FC - DQ | Dissertações de Mestrado e Teses de Doutoramento
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- 3D computational simulation and experimental characterization of polymeric stochastic network materials : case studies in reinforced eucalyptus office paper and nanofibrous materialsPublication . Curto, Joana Maria Rodrigues; Simões, Rogério Manuel dos Santos; Portugal, António Torres GarciaThe properties of stochastic fibrous materials like paper and nanowebs are highly dependent on those fibers from which the network structure is made. This work contributes to a better understanding of the effect of fiber properties on the network structural properties, using an original 3D fibrous material model with experimental validation, and its application to different fibrous materials used in reinforced Eucalyptus office paper and nanofibrous networks. To establish the relationships between the fiber and the final structural material properties, an experimental laboratorial plan has been executed for a reinforced fibrous structure, and a physical based 3D model has been developed and implemented. The experimental plan was dedicated to an important Portuguese material: the reinforced Eucalyptus based office paper. Office paper is the principal Portuguese paper industry product. This paper is mainly produced from Eucalyptus globulus bleached kraft pulp with a small incorporation of a softwood pulp to increase paper strength. It is important to access the contribution of different reinforcement pulp fibers with different biometry and coarseness to the final paper properties. The two extremes of reinforcement pulps are represented by a Picea abies kraft softwood pulp, usually considered the best reinforcement fiber, and the Portuguese pine Pinus pinaster kraft pulp. Fiber flexibility was determined experimentally using the Steadman and Luner method with a computerized acquisition device. When comparing two reinforcement fibers, the information about fiber flexibility and biometry is determinant to predict paper properties. The values presented correspond to the two extremes of fibers available as reinforcement fibers, regarding wall thickness, beating ability and flexibility values. Pinus pinaster has the thickest fiber wall, and consequently it is less flexible than the thinner wall fibers: Pinus sylvestris and Picea abies. Experimental results for the evolutions of paper properties, like paper apparent density, air permeability, tensile and tear strength, together with fiber flexibility for the two reinforcement fibers, constitute valuable information, also applicable for other reinforcement fibers, with fiber walls dimensions in this range. After having quantified the influence of fiber flexibility, we identified that this is as a key physical property to be included in our structural model. Therefore, we chose to develop a 3D network model that includes fiber bending in the z direction as an important parameter. The inclusion of fiber flexibility was done for the first time by Niskanen, in a model known as the KCL-Pakka model. We propose an extension of this model, with improvements on the fiber model, as well as an original computational implementation. A simulator has been developed from scratch and the results have been validated experimentally using handmade laboratory structures made from Eucalyptus fibers (hardwood fibers), and also Pinus pinaster, Pinus Sylvestris and Picea abies fibers, which are representative reinforcement fibers. Finally, the model was modified and extended to obtain an original simulator to nanofibrous materials, which is also an important innovation. In the network model developed in this work, the structure is formed by the sequential deposition of fibers, which are modeled individually. The model includes key papermaking fiber properties like morphology, flexibility, and collapsibility and process operations such as fiber deposition, network forming or densification. For the first time, the model considers the fiber microstructure level, including lumen and fiber wall thickness, with a resolution up to 0.05μm for the paper material case and 0.05nm for the nanofibrous materials. The computational simulation model was used to perform simulation studies. In the case of paper materials, it was used to investigate the relative influence of fiber properties such as fiber flexibility, dimensions and collapsibility. The developed multiscale model gave realistic predictions and enabled us to link fiber microstructure and paper properties. In the case of nanofibrous materials, the 3D network model was modified and implemented for Polyamide-6 electrospun and cellulose nanowebs. The influence of computational fiber flexibility and dimensions was investigated. For the Polyamide-6 electrospun network experimental results were compared visually with simulation results and similar evolutions were observed. For cellulose nanowebs the simulation study used literature data to obtain the input information for the nanocellulose fibers. The design of computer experiments was done using a space filling design, namely the Latin hypercube sampling design, and the simulations results were organized and interpreted using regression trees. Both the experimental characterization, and computational modeling, contributed to study the relationships between the polymeric fibers and the network structure formed.
