Development of triplet repeat primed PCR (TP-PCR) technique as a support of molecular test in Machado-Joseph disease

Dissertação de Mestrado, Ciências Biomédicas, 3 de Fevereiro de 2016, Universidade dos Açores.

Host-symbiont interactions in the deep-sea vent mussel Bathymodiolus azoricus : a molecular approach

Tese de Doutoramento, Ciências do Mar, especialidade de Biologia Marinha, 19 de Dezembro de 2015, Universidade dos Açores.

Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources

Clinical and environmental samples from Portugal were screened for the presence of Aspergillus and the distributions of the species complexes were determined in order to understand how their distributions differ based on their source. Fifty-seven Aspergillus isolates from clinical samples were collected from 10 health institutions. Six species complexes were detected by internal transcribed spacer sequencing; Fumigati, Flavi, and Nigri were found most frequently (50.9%, 21.0%, and 15.8%, respectively). β-tubulin and calmodulin sequencing resulted in seven cryptic species (A. awamorii, A. brasiliensis, A. fructus, A. lentulus, A. sydowii, A. tubingensis, Emericella echinulata) being identified among the 57 isolates. Thirty-nine isolates of Aspergillus were recovered from beach sand and poultry farms, 31 from swine farms, and 80 from hospital environments, for a total 189 isolates. Eleven species complexes were found in these 189 isolates, and those belonging to the Versicolores species complex were found most frequently (23.8%). There was a significant association between the different environmental sources and distribution of the species complexes; the hospital environment had greater variability of species complexes than other environmental locations. A high prevalence of cryptic species within the Circumdati complex was detected in several environments; from the isolates analyzed, at least four cryptic species were identified, most of them growing at 37ºC. Because Aspergillus species complexes have different susceptibilities to antifungals, knowing the species-complex epidemiology for each setting, as well as the identification of cryptic species among the collected clinical isolates, is important. This may allow preventive and corrective measures to be taken, which may result in decreased exposure to those organisms and a better prognosis.

Neural-network approach to modeling liquid crystals in complex confinement

Finding the structure of a confined liquid crystal is a difficult task since both the density and order parameter profiles are nonuniform. Starting from a microscopic model and density-functional theory, one has to either (i) solve a nonlinear, integral Euler-Lagrange equation, or (ii) perform a direct multidimensional free energy minimization. The traditional implementations of both approaches are computationally expensive and plagued with convergence problems. Here, as an alternative, we introduce an unsupervised variant of the multilayer perceptron (MLP) artificial neural network for minimizing the free energy of a fluid of hard nonspherical particles confined between planar substrates of variable penetrability. We then test our algorithm by comparing its results for the structure (density-orientation profiles) and equilibrium free energy with those obtained by standard iterative solution of the Euler-Lagrange equations and with Monte Carlo simulation results. Very good agreement is found and the MLP method proves competitively fast, flexible, and refinable. Furthermore, it can be readily generalized to the richer experimental patterned-substrate geometries that are now experimentally realizable but very problematic to conventional theoretical treatments.

Biomechanical modeling and simulation of the spider crab (maja brachydactyla)

Uma linha de pesquisa e desenvolvimento na área da robótica, que tem recebido atenção crescente nos últimos anos, é o desenvolvimento de robôs biologicamente inspirados. A ideia é adquirir conhecimento de seres biológicos, cuja evolução ocorreu ao longo de milhões de anos, e aproveitar o conhecimento assim adquirido para implementar a locomoção pelos mesmos métodos (ou pelo menos usar a inspiração biológica) nas máquinas que se constroem. Acredita-se que desta forma é possível desenvolver máquinas com capacidades semelhantes às dos seres biológicos em termos de capacidade e eficiência energética de locomoção. Uma forma de compreender melhor o funcionamento destes sistemas, sem a necessidade de desenvolver protótipos dispendiosos e com longos tempos de desenvolvimento é usar modelos de simulação. Com base nestas ideias, o objectivo deste trabalho passa por efectuar um estudo da biomecânica da santola (Maja brachydactyla), uma espécie de caranguejo comestível pertencente à família Majidae de artrópodes decápodes, usando a biblioteca de ferramentas SimMechanics da aplicação Matlab / Simulink. Esta tese descreve a anatomia e locomoção da santola, a sua modelação biomecânica e a simulação do seu movimento no ambiente Matlab / SimMechanics e SolidWorks.

