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The present work reports on a new approach based on electroactive microenvironments to mitigate skeletal muscle cancer. For that, piezoelectric films based on poly(vinylidene fluoride) have been applied to evaluate the influence of mechano- and/or electrical stimuli on rhabdomyosarcoma (RMS) proliferation. Human embryonal rhabdomyosarcoma (RD) cells were cultured on PVDF pristine films with different surface charge (non-poled, poled+ and poled-) and magnetic composites (10% and 20% Fe3O4, and 20% CFO filler content) to allow magneto-mechanical and magnetoelectrical stimulation films. Electrospun PVDF pristine (oriented and randomly) and magnetic (10% Fe3O4) fiber mats were also evaluated to take into consideration the morphology effect on cell response. It was found that the mechanical stimuli enhance RMS proliferation whereas the mechano-electrical decreases it. It was also verified that the RD cells proliferate better on randomly oriented fibers, whereas myoblast cells do it better in oriented ones. The obtained results confirm that electroactive microenvironments can be used to develop novel and effective approaches to deal with RMS cancer, that can be extrapolated to others cancer types., This work has been supported by FCT-Fundacao para a Ciencia e Tecnologia (FCT) under the scope of the strategic funding of UID/FIS/04650/2020 and UIDB/04469/2020 units, and project PTDC/BTMMAT/28237/2017. Vitor Correia thanks FCT for the junior researcher contract (DL57/2016) and Clarisse Ribeiro thanks the FCT for the contract under the Stimulus of Scientific Employment, Individual Support (CEECIND) -3rd Edition (2020.04163.CEECIND). Finally, the authors acknowledge funding by Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the project PID2019-106099RB-C43/AEI/10.13039/501100011033 and from the Basque Government Industry Departments under the ELKARTEK program.

In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in Poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements., We want to acknowledge Dr. João Pedro Silva and Dr. Leon Kluskens, from Departament of Biologic Engineering, University of Minho, for the help provided to work in the department, as well to Sylvie Ribeiro from the Department of Physics of the same university. This work is funded by FEDER funds through the "Programa Operacional Fatores de Competitividade – COMPETE" and by national funds arranged by FCT- Fundação para a Ciência e a Tecnologia, project reference PEST-C/FIS/UI607/2014. The authors also thank funding from Matepro – Optimizing Materials and Processes”, ref. NORTE-07-0124-FEDER-000037”, co-funded by the “Programa Operacional Regional do Norte” (ON.2 – O Novo Norte), under the “Quadro de Referência Estratégico Nacional” (QREN), through the “Fundo Europeu de Desenvolvimento Regional” (FEDER). The authors also thank support from the COST Action MP1206 “Electrospun Nano-fibres for bio inspired composite materials and innovative industrial applications” and MP1301 “New Generation Biomimetic and Customized Implants for Bone Engineering”. JAP, VS, CR and VC thank the FCT for the SFRH/BD/64586/2009 and SFRH/BPD/63148/2009, SFRH/BPD/90870/2012, and SFRH/BPD/97739/2013 grants, respectively. JLGR acknowledges the support of Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2013-46467-C4-1-R (including the FEDER financial support). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

The relative space per player formulated in small-sided and conditioned games can be manipulated either by promoting variations in player numbers or by modifying field dimensions. In this study we analysed how the same relative spaces per player, obtained through manipulations of player numbers and field dimensions, influenced inter-individual coordination. The positional data (GPS, 10 Hz) of 24 U-15 yrs performing in three different relative spaces per player (118, 133 and 152m2) was used. Inter-individual behavioural measures included: (i) effective relative space per player, (ii) radius of free movement; (iii) numerical relations inside each player’s relative space per player; and (iv) players’ spatial distribution variability. Magnitude-based inferences were used to analyse the practical significance of the selected variables. Results showed that manipulations of player numbers elicited more free space in the vicinity of each player. However, more advantageous numerical relations adjacent to each individual player and broader individual spatial distributions on field were observed during manipulations of field dimensions. These findings highlight the complex nature of performance behaviours captured by the co-adaptation of players to surrounding spatial constraints. Sport pedagogists should carefully evaluate the use of player numbers and field dimensions as strategies to simulate constraints of specific game contexts.

Neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are currently the only effective antiviral drugs available worldwide for the management of influenza. The potential development of resistance is continually threatening their use, rationalizing and highlighting the need for a close and sustained evaluation of virus susceptibility. This study aimed to analyze and characterize the phenotypic and genotypic NAIs susceptibility profiles of A(H1N1)pdm09 viruses circulating in Portugal from 2009 to 2010/2011. A total of 144 cases of A(H1N1)pdm09 virus infection from community and hospitalized patients were studied, including three suspected cases of clinical resistance to oseltamivir. Oseltamivir resistance was confirmed for two of the suspected cases. Neuraminidase (NA) H275Y resistant marker was found in viruses from both cases but for one it was only present in 26.2% of virus population, raising questions about the minimal percentage of resistant virus that should be considered relevant. Cross-decreased susceptibility to oseltamivir and zanamivir (2-4 IC50 fold-change) was detected on viruses from two potentially linked community patients from 2009. Both viruses harbored the NA I223V mutation. NA Y155H mutation was found in 18 statistical non-outlier viruses from 2009, having no impact on virus susceptibility. The mutations at NA N369K and V241I may have contributed to the significantly higher baseline IC50 value obtained to oseltamivir for 2010/2011 viruses, compared to viruses from the pandemic period. These results may contribute to a better understanding of the relationship between phenotype and genotype, which is currently challenging, and to the global assessment of A(H1N1)pdm09 virus susceptibility profile and baseline level to NAIs. This work was supported by a research grant from the Portuguese Calouste Gulbenkian Foundation and by doctoral grants from the Portuguese Science and Technology Foundation (SFRH/BD/48532/2008, SFRH/BD/62676/2009, and SFRH/BD/65211/2009).

Made available in DSpace on 2016-08-29T11:03:53Z (GMT). No. of bitstreams: 1 tese_7456_TESE VASTI GONÇALVES DE PAULA CORREIA.pdf: 2781530 bytes, checksum: 880884ce2f8b5ef5d92e624f1241b288 (MD5) Previous issue date: 2014-02-24 O estudo assume como problema de investigação analisar as contribuições da Comunicação Alternativa e Ampliada (CAA) aos processos comunicativos de alunos sem fala articulada no contexto da escola, destacando nesses processos o papel potencializador dos interlocutores. Fundamenta-se na abordagem de linguagem e na noção de enunciado discutidas por Bakhtin e nas contribuições de Vigotski sobre a relação entre desenvolvimento e aprendizagem, postulando que a aquisição e o desenvolvimento da linguagem ocorrem no curso das aprendizagens, ao longo da vida. As análises e reflexões empreendidas evidenciam uma discussão acerca da linguagem que se desloca da dimensão orgânica para a dimensão da constituição do sujeito como humano. Sob essa visão, outros conceitos, como os de língua, fala, interação verbal, dialogia, enunciação, aprendizagem e desenvolvimento são problematizados e também considerados como elementos fundantes e presentes nas relações comunicativas entre os sujeitos sem fala articulada e seus interlocutores. Na primeira etapa, o estudo busca conhecer as formas organizativo-pedagógicas de cinco Secretarias Municipais de Educação da Região Metropolitana de Vitória e da Secretaria de Estado da Educação no que diz respeito à identificação dos alunos com Paralisia Cerebral, sem fala articulada, ao acompanhamento técnico-pedagógico e à formação de professores que atuam na Educação Especial. Na segunda etapa, objetiva conhecer a processualidade da organização do trabalho pedagógico instituída nos contextos escolares e investiga os processos comunicativos em/com dois alunos com severos comprometimentos motores e de fala em duas escolas de Ensino Fundamental, localizadas no município de Serra e de Vitória. Nesta etapa, opta pela pesquisa-ação colaborativo-crítica por contribuir, teórica e metodologicamente, para sustentar os fazeres individuais e coletivos nos lócus de investigação. Os resultados revelam que, institucionalmente, ainda não se conhece quem são e quantos são os alunos com Paralisia Cerebral sem fala articulada no contexto de suas reais necessidades. Esse desconhecimento é atribuído pelas gestoras das Secretarias Municipais de Educação investigadas ao considerarem que, via de regra, são tomadas apenas as informações do Educacenso-INEP. As identificações pontuais, quando ocorrem, são decorrentes de estratégias internas adotadas, sendo uma delas o assessoramento pedagógico das equipes às escolas. No que tange ao ensino, à aprendizagem e à avaliação, o estudo constata que são atravessados por concepções equivocadas sobre os sujeitos com Paralisia Cerebral sustentadas, sobretudo, pela baixa expectativa e pelo pouco esforço quanto à sua escolarização. Constata também que o uso dos recursos de CAA potencializa os processos comunicativos dos alunos investigados e, movimentados pela linguagem, possibilita-lhes enunciar e fixar posições, opiniões e decisões, assegurando-lhes mais autonomia e fluidez do processo comunicacional. As formas de mediação dos interlocutores assim como as dinâmicas dialógicas por eles utilizadas com os alunos se constituem como elementos importantes nos processos de comunicação e interação. A espera do outro, o apoio e o incentivo à reformulação daquilo que se quer expressar, as modificações e alterações no jogo dialógico são exemplos dessa mediação. Quanto às ações de reorganização do trabalho pedagógico, o estudo registra maior articulação e colaboração entre professores da classe, professora da Educação Especial e estagiária no planejamento das aulas, dos conteúdos, com a inserção no notebook para um dos alunos; o uso das pranchas de comunicação, por ambos os alunos e seus interlocutores, como ação inovadora nos contextos escolares; a realização de atividades pelos alunos, com gradativa autonomia, a partir da disponibilização de recursos de TA/CAA (pasta de conteúdos temáticos, figuras imantadas, quadro metálico, ponteira, plano inclinado, notebook); a proposição de ações intencionais de alfabetização, a partir da reorganização de espaços-tempos no cotidiano da escola. Conclui que as discussões teóricas e práticas das questões relacionadas com a linguagem, com os processos cognitivos e com o uso de recursos de TA/CAA alavancam mudanças na concepção dos profissionais das escolas pesquisadas que, ainda, sob uma visão reducionista quanto às formas de comunicação e de interação verbal, impõem limites à escolarização dos alunos com deficiência.

