Strategies of the African swine fever virus to manipulate innate immunity

Dissertation presented to obtain the Ph.D degree in BiologyThe objective of this thesis was to determine the mechanisms and consequences of two non-homologous host evasion genes of the economically important, frequently fatal African Swine Fever Virus (ASFV). In order to survive, large DNA viruses, such as ASFV, typically have multiple genes/strategies for positive and negative modulation of host cell biology and immune responses. The two genes presented here inhibit a major component of innate immunity, the Interferon (IFN) response, and so function to the benefit of the virus. The conserved I329L gene was recently reported to impair the cellular responses controlled by TLR3 that lead to both IFN-β secretion and NF-κB activation. Here, this observation is extended by demonstrating that I329L not only inhibits both induction and secretion of IFN-β, but also inhibits TLR4 stimulated activation. The I329L protein was also biochemically demonstrated to target the adaptor protein TRIF, consistent with the observed inhibition of both TLR3 and TLR4 pathways. To further characterize the modulation of the type I IFN response by I329L, as well as to assess the role of each domain, truncation mutants expressing either the ectodomain or the intracellular domain were designed and tested by luciferase reporter assays.(...)FCT SFRH/BD/16349/200

Efficient extraction of carotenoids from sargassum muticum using aqueous solutions of Tween 20

The replacement of synthetic compounds by natural products witnesses an increasing demand from the pharmaceutical, cosmetic, food and nutraceutical industries. Included in the set of natural raw materials that are poorly explored are the macroalgae. Despite the detailed characterization and identification of most relevant biomolecules that are present in the main macroalgae species, there remains a lack of efficient and economically viable processes available to meet the needs of the markets. In this work, an efficient and single-step process, based on aqueous solutions of Tween 20, to recover carotenoids from Sargassum muticum, an invasive brown macroalgae species present in the Portuguese coast, is proposed and optimized allowing an extraction yield of 2.78 ± 0.4 mgcarotenoids.gdried mass-1, which is shown to increase the extraction efficiency by 38% when compared with traditional methods.publishe

Produção de lipase por fermentação extractiva com líquidos iónicos

Doutoramento em Engenharia QuímicaNovos processos fermentativos, designados por processos de Fermentação Extractiva, são caracterizados por apresentarem etapas de produção e extracção em simultâneo. A extracção líquido-líquido como técnica de separação é amplamente usado na indústria química pela sua simplicidade, baixo custo e facilidade de extrapolação de escala. No entanto o uso de solventes orgânicos nestes processos potencia os riscos ocupacionais e ambientais. Neste contexto, o uso de sistemas de duas fases aquosas baseados em líquidos iónicos, apresenta-se como uma técnica eficaz para a separação e purificação de produtos biológicos. Este trabalho apresenta um estudo integrado sobre o uso de líquidos iónicos não aromáticos foram determinados. A capacidade para a formação de sistemas de duas fases foi estudada para uma vasta gama de líquidos iónicos hidrofílicos com diferentes aniões, catiões e cadeias alqúilicas. A capacidade de separação e purificação de um largo conjunto de líquidos iónicos foi posteriormente investigada, recorrendo-se ao uso de várias biomoléculas modelo de diferentes graus de complexidade, um amino-acido (L-triptofano) e duas enzimas lipolíticas (enzima produzida pela bactéria Bacillus sp. e Candida antarctica lipase B – CaLB). Esta última foi ainda usada para um estudo de biocompatibilidade, tendo sido determinado o efeito de diferentes LIs hidrofílicos na sua actividade enzimática. Este trabalho mostra um estudo ecotoxicológico duma vasta gama de líquidos iónicos e espécies aquáticas, inseridas em diversos níveis tróficos. A bioacumulação foi investigada através do estudo dos coeficientes de distribuição 1-octanol-água (Dow).Novel fermentation processes characterized by the simultaneously production and separation are known as Extractive Fermentations. Liquid-liquid extraction is a separation technique that can be used for this purpose. These processes are widely used in the chemical industry owing to its simplicity, low costs and ease of scale-up. However, most extraction processes include the use of organic solvents, which have both environmental and occupational risks associated to their use. This makes these extraction systems inappropriate for the development of environmental-friendly technologies. In this context, the use of aqueous two-phase systems (ATPS) based in Ionic Liquids (ILs) appears as an effective and viable method for the separation and purification of biological products. The present work reports an integrated study on the use of ILs as alternative solvents in Fermentation processes. Original data on the solubility of nonaromatic ILs in water are reported. The ability of several ILs for the formation of aqueous two-phase systems was studied for a wide range of hydrophilic ILs with different anions, cations and alkyl chains. The separation and purification capacity of a large set of ILs was then investigated, using different biomolecules as models with distinct structural complexities, one amino-acid (Ltryptophan) and two lipolytic enzymes (an enzyme produced by the bacterium Bacillus sp. and Candida antarctica lipase B – CaLB). CaLB was also used on a study of biocompatibility, where the effect of various hydrophilic ILs on its enzymatic activity was addressed. Finally, this work reports an ecotoxicological study for a large number of ILs and aquatic species, included in different trophic levels. The bioaccumulation data was investigated by the study of the 1-octanol-water distribution coefficients (Dow)