Three-dimensional modeling of tongue during speech using MRI data

The tongue is the most important and dynamic articulator for speech formation, because of its anatomic aspects (particularly, the large volume of this muscular organ comparatively to the surrounding organs of the vocal tract) and also due to the wide range of movements and flexibility that are involved. In speech communication research, a variety of techniques have been used for measuring the three-dimensional vocal tract shapes. More recently, magnetic resonance imaging (MRI) becomes common; mainly, because this technique allows the collection of a set of static and dynamic images that can represent the entire vocal tract along any orientation. Over the years, different anatomical organs of the vocal tract have been modelled; namely, 2D and 3D tongue models, using parametric or statistical modelling procedures. Our aims are to present and describe some 3D reconstructed models from MRI data, for one subject uttering sustained articulations of some typical Portuguese sounds. Thus, we present a 3D database of the tongue obtained by stack combinations with the subject articulating Portuguese vowels. This 3D knowledge of the speech organs could be very important; especially, for clinical purposes (for example, for the assessment of articulatory impairments followed by tongue surgery in speech rehabilitation), and also for a better understanding of acoustic theory in speech formation.

Analysis of tongue shape and motion in speech production using statistical modeling

The mechanisms of speech production are complex and have been raising attention from researchers of both medical and computer vision fields. In the speech production mechanism, the articulator’s study is a complex issue, since they have a high level of freedom along this process, namely the tongue, which instigates a problem in its control and observation. In this work it is automatically characterized the tongues shape during the articulation of the oral vowels of Portuguese European by using statistical modeling on MR-images. A point distribution model is built from a set of images collected during artificially sustained articulations of Portuguese European sounds, which can extract the main characteristics of the motion of the tongue. The model built in this work allows under standing more clearly the dynamic speech events involved during sustained articulations. The tongue shape model built can also be useful for speech rehabilitation purposes, specifically to recognize the compensatory movements of the articulators during speech production.

Comparação da fixação em formalina e em fixador molecular e sua influência em técnicas histoquímicas em tecido hepático

A Formalina 10% Tamponada (FNT 10%) é considerada o fixador de eleição nos Laboratórios de Anatomia Patológica. Contudo, a exposição a esta substância acarreta riscos para a saúde dos técnicos. A International Agency for Research on Cancer (IARC) classificou-a como cancerígeno humano e estudos relevam uma correlação positiva entre a exposição a formaldeído e o desenvolvimento de leucemia e leucemia mielóide. Torna-se relevante alterar o processo de fixação substituindo a FNT 10% por outros fixadores melhorando estas questões. Como tal, desenvolveram-se fixadores à base de outros compostos químicos que não formaldeído, como o Fixador Molecular Universal (UMFIX) que tem como base metanol e polietilenoglicol e que é usualmente utilizado com um processador rápido de micro-ondas. Pretende-se comparar as diferenças entre a fixação por FNT 10% e a fixação por UMFIX, em tecido hepático processado em processador rápido de micro-ondas, para as colorações de Hematoxilina-Eosina (H&E), Tricrómio de Gomori, Reticulina e PAS, através da qualidade final das lâminas testadas. A coloração de H&E foi a única que apresentou diferenças estatisticamente significativas entre os dois fixadores (p=0,032; α<0,05; Mann-Whitney). O UMFIX apresenta-se como um substituto da FNT 10% para o processamento rápido em micro-ondas, pois além de apresentar lâminas com uma qualidade final semelhante ou superior às da FNT 10%, ultrapassa os riscos referidos.

