Catalytic routes to convert saccharides to furanic aldehydes

The conversion of plant biomass-derived carbohydrates (preferably non-edible) into added-value products is envisaged to be at the core of the future biorefineries. Carbohydrates are the most abundant natural organic polymers on Earth. This work deals with the chemical valorisation of plant biomass, focusing on the acid-catalysed conversion of carbohydrates (mono and polysaccharides) to furanic aldehydes, namely 2-furaldehyde (Fur) and 5-hydroxymethyl-2-furaldehyde (Hmf), which are valuable platform chemicals that have the potential to replace a variety of oil derived chemicals and materials. The investigated reaction systems can be divided into two types depending on the solvent used to dissolve the carbohydrates in the reaction medium: water or ionic liquid-based systems. The reaction temperatures were greater than 150 ºC when the solvent was water, and lower than 150 º C in the cases of the ionic liquid-based catalytic systems. As alternatives to liquid acids (typically used in the industrial production of Fur), solid acid catalysts were investigated in these reaction systems. Aiming at the identification of (soluble and insoluble) reaction products, complementary characterisation techniques were used namely, FT-IR spectroscopy, liquid and solid state NMR spectroscopy, TGA, DSC and GC´GC-ToFMS analyses. Complex mixtures of soluble reaction products were obtained and different types of side reactions may occur. The requirements to be put on the catalysts for these reaction systems partly depend on the type of carbohydrates to be converted and the reaction conditions used. The thermal stability is important due to the fact that formation of humins and catalyst coking phenomena are characteristically inherent to these types of reactions systems leading to the need to regenerate the catalyst which can be effectively accomplished by calcination. Special attention was given to fully inorganic nanoporous solid acids, amorphous or crystalline, and consisting of nano to micro-size particles. The investigated catalysts were silicoaluminophosphates, aluminosilicates and zirconium-tungsten mixed oxides which are versatile catalysts in that their physicochemical properties can be fine-tuned to improve the catalytic performances in the conversion of different substrates (e.g. introduction of mesoporosity and modification of the acid properties). The catalytic systems consisting of aluminosilicates as solid acids and water as solvent seem to be more effective in converting pentoses and related polysaccharides into Fur, than hexoses and related polysaccharides into Hmf. The investigated solid acids exhibited fairly good hydrothermal stabilities. On the other hand, ionic liquid-based catalytic systems can allow reaching simultaneously high Fur and Hmf yields, particularly when Hmf is obtained from D-fructose and related polysaccharides; however, catalyst deactivation occurs and the catalytic reactions take place in homogeneous phase. As pointed out in a review of the state of the art on this topic, the development of truly heterogeneous ionic liquid-based catalytic systems for producing Fur and Hmf in high yields remains a challenge.