The GEOPLANO/LNEC consortium tested an emerging in situ methodology of soil and groundwater remediation using zero-valent iron nanoparticles (nZVI), in a brownfield site located south of Lisbon. The area where the pilot tests were conducted has an industrial background with a strong presence of heavy metals, sulfates and nitrates. The implementation of the in situ testing was preceded by the physical and chemical characterization of soils, groundwater and lixiviates produced by contaminated soils. The effect of different nZVI dosing was tested at laboratory. The in situ tests were executed with three different concentrations of nZVI solutions. After the nZVI injection, a program for monitoring the groundwater quality and the concentrations of contaminants was implemented. Geoplano DG/NGEA 2013 13 -14

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Background and Aims: The ability of Influenza A (infA) viruses to counteract and manipulate the host response to infection is well acknowledged. In addition, the activation of the PI3K/Akt survival pathway by the infA NS1 protein has been described as one of the strategies to delay the apoptotic response of the infected cell. As influenza B (infB) viruses differ genetically and phenotypically from infA viruses, namely at the NS protein level, we aimed to compare general apoptosis and survival pathways induced by each influenza type. Methods: MDCK-SIAT1 cells were infected with infA(H1N1)pdm09 virus A/Portugal/82/2009 (APT82) and infB virus B/Lisboa/08/2006 (BLx08). Activities of caspase-3 -7 -8 and -9 were measured at several time points post-infection (hpi). Total levels of Akt, pAkt, NF-κB, IκB, p53 and β-actin were also examined by Western blot. Results: Our results indicate that the apoptosis process induced by BLx08 was associated with activation of both intrinsic, caspase-9-dependent and extrinsic, caspase-8-dependent pathways as early as at 8hpi. In contrast, APT82-induced apoptosis only involved the activation of the intrinsic pathway, and occurred at 32hpi. Surprisingly, our data show that the activation of the survival pathway PI3K/Akt was significantly increased upon BLx08 infection when compared with APT82 infection. In fact, increased levels of pAkt were observed at the same time of caspase activation in the early phase of BLx08 infection. This, however, did not result in increased downstream NFκB activation, since its inhibitor IκB was also markedly upregulated. Increased p53 levels associated with APT82 infection may also explain the delayed apoptosis response in infA, assessed by caspase activity, as it may require transcriptional activation that it is deregulated and directed to viral replication. Conclusion: InfB and infA infection differs in time and levels of activation of apoptosis and survival signaling pathways. PI3K/Akt activation in infB is not sufficient to inhibit apoptosis. Further studies will clarify this difference and shed light into the use of cellular mechanisms as new ways to fight influenza.