- Alternative specimens in forensic toxicology: analysis of drugs of abusePublication . Margalho, Cláudia Isabel Reis; Rivadulla Lamas, Manuel Lopez; Gallardo Alba, Maria Eugénia; Gonçalves, Francisco Corte RealThe main reason for this study’s development lies essentially in the fact that it gathers two areas of great interest and increasing progress in the forensic toxicology field: the postmortem alternative matrices and the new psychoactive substances. Firstly, it is possible to verify that alternative matrices have been playing an important role in the forensic toxicology field throughout the last decade. This relates with the fact they can be used as either a blood/urine complementary or in substitution when their collection is unavailable by several factors. In addition, Portugal and the rest of the world experienced a new emergent trend regarding the consumption of “new psychoactive substances”. This phenomenon brought about major public health injuries. The objective of the presented work was a contribution to the development of new analytical methods for the quantitative determinations of several new psychoactive substances in various biological matrices such as whole blood, plasma, vitreous humor and pericardial fluid. The substances on which we conducted our study were: salvinorin a (main active component of the plant salvia divinorum); some cathinones such as cathine, ephedrine, methcathinone, PMA, mephedrone, methedrone and some phenethylamines belonging to the D series, like the DOM and DOB and those that belong to the 2C-X series like 2C-H, 2C-B, 2C-I, 2C-T-2, 2C-T-4 and 2C-T-7. The mixed-mode was used (cathinones and phenethylamines) and reversed-phase (salvinorin a) adsorbents in the solid phase extraction for preparing samples of 250 μL of blood, plasma and pericardial fluid and 100 μL for vitreous humor. The obtained extracts were further analysed by gas chromatography coupled to mass spectrometry in the SIM mode. To enable the detectability and the separation of the studied cathinones and phenethylamines a fast microwave derivatization procedure was previously applied, using the MBTFA reagent. Thus, in order to minimize the time required for classical techniques of derivatization in the analysis of drugs of abuse (over 30 minutes) a microwave procedure was optimized which lasts only 90 seconds. The method was applied to all substances with the exception of salvinorin a. Stable trifluoroacetyl derivatives were obtained with this optimized methodology. Once confidence in the interpretation of the analytical results was attained (as a prerequisite in forensic and clinical toxicology), it was necessary to ensure that the methods developed were suitable for its intended purposes. Thus, the proposed methodologies were fully validated, in all biological matrices studied according to internationally accepted recommendations. The methods were found to be selective for all tested compounds in all matrices studied. Linearity was achieved from 5-100 ng/mL for salvinorin a and from 5-600 ng/mL for cathinones and phenethylamines. The extraction efficiencies were between 80-100% for salvinorin a and 77-113% for cathinones and phenethylamines, in all the biological matrices studied. All results obtained for the parameters repeatability and intermediate precision presented CV<20% and accuracy were within the acceptance interval of ±20% of the nominal values at all concentration levels. The methods presented limits of quantitation of 5 ng/mL for each compound, in the all biological matrices analysed. The developed methodologies have been applied to authentic samples from autopsies performed in the Clinical and Forensic Pathology Service and in the medico-legal Offices belonging to the delegation of the centre for the National Institute of Legal Medicine and Forensic sciences, I.P. These procedures can be a useful tool in forensic toxicology laboratories. The major contributions are the complementation of the information possibly provided by each one of those fluids regarding drugs’ consumption and the prevention of situations where the blood is not available due to several factors of each individual case. In addition, it was also intended to implement the developed methodologies in routine analysis on the Laboratory of Chemistry and Forensic Toxicology of the centre branch of the National Institute of Legal Medicine and Forensic Sciences.
- Desenvolvimento de novos biomateriais para aplicação na área de engenharia de tecidosPublication . Correia, Tiago Ruivo; Correia, Ilídio Joaquim Sobreira; Calvinho, Paula Cristina Nunes FerreiraO envelhecimento da população tem provocado um aumento do número de lesões ósseas e de doenças cardiovasculares que afetam a população mundial. As lesões ósseas têm uma maior incidência na população mais idosa que apresenta uma menor densidade óssea. Por outro lado, esta população está também predisposta a doenças cardiovasculares que ocorrem devido a quadros clínicos como a calcificação dos vasos sanguíneos, o aparecimento de arteriosclerose e o aumento de radicais livres na corrente sanguínea. Atualmente, as terapias disponíveis para o tratamento deste tipo de patologias têm uma eficácia limitada. Em meio clínico os tratamentos preferencialmente usados consistem em autoenxertos devido à aceitação pelo hospedeiro. No entanto, devido à baixa disponibilidade deste tipo de enxertos, os investigadores da área de Engenharia de Tecidos têm procurado desenvolver biomateriais para aplicação nas diversas áreas da Medicina Regenerativa. Neste contexto, estruturas temporárias tridimensionais (3D) denominadas de scaffolds têm sido estudados com o intuito de serem utilizados como soluções terapêuticas para o tratamento do tecido ósseo, de forma a promover a reparação deste tecido, conferindo-lhe características físicas e biológicas essenciais ao seu correto funcionamento. Da mesma forma, no tratamento de doenças vasculares tem-se recorrido a soluções que se baseiam em redes nanofibrosas ocas com arquitetura semelhante à de um vaso sanguíneo. Na presente tese foram efetuados diferentes estudos que tinham como objetivo a produção de biomateriais para posterior aplicação na área da regeneração óssea e do tecido vascular. As estruturas 3D produzidas foram caracterizadas recorrendo a testes físico-químicos e biológicos. Os resultados obtidos revelaram que os biomateriais aqui produzidos possuem propriedades adequadas para a sua futura aplicação na Medicina Regenerativa.