Brazilian savanna forest : conservation, medicinal reservoir and bioprospecting

This study aimed to analyse the Brazilian savanna forest from a Legal Reserve (LR) area from a perspective of conservation, reservoir of organic carbon and medicinal biomass for a prospective use of native medicinal plants. An ethnobotanical and ethnopharmacological survey was carried out close to a community settled in the rural area in the south of Tocantins, being selected 9 of the most cited species (cajuí- Anacardium othonianum; inharé-Brosimum gaudichaudii; jatobá-Hymenaeae courbaril; jenipapo-Genipa americana, aroeira-Myracrodruon urundeuva; negramina-Siparuna guianensis; barbatimão- Stryphnodendron obovatum; assa peixe-Vernonia brasiliana, embaúba-Cecropia pachystachya). Crude foliar extracts were subjected to a preliminary phytochemical prospection and triage of secondary metabolites with antimicrobial activity of potential interest in health and familiar agriculture. Phenolic compounds, terpenes and flavonoids were detected in the extracts of most species, which suggests the presence of antimicrobial, antioxidant and anti-insect activities. It was evident the need to better know the LR as a reservoir of medicinal biomass in an area under ecological tension where 35% (610ha) of the property is LR and should be protected by law. Therefore, a forest inventory of live woody species was performed using the allometric or indirect method. This identified a rare remnant of Semidecidual Seasonal Forest amidst the largest world savannah, the Cerrado biome. An analysis of the forest average productivity per basal area (m².ha), aerial live biomass (ton.ha-1) and carbon stock was carried out. The forest fragment was considered relatively rich in species and diversity, although showing signs of disturbance and dominance by a few species. Its horizontal structure suggests biotic regeneration conditions. It is an important reservoir of medicinal plants. Of the families (57.5%) presenting medicinal species, 19 from a total of 33 are represented in the area and contain 44% (27) of the total species (61) and 63% (432) of the total individuals catalogued. Medicinal species have ecological importance for the equilibrium of the local flora and represent 80% of the 10 species with higher Importance Value Index (IVI): Tetragastris altissima, Chrysophyllum marginatum, Oenocarpus distichus, Sclerolobium paniculatum, Simarouba versicolor, Alibertia macrophylla, Siparuna guianensis, Maprounea guianensis, Licania parvifolia e Physocalymma scaberrimum. Medicinal productivity was high for this type of phytophysionomy: 183,2 ton. ha-1 of biomass and 91,51 ton. ha-1 of carbon representing 66% of the total biomass and carbon of this Cerrado forest. From this stage S. guianensis (Siparunaceae) was selected for performing bioassays in order to verify its biological activity against microorganisms of health and agricultural relevance. This is a native aromatic medicinal plant recommended as priority for conservation, with local popular medicinal validation and availability of medicinal feedstock (3300 Kg.ha-1), with the foliar fraction giving 38Kg/ha of crude extract and 5L/ha of essential oil. Foliar crude extracts and essential oil were obtained and tested in vitro using a disk diffusion bioassay. Different concentrations of these natural products were tested against gram-positive bacteria (Staphylococcus aureus ATCC 29213), gram-negative bacteria (Escherichia coli ATCC 25922 and ATCC 35218; Pseudomonas aeruginosa ATCC 10145) and fungi (Candida albicans ATCC 6258 e Fusarium oxysporum). The essential oil inhibited the growth of S. aureus in its crude concentration (380μg.mL-1), as well as diluted to half (190μg.mL-1) and a quarter strength (95μg.mL-1). It’s likely that such action is due to sesquiterpenes major components, such as bisabolol and bisabolene (10.35%), measured by gas chromatography (GC-MS, GC-FID). Extracts did not exhibit any antimicrobial activity against the microorganisms tested. The native medicinal plants prospective market is an alternative that favours the conservation of biodiversity while generating benefits for the development of sustainable family productive activities within local ecosystems instead of the current inappropriate uses. This strengthens conservation policies of Legal Reserve in rural settlements and is in agreement with public policy on global warming and climate changes.

Mercaptans extraction from jet-fuel streams using ionic liquids

Desulfurization is one of the most important processes in the refining industry. Due to a growing concern about the risks to human health and environment, associated with the emissions of sulfur compounds, legislation has become more stringent, requiring a drastic reduction in the sulfur content of fuel to levels close to zero (< 10 ppm S). However, conventional desulfurization processes are inefficient and have high operating costs. This scenario stimulates the improvement of existing processes and the development of new and more efficient technologies. Aiming at overcoming these shortcomings, this work investigates an alternative desulfurization process using ionic liquids for the removal of mercaptans from "jet fuel" streams. The screening and selection of the most suitable ionic liquid were performed based on experimental and COSMO-RS predicted liquid-liquid equilibrium data. A model feed of 1-hexanethiol and n-dodecane was selected to represent a jet-fuel stream. High selectivities were determined, as a result of the low mutual solubility between the ionic liquid and the hydrocarbon matrix, proving the potential use of the ionic liquid, which prevents the loss of fuel for the solvent. The distribution ratios of mercaptans towards the ionic liquids were not as favorable, making the traditional liquid-liquid extraction processes not suitable for the removal of aliphatic S-compounds due to the high volume of extractant required. This work explores alternative methods and proposes the use of ionic liquids in a separation process assisted by membranes. In the process proposed the ionic liquid is used as extracting solvent of the sulfur species, in a hollow fiber membrane contactor, without co-extracting the other jet-fuel compound. In a second contactor, the ionic liquid is regenerated applying a sweep gas stripping, which allows for its reuse in a closed loop between the two membrane contactors. This integrated extraction/regeneration process of desulfurization produced a jet-fuel model with sulfur content lower than 2 ppm of S, as envisaged by legislation for the use of ultra-low sulfur jet-fuel. This result confirms the high potential for development of ultra-deep desulfurization application.

