Digital microfluidic devices: the role of the dielectric layer

Digital microfluidics (DMF) is a field which has emerged in the last decade as a re-liable and versatile tool for sensing applications based on liquid reactions. DMF allows the discrete displacement of droplets, over an array of electrodes, by the application of voltage, and also the dispensing from a reservoir, mixing, merging and splitting fluidic operations. The main drawback of these devices is due to the need of high driving volt-ages for droplet operations. In this work, alternative dielectric layers combinations were studied aiming the reduction of these driving voltages. DMF chips were designed, pro-duced and optimized according to the theory of electrowetting-on-dielectric, adopting different combinations of parylene-C and tantalum pentoxide (Ta2O5) as dielectric ma-terials, and Teflon as hydrophobic layer. With both devices’ configurations, i.e., Parylene as single dielectric, and multilayer chips combining Parylene and Ta2O5, it was possible to perform all the fluidic opera-tions in the microliter down to hundreds of nanoliters range. Multilayer chips presented significant reduction on driving voltages for droplet op-erations in silicone oil filler medium: from 70 V (parylene only) down to 30 V (parylene/Ta2O5) for dispensing; and from 50 V (parylene only) down to 15 V (parylene/Ta2O5) for movement. Peroxidase colorimetric reactions were successfully performed as proof-of-concept, using multilayer configuration devices.

Functional genomic analysis of heat stress in Vitis vinifera

Grapevine (Vitis vinifera) is one of most agro-economically important fruit crops worldwide, with a special relevance in Portugal where over 300 varieties are used for wine production. Due to global warming, temperature stress is currently a serious issue affecting crop production especially in temperate climates. Mobile genetic elements such as retrotransposons have been shown to be involved in environmental stress induced genetic and epigenetic modifications. In this study, sequences related to Grapevine Retrotransposon 1 (Gret1) were utilized to determine heat induced genomic and transcriptomic modifications in Touriga Nacional, a traditional Portuguese grapevine variety. For this purpose, growing canes were treated to 42 oC for four hours and leaf genomic DNA and RNA was utilized for various techniques to observe possible genomic alterations and variation in transcription levels of coding and non-coding sequences between non-treated plants and treated plants immediately after heat stress (HS-0 h) or after a 24 hour recovery period (HS-24 h). Heat stress was found to induce a significant decrease in Gret1 related sequences in HS-24 h leaves, indicating an effect of heat stress on genomic structure. In order to identify putative heat induced DNA modifications, genome wide approaches such as Amplified Fragment Length Polymorphism were utilized. This resulted in the identification of a polymorphic DNA fragment in HS-0 h and HS-24 h leaves whose sequence mapped to a genomic region flanking a house keeping gene (NADH) that is represented in multiple copies in the Vitis vinifera genome. Heat stress was also found to affect the transcript levels of various non-coding and gene coding sequences. Accordingly, quantitative real time PCR results established that Gret1 related sequences are up regulated immediately after heat stress whereas the level of transcript of genes involved in identification and repair of double strand breaks are significantly down regulated in HS-0 h plants. Taken together, the results of this work demonstrated heat stress affects both genomic integrity and transcription levels.

Numerical simulation of blast effects on fibre grout RC panels

The main purpose of the present dissertation is the simulation of the response of fibre grout strengthened RC panels when subjected to blast effects using the Applied Element Method, in order to validate and verify its applicability. Therefore, four experimental models, three of which were strengthened with a cement-based grout, each reinforced by one type of steel reinforcement, were tested against blast effects. After the calibration of the experimental set-up, it was possible to obtain and compare the response to the blast effects of the model without strengthening (reference model), and a fibre grout strengthened RC panel (strengthened model). Afterwards, a numerical model of the reference model was created in the commercial software Extreme Loading for Structures, which is based on the Applied Element Method, and calibrated to the obtained experimental results, namely to the residual displacement obtained by the experimental monitoring system. With the calibration verified, it is possible to assume that the numerical model correctly predicts the response of fibre grout RC panels when subjected to blast effects. In order to verify this assumption, the strengthened model was modelled and subjected to the blast effects of the corresponding experimental set-up. The comparison between the residual and maximum displacements and the bottom surface’s cracking obtained in the experimental and the numerical tests yields a difference of 4 % for the maximum displacements of the reference model, and a difference of 4 and 10 % for the residual and maximum displacements of the strengthened model, respectively. Additionally, the cracking on the bottom surface of the models was similar in both methods. Therefore, one can conclude that the Applied ElementMethod can correctly predict and simulate the response of fibre grout strengthened RC panels when subjected to blast effects.