- Layer-by-Layer deposition of bioactive polyelectrolytes with incorporation of antimicrobial agents as a new strategy to develop bioactive textilesPublication . Gomes, Ana Paula de Ascensão Rosa; Gouveia, Isabel Cristina Aguiar de Sousa e Silva; Mano, João Filipe Colardelle da Luz; Queiroz, João António de Sampaio RodriguesPolyelectrolyte multilayer coatings have become a new and general way to functionalize a variety of materials. Particularly, the Layer-by-Layer (LbL) method is a technique developed for the coating of solid surfaces. The LbL technique presents a unique mean to construct surface coatings that can conform to a variety of biomaterial surfaces and serve as matrices enabling controlled delivery of bioactive molecules from surface. As the deposition process is achieved in aqueous medium, incorporation of active agents is possible since the coatings obtained by LbL are less densely packed and this is advantageous for diffusion through the coating. The coating is constructed by the alternate adsorption of oppositely charged polyelectrolytes at the surface of the material, easily obtained when it is dipped in polyelectrolyte solutions. A deposition cycle creates a layer, and these cycles can be repeated as often as needed. This study aims to obtain novel bioactive textiles with potential application as wound-dressings. The biopolymers chosen for the functionalization of cotton (substrate), were chitosan (CH) and alginate (ALG). The multilayer coating of cotton with CH and ALG is constructed by the adsorption of CH and ALG with opposite charge on the surface of cotton substrates. The successive deposition of multilayers of CH and ALG was analyzed by three different techniques. Contact angle between a water droplet and the surface of the sample, cationic dye staining method and analysis by ATR-FTIR (Fourier Transform Infrared spectroscopy with Attenuated Total Reflection). These techniques showed that there was alternating deposition between CH and ALG and the presence of electrostatic bonds between the layers. In order to evaluate the antibacterial activity of the functionalized cotton, the Japanese standard JIS L 1902:2002 for the halo method (qualitative assay), and the absorption method (quantitative test) were assessed. These tests revealed an antibacterial effect on the functionalized cotton for both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Klebsiella pneumoniae). In addition a method was optimized for incorporating L-cysteine (L-cys) between the layers of CH and ALG deposited on cotton samples by the LbL, in order to obtain a better antimicrobial effect. Several strategies were used and the best results were obtained by the method where the ALG turns into a gel in the presence of calcium, since L-cys can be incorporated directly between the layers of CH and ALG without any covalent bond. Thus, the bioactive L-cys agent was immobilized without losing its bioactive characteristics. These new samples were analyzed for the antibacterial activity against Staphylococcus aureus and Klebsiella pneumoniae according with the previously used standard, and the results showed an increase in the antibacterial effect due to the presence of L-cys. This new coating method has the great advantage to able to select other types of bioactive agents without needing further optimization. In this way, L-Cys was replaced by antimicrobial peptides (AMPs). The reason for the use of AMPs is related with the continuous use of antibiotics which resulted in multiresistant bacterial strains all over the world. Consequently, there is an urgent need to search for alternatives for antibiotics. The AMPs are the new generation of antimicrobials. Four AMPs of different features were used. The depth in which each AMPs is incorporated between the layers was determined by energy dispersive analysis of X-rays (Energy Dispersive X-ray EDS). Results showed, that all AMPs used have a higher antimicrobial effect when compared with previous samples (with and without L-Cys) for both microorganisms and are non-cytotoxic to normal human dermal fibroblasts at the tested Concentrations. This confirms that this new functionalization approach of cotton coated with layers of CH and ALG by the LbL technique with incorporated AMPs leads to good antibacterial and cytotoxicity results, which make them suitable to be used as wound dressings.
