolated and purified by two different methods, ultracentrifugation and the commercial kit ExoQuick™ Solution, and further characterized using Western Blot analysis. ExoQuick™ Solution was proven to be the most efficient method for exosome isolation and it was revealed that MDA cells secrete more exosomes. Furthermore, the isolated MCF7-derived exosomes were placed together with a normal bronchial/tracheal epithelial cell line (BTEC) for an additional assay, which aimed to observe the uptake of exosomes by other cells and the exosomes’ capability of promoting cell-cell communication. This observation was made based on alterations in the expression levels of c-Myc and miR-21 genes and the fact that they both have an increased expression in BTEC cells incubated with tumor-derived exosomes when compared to control cells (without incubation with the exosomes) lead us to the conclusion that the exosome uptake and exchange of information between the exosomes and the normal cells did occurred.

Analysis of chromium behaviour and speciation during the electrodialytic process

The interest in chromium (Cr) arises from the widespread use of this heavy metal in various industrial processes that cause its release as liquid, solid and gaseous waste into the environment. The impact of Cr on the environment and living organisms primarily depends on its chemical form, since Cr(III) is an essential micronutrient for humans, other animals and plants, and Cr(VI) is highly toxic and a known human carcinogen. This study aimed to evaluate if the electrodialytic process (ED) is an appropriate treatment for Cr removal, through a critical overview of Cr speciation, before and after the ED experiments, to assess possible Cr(III)-Cr(VI) interconversions during the treatment. ED was the treatment technique applied to two types of matrices containing Cr: chromate copper arsenate (CCA) contaminated soil and municipal solid waste incineration (MSWI) fly ash. In order to study Cr remediation, three EDR set-ups were used: a new set-up, the combined cell (2/3C or 3/2C), with three compartments, alternating current between two anodes and different initial experimental conditions, one set-up with three compartments (3C cell) and the other set-up with two compartments (2C cell). The Cr removal rates obtained in this study were between 10-36% for the soil, and 1-13% for the fly ash. The highest Cr removal rates were achieved in the 26 days experiments: 36% for the soil, 13% for the fly ash. Regarding the 13 days experiments, the highest Cr removal rates were attained with the 2/3C set-up: 24% for the soil, 5% for the fly ash. The analysis of Cr(VI) was performed before and after ED experiments to evaluate eventual changes in Cr speciation during the treatment. This analysis was conducted by two methods: USEPA Method 3060A, for the extraction of Cr(VI); and Hach Company Method 8023, for the detection of Cr(VI). Despite the differences in Cr total concentration, both matrices presented a similar speciation, with Cr(III) being the main species found and Cr(VI) less than 3% of Cr total, before and after the treatment. For fly ash, Cr(VI) was initially below the detection limit of the method and remained that way after the treatment. For soil, Cr(VI) decreased after the treatment. Oxidation of Cr(III) to Cr(VI) did not occur during the ED process since there was no increase in Cr(VI) in the matrices after the treatment. Hence, the results of this study indicate that ED is an appropriate technique to remediate matrices containing Cr because it contributes to Cr removal, without causing Cr(III)-Cr(VI) interconversions.