Equilíbrio gás líquido a altas pressões

Mestrado em Medição de Equilíbrios Gás Líquido a Altas PressõesEste trabalho tem como objectivo a medição da solubilidade de gases em líquidos a altas pressões. Para a realização experimental foi usada uma célula de altas pressões de volume variável e um método de observação directa para a detecção das transições de fase. As condições usadas neste trabalho experimental foram temperaturas entre 276.00 e 373.15 K e pressões ate 100 MPa. Numa primeira fase foram efectuadas medições de solubilidade de metano em anilina pura e para uma solução aquosa de composição mássica igual a 5% em anilina. A anilina é fundamentalmente usada em processos como a produção de borracha, de poliuretanos, pigmentos e tintas, fármacos, herbicidas e fungicidas. É usualmente produzida através de processos de redução do nitrobenzeno por reacção com o HCl. De facto, esta síntese é um processo químico complexo onde um grande número de processos compete entre si. Alterações nas condições do processo com a possível formação de produtos intermediários podem afectar a eficiência do processo. Para a sua melhoria foi sugerido que o hidrogénio usado fosse simultaneamente produzido e gasto no reactor principal. Neste caso e para a produção do mesmo, era necessário adicionar metano e água ao reactor. Tendo por base a ideia de que as reacções onde o hidrogénio é reagente e produto ocorrem em simultâneo, torna-se clara a importância do estudo da solubilidade do metano em anilina pura e em soluções aquosas desta. Numa segunda fase foi estudada a solubilidade do dióxido de carbono em soluções aquosas de tri-iso-butil(metil)fosfónio tosilato, com composições molares são de 4, 8 e 12% em líquido iónico. Este pertencente à família dos fosfónios. Possui uma viscosidade e densidade elevadas, é térmica e quimicamente estável e ainda possui uma elevada polaridade. Apresenta uma miscibilidade completa em água e nos solventes mais usuais, como o diclorometano e tolueno, não sendo no entanto míscivel em hexano. O tri-isobutil( metil)fosfónium tosilato é usado como solvente nos processos de hidroformilação de olefinas e ainda em processos de captura e conversão de dióxido de carbono. Neste trabalho experimental, a temperatura e a pressão foram inicialmente aumentadas até o sistema atingir o equilíbrio. A pressão é diminuída lentamente até se verificar o aparecimento/desaparecimento da última bolha de gás. A pressão à qual a última bolha de gás desaparece representa a pressão de equilíbrio para aquela temperatura. Este procedimento foi efectuado para vários sistemas e várias temperaturas.The aim of this work is the measurement of the solubilities of different gases in different liquids systems at high pressures. A variable volume high pressure cell was used to perform the measurements based on the visual synthetic method for the detection of phase transitions. The conditions used in this work were temperatures ranging from 276.00 to 373.15 K and pressures up to 100 MPa. The first step of this work was the measurement of methane’s solubility in pure aniline and for one aqueous solution of 5% (w/w) in aniline. The main applications of aniline are the manufacture of polyurethane, rubber, dyes and pigments, drugs and agrochemicals (herbicides and fungicides). This compound is normally produced by reduction of nitrobenzene by reaction with HCl. In fact, that synthesis is a complex chemical process where a number of competing processes contribute to the final product. Changes in process conditions with the possible formation of intermediates can act as catalyst poisons that change the process’s efficiency. For the improvement of the production process it was suggested that the hydrogen, used for the reduction of the nitrobenzene, could be simultaneously generated and used in the main reactor. In this case for the in situ production of hydrogen it would be necessary the addition of methane and water in the reactor. Having in mind that these reactions occur simultaneously, it becomes clear the importance of studying the solubility of methane in aniline and in its aqueous solutions. In the second step the solubility of carbon dioxide in aqueous solutions of triiso- butyl(methyl)phosphonium tosylate with molar compositions of 4, 8 and 12% molar of ionic liquid, was studied. This compound belongs to the phosphonium - based ionic liquids family. It is characterized by a high viscosity and density. It is thermal and chemically stable and has a high polarity. This compound is totally miscible with water as well as with common organic solvents such as dicloromethane and toluene but is not miscible with hexane. The main applications of Tri-iso-butyl(methyl)phosphonium tosylate are like solvents in the hydroformylation of olefins and in processes of capture and conversion of carbon dioxide. In this experimental work, temperature and pressure were slowly increased until the system becomes homogeneous. After that, the pressure is slowly decreased until the appearance/disappearance of the last bubble of gas. The pressure at witch the last bubble disappears represents the equilibrium pressure for that temperature. This procedure was repeated for several systems and several temperatures