Towards modification of Medicago truncatula epigenome: genome editing with engineered nucleases

Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula.

P53 and Rb tumor suppressor gene alterations in gastric cancer

Inactivation of tumor suppressor genes has been frequently observed in gastric carcinogenesis. Our purpose was to study the involvement of p53, APC, DCC, and Rb genes in gastric carcinoma. METHOD: Loss of heterozygosity of the p53, APC, DCC and Rb genes was studied in 22 gastric cancer tissues using polymerase chain reaction; single-strand conformation polymorphism of the p53 gene exons 5-6 and exons 7-8 was studied using 35S-dATP, and p53 expression was detected using a histological immunoperoxidase method with an anti-p53 clone. RESULTS AND DISCUSSION: No loss of heterozygosity was observed in any of these tumor suppressor genes; homozygous deletion was detected in the Rb gene in 23% (3/13) of the cases of intestinal-type gastric carcinoma. Eighteen (81.8%) cases showed band mobility shifts in exons 5-6 and/or 7-8 of the p53 gene. The presence of the p53 protein was positive in gastric cancer cells in 14 cases (63.6%). Normal gastric mucosa showed negative staining for p53; thus, the immunoreactivity was likely to represent mutant forms. The correlation of band mobility shift and the immunoreactivity to anti-p53 was not significant (P = .90). There was no correlation of gene alterations with the disease severity. CONCLUSIONS: The inactivation of Rb and p53 genes is involved in gastric carcinogenesis in our environment. Loss of the Rb gene observed only in the intestinal-type gastric cancer should be further evaluated in association with Helicobacter pylori infection. The p53 gene was affected in both intestinal and diffuse histological types of gastric cancer.

Fatigue life assessment of endodontic instruments subjected to multi planar curvature

This present study aimed to investigate the fatigue life of unused (new) endodontic instruments made of NiTi with control memory by Coltene™ and subjected to the multi curvature of a mandibular first molar root canal. Additionally, the instrument‟s structural behaviour was analysed through non-linear finite element analysis (FEA). The fatigue life of twelve Hyflex™ CM files was assessed while were forced to adopt a stance with multiple radius of curvature, similar to the ones usually found in a mandibular first molar root canal; nine of them were subjected to Pecking motion, a relative movement of axial type. To achieve this, it was designed an experimental setup with the aim of timing the instruments until fracture while worked inside a stainless steel mandibular first molar model with relative axial motion to simulate the pecking motion. Additionally, the model‟s root canal multi-curvature was confirmed by radiography. The non-linear finite element analysis was conducted using the computer aided design software package SolidWorks™ Simulation, in order to define the imposed displacement required by the FEA, it was necessary to model an endodontic instrument with simplified geometry using SolidWorks™ and subsequently analyse the geometry of the root canal CAD model. The experimental results shown that the instruments subjected to pecking motion displayed higher fatigue life values and higher lengths of fractured tips than those with only rotational relative movement. The finite element non-linear analyses shown, for identical conditions, maximum values for the first principal stress lower than the yield strength of the material and those were located in similar positions to the instrument‟s fracture location determined by the experimental testing results.

Nanomateriais manufaturados : avaliação de segurança através da caracterização dos seus efeitos genéticos