Post-fire vegetation regeneration in Portugal : implications for management

This thesis aims at improving the knowledge on the post-fire vegetation regeneration. For that, forests and shrublands were studied, after forest fires and experimental fires. Maritime Pine (Pinus pinaster) recruitment after fire was studied. Fire severity was evidenced as a major effect on this process. High crown fire severity can combust the pines, destroying the seed bank and impeding post fire pine recruitment. However, crown combustion also influences the post-fire conditions on the soil surface, since high crown combustion (HCC) will decrease the postfire needle cast. After low crown combustion (LCC) (scorched rather than torched crowns), a considerable needle cover was observed, along with a higher density of pine seedlings. The overall trends of post-fire recruitment among LCC and HCC areas could be significantly attributed to cover by needles, as well by the estimation of fire severity using the diameters of the burned twigs (TSI). Fire increased the germination from the soil seed bank of a Pinus pinaster forest, and the effects were also related with fire severity. The densities of seedlings of the dominant taxa (genus Erica and Calluna vulgaris) were contrastingly affected in relation to the unburned situation, depending on fire severity, as estimated from the degree of fire-induced crown damage (LCC/HCC), as well as using a severity index based on the diameters of remaining twigs (TSI). Low severity patches had an increase in germination density relatively to the control, while high severity patches suffered a reduction. After an experimental fire in a heathland dominated by Pterospartum tridentatum, Erica australis and E. umbellata, no net differences in seedling emergence were observed, in relation to the pre-fire situation. However, rather than having no effect, the heterogeneity of temperatures caused by fire promoted caused divergent effects over the burned plot in terms of Erica australis germination – a progressive increased was observed in the plots were maximum temperature recorded ranged from 29 to 42.5ºC and decreased in plots with maximum temperature ranging from 51.5 to 74.5ºC. In this heathland, the seed density of two of the main species (E. australis and E. umbellata) was higher under their canopies, but the same was not true for P. tridentatum. The understory regeneration in pine and eucalypt stands, 5 to 6 years post fire, has been strongly associated with post-fire management practices. The effect of forest type was, comparatively, insignificant. Soil tilling, tree harvesting and shrub clearance, were linked to lower soil cover percentages. However, while all these management operations negatively affected the cover of resprouters, seeders were not affected by soil tilling. A strong influence of biogeographic region was identified, suggesting that more vulnerable regions may suffer higher effects of management, even under comparatively lower management pressure than more productive regions. This emphasizes the need to adequate post-fire management techniques to the target regions.

Desenvolvimento de um carro elétrico puro: instrumentação e registos energéticos

Desde meados do século XX os avanços na indústria automóvel trazem a associação da eletrónica sendo esta cada vez mais necessária. Com este trabalho pretende-se construir um computador de bordo de um veículo elétrico dando continuação a uma dissertação anterior, que consistiu em um Controlador de Motor de carro elétrico com capacidade de travagem regenerativa. Este Computador deve ser de baixo custo e é destinado à instrumentação, apresentação de balanços energéticos e funções básicas de diagnóstico do estado do veículo. Foi igualmente um objetivo desenvolver uma Interface a partir de um LCD para este Computador de Bordo. No modelo de Controlador de Motor disponível foram aplicadas alterações e adições na instrumentação de forma a conseguir um diagnóstico do veículo mais preciso e extenso. Para o computador ter conhecimento do consumo e outros parâmetros relacionados com o motor e respetivo Controlador, foi necessário estabelecer uma comunicação entre ambas unidades. Antes de se implementar um protocolo de comunicação realizou-se uma pesquisa por protocolos usados na indústria automóvel, com o intuito de saber qual o mais apropriado para o presente trabalho. A tarefa seguinte consistiu em uma pesquisa por hardware com o qual desenvolver o Computador e sua Interface. Os balanços energéticos implicaram o desenvolvimento de métodos de cálculo, efetuados com os parâmetros transmitidos pelo Controlador de Motor. No final da dissertação demonstra-se todas as funcionalidades do Computador de Bordo desenvolvido e como este é utilizado.

