Development of novel benzimidazole- based COX Inhibitors new synthetic strategies and screening of heterocyclic structures

Dissertação para obtenção do Grau de Doutor em Química, especialidade Química Orgânica

Multi-Enzymatic Systems for Cleaning-up Synthetic Dyes from the Environment

Environmental pollution is one of the major and most important problems of the modern world. In order to fulfill the needs and demands of the overgrowing human population, developments in agriculture, medicine, energy sources, and all chemical industries are necessary (Ali 2010). Over the last century, the increased industrialization and continued population growth led to an augmented production of environmental pollutants that are released into air, water, and soil, with significant impact in the degradation of various ecosystems (Ali 2010, Khan et al. 2013).(...)

Staying under the radar - the multifunctional LANA Protein

Many viruses have developed numerous strategies to recruit and take advantage of cellular protein degradation pathways to evade the cellular viral immune system. One such virus is the Kaposi´s Sarcoma associated herpesvirus (KSHV), first discovered in Kaposi´s Sarcoma lesions found in AIDS patients. Latency-Associated Nuclear Antigen (LANA) is a KSHV multifunctional protein responsible for tethering viral DNA to the chromosome ensuring maintenance and segregation of the viral genome during cell division. Besides its main role of viral maintenance, LANA also physically interacts with several host proteins to modulate cell functions. One such function is to recruit the EC5S ubiquitin-ligase complex by interacting with Elongin BC complex and Cullin 5 protein, which in turn ubiquitinate substrates such as NF-κB and p53 to allow persistent viral infection. Like any other post-translation modifications, ubiquitination is reversible through deubiquitination enzymes (DUBs). LANA also interacts with ubiquitin specific protease 7 (USP7), a deubiquitination enzyme involved in regulation of several proteins including p53. Interaction with USP7 is made through a conserved peptide motif, which is also present in LANA. This work addresses the role of LANA in the recruitment and modulation of the ubiquitination and deubiquitination pathways. Despite the continued efforts in uncovering new LANA interacting partners to form a functional EC5S ubiquitin-ligase complex, only MHV-68 LANA interacted directly with Elongin BC, other interactions were not direct and may require a linker protein. On the other hand, LANA interaction with USP7 was able to be analysed by X-ray structure determination. In addition to a conserved P/AxxS motif, a novel Glutamine (Gln) residue from KSHV LANA was shown to make a specific interaction with USP7. This Gln residue is also present in other herpesvirus protein and hence it might be a conserved motif within herpesviruses.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

Improvement of a photoautotrophic chassis robustness for synthetic biology applications

Cyanobacteria are photoautotrophic microorganisms with great potential for the biotechnological industry due to their low nutrient requirements, photosynthetic capacities and metabolic plasticity. In biotechnology, the energy sector is one of the main targets for their utilization, especially to produce the so called third generation biofuels, which are regarded as one of the best replacements for petroleum-based fuels. Although, several issues could be solved, others arise from the use of cyanobacteria, namely the need for high amounts of freshwater and contamination/predation by other microorganisms that affect cultivation efficiencies. The cultivation of cyanobacteria in seawater could solve this issue, since it has a very stable and rich chemical composition. Among cyanobacteria, the model microorganism Synechocystis sp. PCC 6803 is one of the most studied with its genome fully sequenced and genomic, transcriptomic and proteomic data available to better predict its phenotypic behaviors/characteristics. Despite suitable for genetic engineering and implementation as a microbial cell factory, Synechocystis’ growth rate is negatively affected by increasing salinity levels. Therefore, it is important to improve. To achieve this, several strategies involving the constitutive overexpression of the native genes encoding the proteins involved in the production of the compatible solute glucosylglycerol were implemented, following synthetic biology principles. A preliminary transcription analysis of selected mutants revealed that the assembled synthetic devices are functional at the transcriptional level. However, under different salinities, the mutants did not show improved robustness to salinity in terms of growth, compared with the wild-type. Nevertheless, some mutants carrying synthetic devices appear to have a better physiological response under seawater’s NaCl concentration than in 0% (w/v) NaCl.

Inspecção de infraestruturas ferroviárias. Avaliação não destrutiva com o radar de prospecção.