Neuraminidase (NA) protein of influenza viruses has the particularity of being under antibody and antiviral drug selective pressure, as it is one of the main surface antigens and the target of neuraminidase inhibitors(NAIs). The aim of this study is to investigate the selective pressure(SP) acting on the NA of seasonal and pandemic influenza viruses. It comprises two objectives: (a)to evaluate the contribution of positive SP for the emergence of NAIs resistant viruses; and (b)to determine the impact of NAIs introduction into clinic and its wide use during pandemic on the SP acting on NA. For the 1st objective it will be analysed the SP acting on the sites associated with NAIs resistance or reduction in susceptibility. The 2nd objective implies a differential evolutionary pressure analysis according to time, with 3 sub-datasets of NA sequences being considered: (1)before worldwide introduction of NAIs into clinic(1999); (2)before wide use of oseltamivir during A(H1N1)2009 pandemic(2009); and (3)from 2009 to date. A large dataset of full-length NA coding sequences will be used for each (sub)type/variant, comprising sequences obtained at national level(since 2000/2001) and sequences available at GISAID and NCBI. A(H1N1)seasonal dataset was already created, including a total of 1523 sequences, from which 94 belong to 1st sub-dataset, 1094 to 2nd and 335 to 3rd. All SP analysis will be performed using the expertise acquired with this workshop. This study may contribute for understanding the role of antiviral drug selective pressure in NAIs resistance, patterns of emergency of resistant viruses and NA evolutio

Com o propósito de dar resposta à necessidade de mitigação de locais contaminados,têm vindo a ser desenvolvidas novas técnicas de reabilitação baseadas na utilização de ferro de valência zero nano particulado (NZVI). Por este motivo, continuam a desenvolver-se ensaios piloto em condições variadas, com o objetivo de suportar a aplicabilidade e a eficácia desta tecnologia. Geoplano / LNEC DG/NGEA 2012 18 a 20 de junho

Background: Genetic changes in influenza surface and internal genes can alter viral fitness and virulence. Mutation trend analysis and antiviral drug susceptibility profiling of A(H1N1)pdm09 viruses is essential for risk assessment of emergent strains and disease management. Objective: To profile genomic signatures and antiviral drug resistance of A(H1N1)pdm09 viruses and to discuss the potential role of mutated residues in human host adaptation and virulence. Study design: A(H1N1)pdm09 viruses circulating in Portugal during pandemic and post-pandemic periods and 2009/2010 season. Viruses were isolated in MDCK-SIAT1 cell culture and subjected to mutation analysis of surface and internal proteins, and to antiviral drug susceptibility profiling. Results: The A(H1N1)pdm09 strains circulating during the epidemic period in Portugal were resistant to amantadine. The majority of the strains were found to be susceptible to oseltamivir and zanamivir, with five outliers to neuraminidase inhibitors (NAIs) identified. Specific mutation patterns were detected within the functional domains of internal proteins PB2, PB1, PA, NP, NS1, M1 and NS2/NEP, which were common to all isolates and also some cluster-specific. Discussion: Modification of viral genome transcription, replication and apoptosis kinetics, changes in antigenicity and antiviral drug susceptibility are known determinants of virulence. We report several point mutations with putative roles in viral fitness and virulence, and discuss their potential to result in more virulent phenotypes. Monitoring of specific mutations and genetic patterns in influenza viral genes is essential for risk assessing emergent strains, disease epidemiology and public health implications.

A hidatidose apresenta uma distribuição cosmopolita e encontra-se entre as zoonoses de maior importância a nível mundial. O parasita Echinococcus granulosus, responsável por esta doença, apresenta uma distribuição cosmopolita afectando diversas espécies de animais e, acidentalmente, o Homem. O objectivo do estudo é a obtenção do estado larvar do E. granulosus (protoescólices) para o estudo da fertilidade dos quistos em diferentes espécies animais em Portugal. MATERIAL E MÉTODOS: Para este estudo foi utilizado material biológico parasitado, obtido em matadouros públicos e privados da zona centro (Castelo Branco, Pedrógão e Guarda), recolhido entre Outubro de 2007 e Março de 2010. Procedeu-se à extracção do conteúdo dos quistos hidáticos (líquido e areia hidáticos). E através da observação ao microscópio óptico, observou-se a presença de protoescólices e/ou ganchos, bem como o seu estado de conservação e a presença de elementos estranhos. As amostras férteis foram conservadas a - 20ºC para posteriores trabalhos. RESULTADOS: Foram analisadas um total de 95 amostras de quistos hidáticos provenientes do fígado e pulmão de ovinos, bovinos e caprinos e estabelecida uma relação entre a fertilidade/infertilidade dos quistos e os órgãos parasitados. Relativamente à espécie hospedeira e faixa etária do animal parasitado, os resultados obtidos não foram estatisticamente significativos. DISCUSSÃO: Foi encontrada uma associação entre a fertilidade dos quistos hidáticos e órgão parasitado, apresentando uma relação semelhante às obtidas em estudos do mesmo género efectuados noutras zonas endémicas.