Endocrine regulation of extracellular matrix proteins in calcified tissue in teleost fish

Tese dout., Biologia, Universidade do Algarve, 2005

Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium

Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008

Population model of the recent subtidal invasion of Sargassum muticum in southern Portugal

Dissertação de Mestrado, Biologia Marinha, Especialização em Ecologia e Conservação, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2007

Variabilidade genómica de Citrus tristeza virus e transformação genética de Citrus aurantium L. com vista à obtenção de resistência

Tese de dout., Biologia, Faculdade de Engenharia de Recursos Naturais, Univ. do Algarve, 2003

The role of lymphotoxin-B receptor signaling in t-cell acute lymphoblastic leukemia development

Dissertação de mest., Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Evolution of the extracellular matrix in deuterostomes and the influence of calcitropic hormones

Tese de dout., Biologia (Biologia Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Identification of genes regulated by mir-363*, a regulator of bone marrow endothelial cell properties

As células estaminais hematopoiéticas residem na medula óssea e possuem capacidade para se auto-renovar e dar origem a todos os tipos de células sanguíneas. O endotélio da medula óssea é constituído por células endoteliais de medula óssea (BMEC) e compreende dois nichos com funções distintas: o nicho osteoblástico e o nicho vascular. O nicho osteoblásctico proporciona condições para a quiescência de células estaminais hematopoiéticas, enquanto no nicho vascular ocorre proliferação e diferenciação das mesmas. Quando ocorre um desequilíbrio na expressão de genes que codificam para proteínas envolvidas na mobilização de células do nicho osteoblástico para o nicho vascular – factores angiócrinos – ocorre uma desestabilização do microambiente medular, que se pode traduzir num processo tumoral. Os microRNAs (miRNAs) são uma classe de RNAs não codificantes, de cadeia simples, que regula a expressão génica. Os miRNAs são sequências endógenas de RNA que possuem entre 19 e 25 nucleótidos de tamanho. Os miRNAs são reguladores da expressão genica, induzindo o silenciamento a nível da pós-transcrição, através da sua ligação com uma sequência específica para a qual possuem afinidade, na região 3’ não traduzida (3’ UTR) dos seus mRNA alvo, conduzindo à inibição da tradução ou à sua degradação. Os miRNAs estão envolvidos na regulação de genes de diversas vias afectando processos fundamentais como hematopoiese, apoptose, proliferação celular e tumorigénese. Os níveis de expressão dos miRNAs estão alterados no cancro, podendo actuar directamente como supressores de tumor ou como oncogenes, sendo neste caso denominados de oncomirs. Os perfis dos níveis de expressão de vários miRNAs foram estudados, tendo-se verificado que se alteram durante o processo de carcinogénese, podendo actuar directamente como supressores de tumor ou como oncogenes, sendo neste caso denominados de oncomirs. Apesar do miR-363* estar envolvido na regulação da expressão de genes que regulam propriedades das células endoteliais e medula óssea, os genes sobre os quais exerce a sua função ainda não foram identificados.O objectivo do presente estudo é a identificação dos genes directamente regulados pelo miR-363* (genes alvo) e a sua relevância para a disfunção medular e a sua caracterização nos síndromes mielodisplásicos. A estratégia usada baseou-se na redução ou aumento forçados dos níveis de miR-363* em células endoteliais e subsequente análise da expressão génica através de microarrays de cDNA do genoma humano. A redução do miR-363* vai implicar o aumento da expressão dos seus genes alvo, assim como o aumento dos níveis do miR-363* vai induzir a degradação e consequente redução dos seus genes alvos. A intersecção dos dados gerados através do estudo da expressão com bases de dados que possuem algoritmos para previsão de genes alvo directos dos miRNAs (miRBase e MicroCosm Targets) permitiu restringir os genes a analisar a sete genes, nomeadamente BST1, ESAM, FCER1G, IKBKG, SELE, THBS3 e TIMP1. A interacção directa destes candidatos a alvos directos do miR-363* foi posteriormente validada. Para tal, as 3’UTR dos genes foram clonadas num vector que contém o gene da luciferase. Uma vez as clonagens realizadas, efectuaram-se ensaios funcionais em células endoteliais, nomeadamente HUVEC, nas quais se co-transfectaram os vectores gerados, anti-miRs ou pre-miRs (para diminuir ou aumentar o nível de miRNA) e o plasmídeo controlo da Renilla para normalização dos ensaios de luciferase. A variação da luminescência obtida em presença do aumento ou redução do miR-363* deu uma forte indicação da regulação directa do miR-363* nesses alvos. No entanto, a confirmação desta interacção directa foi efectuada através de ensaios de mutagénese, nos quais de induziram mutações na 3’UTR nos locais de ligação do miRNA, seguidos dos ensaios funcionais como acima descritos. Esta estratégia sugere que o TIMP1, inibidor da metaloprotease-9 (MMP-9), é regulado directamente pelo miR-363*. Adicionalmente, os níveis de expressão dos alvos directos do miR-363* foram estudados em 17 amostras de aspirados de medula óssea de doentes com síndromes mielodisplásicos. Os síndromes mielodisplásicos são caracterizados como um grupo heterogéneo de condições, que apresentam citopenias (produção deficiente de eritrócitos, leucócitos e/ou megacariócitos) e medula óssea displástica e hipercelular. A escalonagem dos doentes foi feita de acordo com o sistema de prognóstico IPSS elaborado pela Organização Mundial de Saúde, e que consiste numa tabela de risco de progressão de síndromes mielodisplásicos para leucemia mielóide aguda (LMA) e que agrupa os doentes em baixo risco – que compreende os níveis baixo e intermédio 1 – e em alto risco – que compreende os níveis intermédio 2 e alto. Dos genes regulados pelo miR-363*, o destacam-se o TIMP1, estando aumentando em doentes com mau prognóstico, e o THBS3 que apresenta um aumento nos doentes com prognóstico intermédio. Em suma, os estudos realizados permitiram a identificação de genes regulados pelo miR-363* e contribuiram para o conhecimento de como o miR-363* contribui para a disfunção medular, particularmente em síndromes mielodisplásicos, pela desregulação das propriedades endoteliais.