As infraestruturas ferroviárias são cada vez mais, essenciais para o serviço de mobilidade de pessoas e bens em curtas e em longas distâncias. A notável evolução da indústria e da tecnologia impulsionou o desenvolvimento socioeconómico dos países e por sua vez, a densidade das redes de transporte. Com base nesta expansão as entidades gestoras dos caminhos-de-ferro têm como função oferecer cada vez melhores serviços de transporte e, por conseguinte, garantir um elevado nível de segurança e conforto aos passageiros conjugando-os, claro, com os recursos que dispõem. Hoje em dia, as noções de manutenção e conservação das vias férreas estão directamente ligadas com a segurança dos utilizadores. Daqui resulta que as inspeções das vias são cada vez mais importantes para o bom funcionamento das mesmas. Assim, o objectivo desta dissertação consiste em propor uma metodologia para uma melhor avaliação das infraestruturas ferroviárias em tempo real. Ou seja, o intuito será de diminuir o tempo de inspecção das vias férreas, com base na antecipada caracterização dos parâmetros eletromagnéticos dos materiais ferroviários, em especial, do balastro contaminado com solo. Pretendeu-se desenvolver ensaios em laboratório que simulem as várias hipóteses de contaminação do balastro que podem existir numa via tradicional, variando o nível de contaminação do balastro e o teor em água do solo. Posteriormente, através da medição em contínuo da camada de balastro, com o apoio do radar de prospecção, GPR, irão ser estudadas as constantes dieléctricas dos diferentes casos de estudo. A metodologia em estudo procura estabelecer uma relação entre os dados obtidos em laboratório, através do radar de prospecção, e a medição dos parâmetros geométricos de via numa via em serviço, analisando, principalmente, as zonas de deterioração. A utilização do GPR mostrou ser uma ferramenta eficiente para avaliação da condição da via, permitindo observar a evolução ao longo do tempo da contaminação do balastro.

The value creation radar applied to the space engineering sector

This case study illustrates the application of the Value Creation Radar (VCR) to SenSyF, an Earth Observation (EO) system which was developed by Deimos Engenharia S.A. (DME), the Portuguese affiliate of Elecnor Deimos. It describes how a team of consultants adopted the VCR in order to find new market applications for SenSyF, selected the one with the highest potential, and defined a path to guarantee a sustainable market launch. This case study highlights the main challenges of bringing a technology-driven company closer to the market in the pursuit of long-term sustainability, while not compromising its technological capabilities

The global radar applied to the precision farming sector

The case describes the development of MyFARM’s internationalization plan, a service of Deimos Engenharia, under the GloCal Radar. This space engineering company hired Lisbon Consulting Company to undertake the project to overcome its lack of market orientation. The consultants’ analysis revealed Stevens County, Kansas, as the market with the highest potential for MyFARM. A suitable entry strategy and adaptation of the service for the local market was proposed. The case culminates with the Board of Directors discussing the viability of implementing the consultants’ recommendations to start diversifying their sources of revenue streams.

Uso de imagens de radar para o cálculo da produção primária de plantas aquáticas nas várzeas da Amazônia

A área da planície de inundação da Amazônia é estimada em 300 000km² e sua produtividade primária em 1,17 x 10(14) g C yr-1. Deste total de área e produtividade, estimativas sugerem que 43% e 62%, respectivamente, são atribuídos às plantas aquáticas. Estas estimativas variam de acordo com o pulso de inundação. Por exemplo, durante o período de seca as plantas terrestres (herbáceas) geralmente ocupam áreas que apresentam plantas aquáticas na cheia. A área e a produtividade destes ecossistemas são informações essenciais para a compreensão da dinâmica biogeoquímica da Amazônia. Imagens de satélites (radar) combinadas com amostragem de campo foram utilizadas para estimar a biomassa e mapear a área de cobertura de plantas aquáticas emergentes para calcular a produção primária de plantas aquáticas na várzea do baixo Amazonas. A combinação de bandas C e L forneceu a melhor correlação (r=0,82) e um ponto de saturação de biomassa intermediário (620 gm-2) para estimar biomassa aérea. O método de segmentação e classificação por região foi utilizado para classificar combinações de bandas C e L para cada período de nível de água, e forneceu uma precisão de mapeamento maior que 95% para determinação espacial de áreas cobertas por plantas aquáticas. Combinando a distribuição espacial de plantas aquáticas, o modelo para estimativa de biomassa aérea e a porcentagem de biomassa submersa, estimou-se espacialmente uma produção primária líquida anual de 1.9x10(12) g C yr-1 (±28%) para as plantas aquáticas em uma área de 394km².