- Understanding the interaction between pDNA and anion exchange supports under linear and overloaded chromatographic conditionsPublication . Aguilar, Patrícia Pereira; Cabral, Ana Cristina Mendes DiasO elevado potencial da utilização de moléculas de DNA, na forma de plasmídeos, em terapias cujo objetivo é o tratamento ou a cura de várias doenças, tem sido comprovado por inúmeros estudos desenvolvidos recentemente. No entanto, a utilização deste tipo de moléculas em terapias como Vacinas de DNA e na Terapia Génica, exige que a sua produção seja realizada até se atingir a escala da grama e garantindo elevados níveis de pureza do plasmídeo. Para além disso, pretende-se que o processo seja realizado da forma mais económica possível, cumprindo sempre as diretrizes das agências reguladoras como por exemplo a FDA (Food and Drug Administration) e a EMEA (European Agency for the Evaluation of Medical Products). Vários processos têm sido utilizados no processo de purificação de plasmídeos, quer para a sua separação de impurezas como restos celulares, proteínas, DNA genómico, etc., quer para separar as diferentes isoformas em que um plasmídeo se pode encontrar (linear, circular aberto ou superenrolado). Uma das técnicas que tem sido utilizado com sucesso é a cromatografia de troca aniónica. No entanto, o mecanismo de separação de plasmídeos por esta técnica não é, ainda, totalmente compreendido. Para além disso, devido a razões económicas, sugere-se que os processos cromatográficos sejam conduzidos em modo de sobrecarga, ou seja através da sobrecarga da coluna de cromatografia. Contudo, trabalhar em sobrecarga é consideravelmente mais complexo do que trabalhar em cromatografia linear e, até ao momento, não existem modelos adequados que prevejam o mecanismo do processo cromatográfico nestas condições. Deste modo, a previsão do comportamento das moléculas neste caso, bem como do resultado da separação através de cromatografia não é viável, sendo um dos principais obstáculos na conceção e implementação de unidades preparativas à escala industrial. Assim, uma melhor compreensão dos mecanismos subjacentes à cromatografia não linear tem um elevado interesse prático. A análise termodinâmica dos processos de adsorção através da Microcalorimetria de Fluxo (FMC) tem vindo a ser utilizada para obter uma melhor compreensão das forças motrizes, dos mecanismos e das cinéticas envolvidas no processo de adsorção de biomoléculas em diferentes sistemas cromatográficos. Por isso, neste trabalho, através da utilização da microcalorimetria de fluxo, pretende-se compreender o mecanismo de adsorção de um plasmídeo na sua forma linear (ln pVAX1-LacZ) sobre um suporte cromatográfico de troca aniónica (Fast Flow Q-sepharose). Os ensaios foram levados a cabo de modo a que o processo possa ser avaliado em condições lineares e em condições de sobrecarga. Para além do estudo do processo de adsorção tendo em conta a interação entre a biomolécula e o suporte cromatográfico, foi também considerada e avaliada a presença e a influência de efeitos não específicos como por exemplo a reorientação da biomolécula. Para além da microcalorimetria de fluxo, para obter uma melhor compreensão do mecanismo de adsorção, foram também realizados estudos da capacidade de ligação em modo estático (static binding capacity), microcalorimetria de titulação (Isothermal Titration Microcalorimetry) bem como estudos cromatográficos (fast performance chromatography). Os resultados obtidos nos estudos de capacidade de ligação revelaram que o processo de adsorção do plasmídeo segue uma isotérmica de Langmuir, em que a capacidade de ligação da Q-sepharose aumenta com o aumento da concentração de plasmídeo em equilíbrio até se atingir um patamar em que a saturação do suporte cromatográfico é atingida. Os estudos realizados utilizando a microcalorimetria de fluxo foram efetuados nas zonas linear, de transição e de sobrecarga tendo em conta a isotérmica construída. A injeção das amostras for realizada de dois modos diferentes, através da introdução de um pulso de amostra na célula do microcalorímetro ou através da alimentação contínua de amostra na célula. Estes modos de injeção foram conseguidos utilizando loops de diferentes volumes. Todos os termogramas obtidos são compostos por um primeiro pico endotérmico seguido de um pico exotérmico, o qual, em alguns casos específicos (sobrecarga de volume) resulta de picos sobrepostos. Para os picos endotérmicos o processo de dessolvatação foi sugerido como sendo o principal contribuinte enquanto que para o caso dos picos exotérmicos a interação entre o DNA e o suporte cromatográfico e a adsorção secundária foram apontados como os principais contribuidores. Para além disso, a microcalorimetria de fluxo sugere que o processo global de adsorção é exotérmico, o que é esperado uma vez que se trata de uma interação de troca aniónica. No entanto, foram também demonstradas evidências da presença de efeitos não específicos do processo de adsorção, nomeadamente a reorientação e a repulsão electroestática entre as moléculas adsorvidas.