RESUMO - Os nanomateriais manufaturados (NMs), isto é, fabricados deliberadamente para fins específicos, apresentam propriedades físico-químicas únicas como a dimensão, área superficial ou funcionalização, que lhes conferem caraterísticas mecânicas, óticas, elétricas e magnéticas muito vantajosas para aplicações industriais e biomédicas. Efetivamente, a tecnologia baseada nos NMs, ou nanotecnologia, foi identificada como uma key enabling technology, impulsionadora do crescimento económico dos países industrializados, devido ao seu potencial para melhorar a qualidade e desempenho de muitos tipos de produtos e de processos. Contudo, a expansão da utilização de NMs contrasta com a insuficiente avaliação de risco para a saúde humana e para o ambiente, sendo considerados como um risco emergente para a saúde pública. As incertezas sobre a segurança dos NMs para a saúde pública advêm sobretudo de estudos epidemiológicos em humanos expostos a nanomateriais produzidos como consequência dos processos e atividades humanas e da poluição. Uma das principais preocupações relativamente aos efeitos adversos dos NMs na saúde humana é o seu potencial efeito carcinogénico, que é sugerido por alguns estudos experimentais, como no caso dos nanomateriais de dióxido de titânio ou dos nanotubos de carbono. Para avaliar em curto termo as propriedades carcinogénicas de um composto, utilizam-se frequentemente ensaios de genotoxicidade em linhas celulares de mamífero ou ensaios em modelos animais, em que se analisa uma variedade de lesões genéticas potencialmente relacionados com o processo de carcinogénese. No entanto, a investigação sobre as propriedades genotóxicas dos NMs não foi, até hoje, conclusiva. O presente estudo tem por objectivo principal caracterizar os efeitos genotóxicos associados à exposição a nanomateriais manufaturados, de forma a contribuir para a avaliação da sua segurança. Constituíram objectivos específicos deste estudo: i) avaliar a genotoxicidade dos NMs em três tipos de células humanas expostas in vitro: linfócitos humanos primários, linha celular de epitélio brônquico humano (BEAS-2B) e linha celular de adenocarcinoma epitelial de pulmão humano (A549); ii) avaliar a sua genotoxicidade num modelo de ratinho transgénico; iii) investigar alguns mecanismos de acção que poderão contribuir para a genotoxicidade dos nanomateriais, como a contribuição de lesões oxidativas para a genotoxicidade induzida pelos NMs in vitro, e a investigação da sua bioacumulação e localização celular in vivo. Foram analisados os efeitos genotóxicos associados à exposição a duas classes de NMs, dióxido de titânio e nanotubos de carbono de parede múltipla, bem como a um NM de óxido de zinco, candidato a ser utlilizado como controlo positivo de dimensão nanométrica. Os xx NMs utilizados foram previamente caracterizados com detalhe relativamente às suas características físico-químicas e também relativamente à sua dispersão em meio aquoso e no meio de cultura. A metodologia incluiu ensaios de citotoxicidade e de genotoxicidade in vitro, designadamente, ensaios de quebras no DNA (ensaio do cometa) e nos cromossomas (ensaio do micronúcleo) em células humanas expostas a várias concentrações de NMs, por comparação com células não expostas. Também foram realizados ensaios in vivo de quebras no DNA, quebras cromossómicas e ainda um ensaio de mutações em vários órgãos de grupos de ratinhos transgénicos LacZ, expostos por via intravenosa a duas doses de dióxido de titânio. Foi investigada a existência de uma relação dose-resposta após exposição das células humanas ou dos animais a NMs. A contribuição de lesões oxidativas para a genotoxicidade após exposição das células aos NMs in vitro foi explorada através do ensaio do cometa modificado com enzima. Realizaram-se estudos histológicos e citológicos para deteção e localização celular dos NMs nos órgãos-alvo dos ratinhos expostos in vivo. Os resultados demonstraram efeitos genotóxicos em alguns dos NMs analisados em células humanas. No entanto, os efeitos genotóxicos, quando positivos, foram em níveis reduzidos, ainda que superiores aos valores dos controlos, e a sua reprodutibilidade era dependente do sistema experimental utilizado. Para outros NMs, a evidência de genotoxicidade revelou-se equívoca, conduzindo à necessidade de esclarecimento através de ensaios in vivo. Para esse fim, recorreu-se a uma análise integrada de múltiplos parâmetros num modelo animal, o ratinho transgénico baseado em plasmídeo contendo o gene LacZ exposto a um NM de dióxido de titânio, NM-102. Embora tenha sido demonstrada a exposição e a acumulação do NM no fígado, não se observaram efeitos genotóxicos nem no fígado, nem no baço nem no sangue dos ratinhos expostos a esse NM. Neste estudo concluiu-se que algumas formas de dióxido de titânio e nanotubos de carbono de parede múltipla produzem efeitos genotóxicos em células humanas, contribuindo para o conjunto de evidências sobre o efeito genotóxico desses NMs. As diferenças observadas relativamente à genotoxicidade entre NMs do mesmo tipo, mas distintos em algumas das suas características físico-quimicas, aparentemente não são negligenciáveis, pelo que os resultados obtidos para um NM não devem ser generalizados ao grupo correspondente. Para além disso, a genotoxicidade equívoca verificada para o NM-102 em células humanas expostas in vitro, não foi confirmada no modelo in vivo, pelo que o valor preditivo da utilização dos ensaios in vitro para a identificação de NMs com efeitos genotóxicos (e portanto potencialmente carcinogénicos) ainda tem de ser esclarecido antes de ser possível extrapolar as conclusões para a saúde humana. Por sua vez, como a informação aqui produzida pelas metodologias in vitro e in vivo não reflete os efeitos de exposição continua ou prolongada, que poderá conduzir a efeitos genotóxicos distintos, esta xxi deverá ser complementada com outras linhas de evidência relativamente à segurança dos NMs. Perante a incerteza dos níveis de exposição real do organismo humano e do ambiente, a segurança da utilização dos NMs não pode ser garantida a longo prazo e, tendo em conta a elevada produção e utilização destes NMs, são prementes futuros estudos de monitorização ambiental e humana.