Análise do potencial de imagem TerraSAR-X para mapeamento temático no sudoeste da Amazônia brasileira

O presente trabalho tem como objetivo analisar o potencial de imagens SAR polarimétricas do sensor TerraSAR-X, no modo StripMap, para mapear o uso e cobertura da terra na região sudoeste da Amazônia brasileira. No procedimento metodológico imagens de amplitude nas polarizações A HH e A VV, A

Synthetic biology approaches to engineer new pathways for the production of plant secondary metabolites

Secondary metabolites from plants are important sources of high-value chemicals, many of them being pharmacologically active. These metabolites are commonly isolated through inefficient extractions from natural biological sources and are often difficult to synthesize chemically. Therefore, their production using engineered organisms has lately attracted an increased attention. Curcuminoids, an example of such metabolites, are produced in Curcuma longa and exhibit anti-cancer and anti-inflammatory activities. Herein we report the construction of an artificial biosynthetic pathway for the curcuminoids production in Escherichia coli. Different 4-coumaroyl-CoA ligases (4CL) and polyketide synthases (diketide-CoA synthase (DCS), curcumin synthase (CURS) and curcuminoid synthase) were tested. The highest curcumin production (70 mg/L) was obtained by feeding ferulic acid and with the Arabidopsis thaliana 4CL1 and C. longa DCS and CURS enzymes. Other curcuminoids (bisdemethoxy- and demethoxycurcumin) were also produced by feeding coumaric acid or a mixture of coumaric and ferulic acids, respectively. Curcuminoids, including curcumin, were also produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase and 4-coumarate 3-hydroxylase were used. Caffeoyl-CoA O-methyltransferase was used to convert caffeoyl-CoA to feruloyl-CoA. This pathway represents an improvement of the curcuminoids heterologous production. The construction of this pathway in another model organism is being considered, as well as the introduction of alternative enzymes.

Production of medium-chain fatty acids and higher alcohols by a synthetic co-culture grown on carbon monoxide or syngas

Synthesis gas, a mixture of CO, H2, and CO2, is a promising renewable feedstock for bio-based production of organic chemicals. Production of medium-chain fatty acids can be performed via chain elongation, utilizing acetate and ethanol as main substrates. Acetate and ethanol are main products of syngas fermentation by acetogens. Therefore, syngas can be indirectly used as a substrate for the chain elongation process.

Enzymatic biofinishes for synthetic textiles

Fundação para a Ciência e a Tecnologia, Grant SFRH/BPD/46515/2008. Jiajia Fu acknowledges the support of Jiangsu Provincial Natural Science Foundation of China (No. BK2012112) and the National Natural Science Foundation of China under Grant No. 3120113

Synthetic biology approaches to shape bacteriophages towards Pseudomonas aeruginosa biofilm control

Dissertation for Ph.D. degree in Biomedical Engineering.

Synthetic and mechanistic studies of the Thia-Fries Rearrangement

A study has been undertaken of the published literature on the Fries rearrangement, thermal, photo and microwave, since its discovery in 1908. A resume of these publications and especially of those pertaining to the thia-Fries rearrangement of sulfamate esters, has been compiled. Phenyl sulfamate, phenyl N,N-dimethylsulfamate, phenyl N,N-diethylsulfamate and phenyl N,N-di-n-propylsulfamate and many of their substituted compounds have been synthesised and purified, a total of thirty nine esters. The sulfamates have been characterised by mp / bp, infrared, C, H and N microanalysis and mass spectrum. Many of these sulfamates, twenty six in total, have been rearranged to sulfonamides in the thia-Fries rearrangement, and subsequently purified. The products were characterised by mp / bp, infrared, C, H and N microanalysis and mass spectrum. Mechanistic studies of the sulfamates have been investigated, particularly phenyl N,N-dimethylsulfamate. The rearrangement with various catalysts and catalytic ratios, the effect of solvents on the rearrangement and many crossover experiments have been carried out to determine the molecularity i.e. whether it is an inter-, intra- or bimolecular reaction. The microwave induced thia-Fries rearrangement has been examined to determine what effect this irradiation has on the rearrangement. Photo thia-Fries rearrangement has also been investigated.