Effect of polyunsaturated fatty acids (PUFAs) on the proliferation and extracellular matrix mineralization and gene expression of gilthead seabream skeletal cell lines

The aquaculture industry aims at replacing significant amounts of marine fish oil by vegetable oils in fish diet. Dietary lipids have been shown to alter the fatty acid composition of bone compartments, which would impact the local production of factors controlling bone formation. Knowledge on the mechanisms underlying the nutritional regulation of bone metabolism is however scarce in fish. Two in vitro bone-derived cell systems developed from seabream (an important species for aquaculture in the Mediterranean region) vertebra, capable of in vitro mineralization and exhibiting prechondrocyte (VSa13) and pre-osteoblast (VSa16) phenotype, were used to assess the effect of certain polyunsaturated fatty acids (PUFAs; arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids) on cell proliferation, extracellular matrix (ECM) mineralization and gene expression. While all PUFAs promoted morphological changes in both cell lines, VSa16 cell proliferation appeared to be stimulated by PUFAs in a dose dependent manner until 100M, whereas proliferation of VSa13 cells was impaired at concentrations above 10M. AA, EPA and DHA inhibited VSa13 ECM mineralization, alone and in combination, while VSa16 ECM mineralization was only inhibited by AA and EPA. DHA had the opposite effect, increasing mineralization almost by 2 fold. When EFAs were combined, DHA apparently compensated for the inhibitory effect of AA and EPA. Expression of marker genes for bone and lipid metabolisms has been investigated by qPCR and shown to be regulated in pre-osteoblasts exposed to individual PUFAs. Our results show that PUFAs are effectors of fish bone cell lines, altering cell morphology, proliferation and mineralization when added to culture medium. This work also demonstrates the suitability of our in vitro cell systems to get insights into mineralization-related effects of PUFAs in vivo and to evaluate the replacement of fish oils by vegetable oil sources in fish feeds.