Masonry infill walls under blast loading using confined underwater blast wave generators (WBWG)

The vulnerability of the masonry envelop under blast loading is considered critical due to the risk of loss of lives. The behaviour of masonry infill walls subjected to dynamic out-of-plane loading was experimentally investigated in this work. Using confined underwater blast wave generators (WBWG), applying the extremely high rate conversion of the explosive detonation energy into the kinetic energy of a thick water confinement, allowed a surface area distribution avoiding also the generation of high velocity fragments and reducing atmospheric sound wave. In the present study, water plastic containers, having in its centre a detonator inside a cylindrical explosive charge, were used in unreinforced masonry infills panels with 1.7m by 3.5m. Besides the usage of pressure and displacement transducers, pictures with high-speed video cameras were recorded to enable processing of the deflections and identification of failure modes. Additional numerical studies were performed in both unreinforced and reinforced walls. Bed joint reinforcement and grid reinforcement were used to strengthen the infill walls, and the results are presented and compared, allowing to obtain pressure-impulse diagrams for design of masonry infill walls.

Detection of road accident accumulation zones with a visual analytics approach

Nowadays, road accidents are a major public health problem, which increase is forecasted if road safety is not treated properly, dying about 1.2 million people every year around the globe. In 2012, Portugal recorded 573 fatalities in road accidents, on site, revealing the largest decreasing of the European Union for 2011, along with Denmark. Beyond the impact caused by fatalities, it was calculated that the economic and social costs of road accidents weighted about 1.17% of the Portuguese gross domestic product in 2010. Visual Analytics allows the combination of data analysis techniques with interactive visualizations, which facilitates the process of knowledge discovery in sets of large and complex data, while the Geovisual Analytics facilitates the exploration of space-time data through maps with different variables and parameters that are under analysis. In Portugal, the identification of road accident accumulation zones, in this work named black spots, has been restricted to annual fixed windows. In this work, it is presented a dynamic approach based on Visual Analytics techniques that is able to identify the displacement of black spots on sliding windows of 12 months. Moreover, with the use of different parameterizations in the formula usually used to detect black spots, it is possible to identify zones that are almost becoming black spots. Through the proposed visualizations, the study and identification of countermeasures to this social and economic problem can gain new grounds and thus the decision- making process is supported and improved.

Análisis mecánico de albañilería arqueológica de adobe bajo cargas de compresión uniaxial: El caso de Huaca de la Luna

This article presents an experimental and numerical study for the mechanical characterization under uniaxial compressive loading of the adobe masonry of one of the most emblematic archaeological complex in Peru, 'Huaca de la Luna' (100-650AD). Compression tests of prisms were carried out with original material brought to the laboratory. For measuring local deformations in the tests, displacement transducers were used which were complemented by a digital image correlation system which allowed a better understanding of the failure mechanism. The tests were then numerically simulated by modelling the masonry as a continuum media. Several approaches were considered concerning the geometrical modelling, namely 2D and 3D simplified models, and 3D refined models based on a photogrammetric reconstruction. The results showed a good approximation between the numerical prediction and the experimental response in all cases. However, the 3D models with irregular geometries seem to reproduce better the cracking pattern observed in the tests.

Assessment of the efficiency of prefabricated hybrid composite plates (HCPs) for retrofitting of damaged interior RC beam–column joints

The effectiveness of prefabricated hybrid composite plates (HCPs) as a seismic retrofitting solution for damaged interior RC beam-column joints is experimentally studied. HCP is composed of a thin plate made of strain hardening cementitious composite (SHCC) reinforced with CFRP sheets/laminates. Two full-scale severely damaged interior beam-column joints are retrofitted using two different configurations of HCPs. The effectiveness of these retrofitting solutions mainly in terms of hysteretic response, dissipated energy, degradation of secant stiffness, displacement ductility and failure modes are compared to their virgin states. According to these criteria, both solutions resulted in superior responses regarding the ones registered in their virgin states.