Novel potentiometric sensors of molecular imprinted polymers for specific binding of chlormequat

Molecularly imprinted polymers (MIP) were used as potentiometric sensors for the selective recognition and determination of chlormequat (CMQ). They were produced after radical polymerization of 4-vinyl pyridine (4-VP) or methacrylic acid (MAA) monomers in the presence of a cross-linker. CMQwas used as template. Similar nonimprinted (NI) polymers (NIP) were produced by removing the template from reaction media. The effect of kind and amount of MIP or NIP sensors on the potentiometric behavior was investigated. Main analytical features were evaluated in steady and flow modes of operation. The sensor MIP/4-VP exhibited the best performance, presenting fast near-Nernstian response for CMQover the concentration range 6.2×10-6 – 1.0×10-2 mol L-1 with detection limits of 4.1×10-6 mol L-1. The sensor was independent from the pH of test solutions in the range 5 – 10. Potentiometric selectivity coefficients of the proposed sensors were evaluated over several inorganic and organic cations. Results pointed out a good selectivity to CMQ. The sensor was applied to the potentiometric determination of CMQin commercial phytopharmaceuticals and spiked water samples. Recoveries ranged 96 to 108.5%.

Assessment of data-driven modeling strategies for water delivery canals

The aim of this paper is to develop models for experimental open-channel water delivery systems and assess the use of three data-driven modeling tools toward that end. Water delivery canals are nonlinear dynamical systems and thus should be modeled to meet given operational requirements while capturing all relevant dynamics, including transport delays. Typically, the derivation of first principle models for open-channel systems is based on the use of Saint-Venant equations for shallow water, which is a time-consuming task and demands for specific expertise. The present paper proposes and assesses the use of three data-driven modeling tools: artificial neural networks, composite local linear models and fuzzy systems. The canal from Hydraulics and Canal Control Nucleus (A parts per thousand vora University, Portugal) will be used as a benchmark: The models are identified using data collected from the experimental facility, and then their performances are assessed based on suitable validation criterion. The performance of all models is compared among each other and against the experimental data to show the effectiveness of such tools to capture all significant dynamics within the canal system and, therefore, provide accurate nonlinear models that can be used for simulation or control. The models are available upon request to the authors.

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients

Background - Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. Methods - Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. Results - Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. Conclusions - To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.

Multimedia fate modeling and comparative impact on freshwater ecosystems of pharmaceuticals from biosolids-amended soils

This study modeled the impact on freshwater ecosystems of pharmaceuticals detected in biosolids following application on agricultural soils. The detected sulfonamides and hydrochlorothiazide displayed comparatively moderate retention in solid matrices and, therefore, higher transfer fractions from biosolids to the freshwater compartment. However, the residence times of these pharmaceuticals in freshwater were estimated to be short due to abiotic degradation processes. The non-steroidal anti-inflammatory mefenamic acid had the highest environmental impact on aquatic ecosystems and warrants further investigation. The estimation of the solid-water partitioning coefficient was generally the most influential parameter of the probabilistic comparative impact assessment. These results and the modeling approach used in this study serve to prioritize pharmaceuticals in the research effort to assess the risks and the environmental impacts on aquatic biota of these emerging pollutants.

Potential pathogenic fungi assessment through molecular biology in cork industry

Portugal has been the world leader in the cork sector in terms of exports, employing ten thousands of workers. In this working activity, the permanent contact with cork may lead to the exposure to fungi, raising concerns as potential occupational hazards in cork industry. The application of molecular tools is crucial in this setting, since fungal species with faster growth rates may hide other species with clinical relevance, such as species belonging to P. glabrum and A. fumigatus complexes. A study was developed aiming at assessing fungal contamination due to Aspergillus fumigatus complex and Penicillium glabrum complex by molecular methods in three cork industries in the outskirt of Lisbon city.

Caracterização molecular da resistência a antimicrobianos em Escherichia coli produtoras de β-lactamases de espectro alargado isoladas em cães e estudo da partilha de clones bacterianos entre animais de companhia e coabitantes humanos