Insights into molecular and cellular events involved in normal and pathological vertebral development and fusion using zebrafish as model organism

The vertebral column and its units, the vertebrae, are fundamental features, characteristic of all vertebrates. Developmental segregation of the vertebral bodies as articulated units is an intrinsic requirement to guarantee the proper function of the spine. Whenever these units become fused either during development or postsegmentation, movement is affected in a more or less severe manner, depending on the number of vertebrae affected. Nevertheless, fusion may occur as part of regular development and as a physiological requirement, like in the tetrapod sacrum or in fish posterior vertebrae forming the urostyle. In order to meet the main objective of this PhD project, which aimed to better understand the molecular and cellular events underlying vertebral fusion under physiological and pathological conditions, a detailed characterization of the vertebral fusion occurring in zebrafish caudal fin region was conducted. This showed that fusion in the caudal fin region comprised 5 vertebral bodies, from which, only fusion between [PU1++U1] and ural2 [U2+] was still traceable during development. This involved bone deposition around the notochord sheath while fusion within the remaining vertebral bodies occur at the level of the notochord sheath, as during the early establishment of the vertebral bodies. A comparison approach between the caudal fin vertebrae and the remaining vertebral column showed conserved features such as the presence of mineralization related proteins as Osteocalcin were identified throughout the vertebral column, independently on the mineralization patterns. This unexpected presence of Osteocalcin in notochord sheath, here identified as Oc1, suggested that this gene, opposing to Oc2, generally associated with bone formation and mature osteoblast activity, is potentially associated with early mineralization events including chordacentrum formation. Nevertheless, major differences between caudal fin region and anterior vertebral bodies considering arch histology and mineralization patterns, led us to use RA as an inductive factor for vertebral fusion, allowing a direct comparison of equivalent structures under normal and fusion events. This fusion phenotype was associated with notochord sheath ectopic mineralization instead of ectopic perichordal bone formation related with increased osteoblast activity, as suggested in previous reports. Additionally, alterations in ECM content, cell adhesion and blood coagulation were discussed as potentially related with the fusion phenotype. Finally, Matrix gla protein, upregulated upon RA treatment and shown to be associated with chordacentrum mineralization sites in regular development, was further described considering its potential function in vertebral formation and pathological fusion. Therefore with this work we propose zebrafish caudal fin vertebral fusion as a potential model to study both congenital and postsegmentation fusion and we present candidate factors and genes that may be further explored in order to clarify whether we can prevent vertebral fusion.

Searching biocompounds in marine natural extracts with potential use in tumor prevention and treatment

Cancer is a multistage process characterized by three stages: initiation, promotion and progression; and is one of the major killers worldwide. Oxidative stress acts as initiator in tumorigenesis; chronic inflammation promotes cancer; and apoptosis inactivation is an issue in cancer progression. In this study, it was investigated the antioxidant, antiinflammatory and antitumor properties of hexane, ether, chloroform, methanol and water extracts of five species of halophytes: A. macrostachyum, P. coronopus, J. acutus, C. edulis and A. halimus. Antioxidant activity was assessed by DPPH• and ABTS•+ methods, and the total phenolics content (TPC) was evaluated by the Folin-Ciocalteau method. The anti-inflammatory activity of the extracts was determined by the Griess method, and by evaluating the inhibition of NO production in LPS-stimulated RAW- 264.7 macrophages. The cytotoxic activity of the extracts against HepG2 and THP1 cell lines was estimated by the MTT assay, and the results obtained were further compared with the S17 non-tumor cell line. The induction of apoptosis of J. acutus ether extract was assessed by DAPI staining. The highest antioxidant activities was observed in C. edulis methanol and the J. acutus ether extracts against the DPPH• radical; and J. acutus ether and A. halimus ether extracts against the ABTS•+ radical. The methanol extracts of C. edulis and P. coronopus, and the ether extract of J. acutus revealed a high TPC. Generally the antioxidant activity had no correlation with the TPC. The A. halimus chloroform and P. coronopus hexane extracts demonstrated ability to reduce NO production in macrophages (> 50%), revealing their anti-inflammatory capacity. The ether extract of J. acutus showed high cytotoxicity against HepG2 cancer cells, with reduced cellular viability even at the lowest concentrations. This outcome was significantly lower than the obtained with the non-tumor cells (S17). This result was complemented by the induction of apoptosis.