Seismic vulnerability of existing masonry buildings: Nonlinear parametric analysis

Existing masonry structures are usually associated to a high seismic vulnerability, mainly due to the properties of the materials, weak connections between floors and load-bearing walls, high mass of the masonry walls and flexibility of the floors. For these reasons, the seismic performance of existing masonry structures has received much attention in the last decades. This study presents the parametric analysis taking into account the deviations on features of the gaioleiro buildings - Portuguese building typology. The main objective of the parametric analysis is to compare the seismic performance of the structure as a function of the variations of its properties with respect to the response of a reference model. The parametric analysis was carried out for two types of structural analysis, namely for the non-linear dynamic analysis with time integration and for the pushover analysis with distribution of forces proportional to the inertial forces of the structure. The Young's modulus of the masonry walls, Young's modulus of the timber floors, the compressive and tensile non-linear properties (strength and fracture energy) were the properties considered in both type of analysis. Additionally, in the dynamic analysis, the influences of the vis-cous damping and of the vertical component of the earthquake were evaluated. A pushover analysis proportional to the modal displacement of the first mode in each direction was also carried out. The results shows that the Young's modulus of the masonry walls, the Young's modulus of the timber floors and the compressive non-linear properties are the pa-rameters that most influence the seismic performance of this type of tall and weak existing masonry structures. Furthermore, it is concluded that that the stiffness of the floors influences significantly the strength capacity and the collapse mecha-nism of the numerical model. Thus, a study on the strengthening of the floors was also carried out. The increase of the thickness of the timber floors was the strengthening technique that presented the best seismic performance, in which the reduction of the out-of-plane displacements of the masonry walls is highlighted.

Out-of-plane behavior of masonry infill walls

In order to investigate the out-of-plane behaviour of masonry infill walls, quasi-static testing was performed on a masonry infill walls built inside a reinforced concrete frame by means of an airbag system to apply the uniform out-of-plane load to each component of the infill. The main advantage of this testing setup is that the out-of-plane loading can be applied more uniformly in the walls, contrarily to point load configuration. The test was performed under displacement control by selecting the mid-point of the infill as control point. Input and output air in the airbag was controlled by using a software to apply a specific displacement in the control point of the infill wall. The effect of the distance between the reaction frame of the airbag and the masonry infill on the effective contact area was previously analysed. Four load cells were attached to the reaction frame to measure the out-of-plane force. The effective contact area of the airbag was calculated by dividing the load measured in load cells by the pressure inside the airbag. When the distance between the reaction walls and the masonry infill wall is smaller, the effective area is closer to the nominal area of the airbag. Deformation and crack patterns of the infill confirm the formation of arching mechanism and two-way bending of the masonry infill. Until collapse of the horizontal interface between infill and upper beam in RC frame, the infill bends in two directions but the failure of that interface which is known as weakest interface due to difficulties in filling the mortar between bricks of last row and upper beam results in the crack opening trough a well-defined path and the consequent collapse of the infill.

In-plane and out-of plane experimental characterization of rc masonry infilled frames

Seismic investigations of typical south European masonry infilled frames were performed by testing two reduced scale specimens: one in the in-plane direction and another in the out-ofplane direction. Information about geometry and reinforcement scheme of those structures constructed in 1980s were obtained by [1]. The specimen to be tested in the in-plane direction was constructed as double leaf masonry while the specimen for testing in the out-of-plane direction is constructed with only its exterior leaf since the recent earthquakes have highlighted the vulnerability of the external leaf of the infills in out-of-plane direction [2]. The tests were performed by applying the pre-defined values of displacements in the in-plane and out-of-plane directions in the control points. For in-plane testing it was done by hydraulic actuator and for out-of-plane testing through the application of an airbag. Input and output air in the airbag was controlled by using a software to apply a specific displacement in the control point of the infill wall. Mid-point of the infill was assumed as a control point for outof- plane testing. Deformation and crack patterns of the infill confirm the formation of two-way arching mechanism of the masonry infill until collapse of the upper horizontal interface between infill and frame which is known as weakest interface due to difficulties in filling the mortar between bricks of last row and upper beam. This results in the crack opening through a welldefined path and the consequent collapse of the infill.

Numerical approaches for the analysis of timber frame walls

A numerical approach to simulate the behaviour of timber shear walls under both static and dynamic loading is proposed. Because the behaviour of timber shear walls hinges on the behaviour of the nail connections, the force-displacement behaviour of sheathing-to-framing nail connections are first determined and then used to define the hysteretic properties of finite elements representing these connections. The model nails are subsequently implemented into model walls. The model walls are verified using experimental results for both monotonic and cyclic loading. It is demonstrated that the complex hysteretic behaviour of timber shear walls can be reasonably represented using model shear walls in which nonlinear material failure is concentrated only at the sheathing-to-framing nail connections.