Carotenoid production from microalgae: the portuguese scenario

Microalgae have an outstanding capacity to efficiently produce value-added compounds. They have been inspiring researchers worldwide to develop a blue biorefinery, supporting the development of the bioeconomy, tackling the environmental crisis, and mitigating the depletion of natural resources. In this review, the characteristics of the carotenoids produced by microalgae are presented and the downstream processes developed to recover and purify them are analyzed, considering their main applications. The ongoing activities and initiatives taking place in Portugal regarding not only research, but also industrialization under the blue biorefinery concept are also discussed. The situation reported here shows that new techniques must be developed to make microalgae production more competitive. Downstream pigment purification technologies must be developed as they may have a considerable impact on the economic viability of the process. Government incentives are needed to encourage a constructive interaction between academics and businesses in order to develop a biorefinery that focuses on high-grade chemicals.publishe

Estimation of stand crown cover using a generalized crown diameter model: application for the analysis of portuguese cork oak stands stocking evolution

A generalized non-linear tree crown diameter model was developed with the aim of allowing the computation of tree crown diameter over a large range of tree dimensions, and allowing its inclusion in forest growth and yield models. The model was formulated to provide biological meaning to the predicted values. Due to the nested structure of the data analyzed (trees within stands), both mixed- and fixed-effect models were developed. Since tree crown diameter is not frequently measured in forest inventories, the validation of the mixed model was carried out by considering the population specific response. The results demonstrate that when the measurements required for the mixed model calibration are not available, the use of the fixed effect model results in less biased and more accurate estimates. The fixed model was applied to the data from the two last Portuguese National Forest Inventories (NFI) to analyze the change in stand crown cover and assess the stocking evolution of cork oak stands in Portugal between 1996 and 2006. Results showed an increase in the frequency of stands with crown cover lower than 20%, as well as a decrease in the frequency of stands with crown cover between 20 and 40%. Average crown cover values were significantly different in the two NFI, with a decrease from 28.0 to 26.5% over the considered periodinfo:eu-repo/semantics/publishedVersio