A pressão seletiva originada pelo uso excessivo de antimicrobianos na medicina humana e veterinária tem contribuído para a emergência de estirpes bacterianas multirresistentes, sendo os estudos mais escassos relativamente à sua presença nos animais de companhia. Porque os animais e os seus proprietários partilham o mesmo espaço habitacional, apresentando comportamentos de contacto próximo, existe uma hipótese elevada de transferência microbiana inter-espécie. Ante esta possibilidade é importante escrutinar o papel dos animais de companhia enquanto reservatórios de estirpes e de genes de resistência, bem como a sua envolvência na disseminação de estirpes bacterianas multirresistentes. Importa também, investigar o papel das superfícies e objetos domésticos partilhados por ambos, como potenciadores deste fenómeno. O objetivo deste trabalho foi, identificar o filogrupo e fazer a caracterização molecular dos genes que conferem resistência aos β-lactâmicos e às quinolonas, em quarenta isolados de Escherichia coli produtoras de β-lactamases de espectro alargado (ESBL), obtidas em zaragatoas fecais de cães consultados no Hospital Veterinário do ICBAS-UP. Complementarmente pretendeu-se inferir sobre a partilha de clones de Escherichia coli e Enterococcus spp. com elevadas resistências, em cinco agregados familiares (humanos e seus animais de companhia) assim como avaliar a potencial disseminação de estirpes multirresistentes no ambiente doméstico. Previamente foram recolhidas zaragatoas de fezes, pelo e mucosa oral dos animais e em alguns casos, dos seus proprietários, e ainda do ambiente doméstico. As zaragatoas foram processadas e as estirpes isoladas com base em meios seletivos. Foram realizados testes de suscetibilidade antimicrobiana de modo a estabelecer o fenótipo de resistência de cada isolado. O DNA foi extraído por varias metodologias e técnicas de PCR foram utilizadas para caracterização de filogrupos (Escherichia coli) e identificação da espécie (Enterococcus spp.). A avaliação da proximidade filogenética entre isolados foi efetuada por ERIC PCR e PFGE. No conjunto de quarenta isolados produtores de ESBL e/ou resistentes a quinolonas verificou-se que 47,5% pertenciam ao filogrupo A, havendo uma menor prevalência do filogrupo D (25,0%), B1 (17,5%), e B2 (10,0%).A frequência de resistência nestes isolados é factualmente elevada, sendo reveladora de uma elevada pressão seletiva. Com exceção de dois isolados, os fenótipos foram justificados pela presença de β-lactamases. A frequência da presença de genes foi: 47% blaTEM, 34% blaSHV, 24% blaOXA , 18% blaCTX-M-15, 8% blaCTX-M-2, 3% blaCTX-M-9. Nos isolados resistentes às quinolonas verificou-se maioritariamente a presença de mutações nos genes cromossomais gyrA e parC, e em alguns casos a presença de um determinante de resistência mediado por plasmídeo – qnrS. Nos cinco “agregados familiares” (humanos e animais) estudados foi observada uma partilha frequente de clones de E. coli e Enterococcus faecalis com múltiplas resistências, isolados em fezes e mucosa oral de cães e gatos e fezes e mãos dos respetivos proprietários, evidenciando-se assim uma possível transferência direta entre coabitantes (agregados A, C, D, E). Ficou também comprovado com percentagens de similaridade genotípica superiores a 94% que essa disseminação também ocorre para o ambiente doméstico, envolvendo objetos dos animais e de uso comum (agregados A, E). Os resultados obtidos reforçam a necessidade de um uso prudente dos antimicrobianos, pois elevados padrões de resistências terão um impacto não só na qualidade de vida dos animais mas também na saúde humana. Adicionalmente importa sensibilizar os proprietários para a necessidade de uma maior vigilância relativamente às formas de interação com os animais, bem como para a adoção de medidas higiénicas cautelares após essa mesma interação.

Modeling for control design of a multi-reach water canal

This article addresses the problem of obtaining reduced complexity models of multi-reach water delivery canals that are suitable for robust and linear parameter varying (LPV) control design. In the first stage, by applying a method known from the literature, a finite dimensional rational transfer function of a priori defined order is obtained for each canal reach by linearizing the Saint-Venant equations. Then, by using block diagrams algebra, these different models are combined with linearized gate models in order to obtain the overall canal model. In what concerns the control design objectives, this approach has the advantages of providing a model with prescribed order and to quantify the high frequency uncertainty due to model approximation. A case study with a 3-reach canal is presented, and the resulting model is compared with experimental data. © 2014 IEEE.