Microalgae as Contributors to Produce Biopolymers

Biopolymers are very favorable materials produced by living organisms, with interesting properties such as biodegradability, renewability, and biocompatibility. Biopolymers have been recently considered to compete with fossil-based polymeric materials, which rase several environmental concerns. Biobased plastics are receiving growing interest for many applications including electronics, medical devices, food packaging, and energy. Biopolymers can be produced from biological sources such as plants, animals, agricultural wastes, and microbes. Studies suggest that microalgae and cyanobacteria are two of the promising sources of polyhydroxyalkanoates (PHAs), cellulose, carbohydrates (particularly starch), and proteins, as the major components of microalgae (and of certain cyanobacteria) for producing bioplastics. This review aims to summarize the potential of microalgal PHAs, polysaccharides, and proteins for bioplastic production. The findings of this review give insight into current knowledge and future direction in microalgal-based bioplastic production considering a circular economy approach. The current review is divided into three main topics, namely (i) the analysis of the main types and properties of bioplastic monomers, blends, and composites; (ii) the cultivation process to optimize the microalgae growth and accumulation of important biobased compounds to produce bioplastics; and (iii) a critical analysis of the future perspectives on the field.publishe

Separation and purification of biomacromolecules based on microfluidics

Separation and purification of biomacromolecules either in biopharmaceuticals and fine chemicals manufacturing, or in diagnostics and biological characterization, can substantially benefit from application of microfluidic devices. Small volumes of equipment, very efficient mass and heat transfer together with high process control result in process intensification, high throughputs, low energy consumption and reduced waste production as compared to conventional processing. This review highlights microfluidics-based separation and purification of proteins and nucleic acids with the focus on liquid-liquid extractions, particularly with biocompatible aqueous two-phase systems, which represent a cost-effective and green alternative. A variety of microflow set-ups are shown to enable sustainable and efficient isolation of target biomolecules both for preparative, as well as for analytical purposes.publishe

Synthesis and characterization of analogues of glycine-betaine ionic liquids and their use in the formation of aqueous biphasic systems

A series of novel analogues of glycine-betaine ionic liquids (AGB-ILs), viz. 1-(4-ethoxy-4-oxobutyl)-1-methylpyrrolidin-1-ium, N,N,N-tri(n-butyl)(4-ethoxy-4-oxobutyl)-1-phosphonium and N,N,N-trialkyl(4-ethoxy-4-oxobutyl)-1-aminium cations with ethyl, n-propyl and n-butyl alkyl chains, combined with the bromide anion, have been synthesized and characterized. Their synthesis and characterization by spectroscopic methods and elemental analysis is here reported. These ILs were further characterized in what concerns their thermal properties and ecotoxicity against Allvibrio fischeri, and compared with the commercial tetra(n-butyl)ammonium and tetra(n-butyl)phosphonium bromide. The novel AGB-ILs described in this work have low melting points, below 100 °C, display high degradation temperatures (180–310 °C), and low toxicity as shown by being harmless or practically harmless towards the marine bacteria Allvibrio fischeri. Finally, the ability of the synthesized AGB-ILs to form aqueous biphasic systems with potassium citrate/citric acid (at pH 7) was evaluated, and the respective ternary phase diagrams were determined. It is shown that the increase of the cation alkyl chain length facilitates the creation of ABS, and that phosphonium-based ILs present a slightly better separation performance in presence of aqueous solutions of the citrate-based salt.publishe

Stakeholders sustainable management in agriculture: lessons from participatory processes

XXIII International Conference Society for Human Ecology, «Navigating complexity: human-environmental solutions for a challenging future», Lisbon 7-10July 2018.Stakeholders are nowadays encouraged to become actively involved in the sustainable management of the territory and, in rural areas such as Castro Verde which is Natura 2000 Special Protection Area and nowadays a UNESCO Biosphere Reserve, as well as in the all country, equilibrium between nature conservation and farm production systems is a key factor for sustainability. This paper focuses on the contribution of local actors to evaluate the agro-environmental commitments applied during the last Portuguese Rural Development Program (PRODER/2007-2013). It aims to propose adjustments and new ideas to improve agricultural sustainable practices by assessing the existing commitments and supporting new public strategies regarding the new 2014-2020 programming cycle of the European Agricultural Fund for Rural Development. This was accomplished through the participation of local stakeholders in a set of five workshops, each one in different agricultural region of Portugal. The three-hour workshops were based on interactive techniques as drivers to generate debate, promote knowledge exchanges and produce new knowledge to be presented to policy makers