Structural, physical, and chemical modifications induced by microwave heating on native agar-like galactans

Native agars from Gracilaria vermiculophylla produced in sustainable aquaculture systems (IMTA) were extracted under conventional (TWE) and microwave (MAE) heating. The optimal extracts from both processes were compared in terms of their properties. The agars’ structure was further investigated through Fourier transform infrared and NMR spectroscopy. Both samples showed a regular structure with an identical backbone, β-D-galactose (G) and 3,6-anhydro-α-L-galactose (LA) units; a considerable degree of methylation was found at C6 of the G units and, to a lesser extent, at C2 of the LA residues. The methylation degree in the G units was lower for MAEopt agar; the sulfate content was also reduced. MAE led to higher agar recoveries with drastic extraction time and solvent volume reductions. Two times lower values of [η] and Mv obtained for the MAEopt sample indicate substantial depolymerization of the polysaccharide backbone; this was reflected in its gelling properties; yet it was clearly appropriate for commercial application in soft-texture food products.

The evidence of conventional physiotherapeutic interventions after exercise-induced muscle damage: systematic review and meta-analysis

Abstract Introduction: Exhaustive and/or unaccustomed exercise, mainly those involving eccentric muscle actions, induces temporary muscle damage, evidenced by Delayed Onset Muscle Soreness. Different strategies to recover the signs and symptoms of this myogenic condition have been studied by researchers, as a result a significant number of articles on this issue have been published. Purpose: A systematic review was conducted to assess the evidence of the physiotherapeutic interventions of exercise-induced muscle damage. Methods: The electronic data bases were searched, including MEDLINE (1996-2011), CINHAL (1982- 2011), EMBASE (1988-2011), PEDro (1950-2011), and SPORTDiscus (1985-2011). Systematic review was limited to randomized control trials (RCTs) studies, written in English or Portuguese, which included physiotherapeutic interventions, namely massage, cryotherapy, stretching and low-intensity exercise, on adult human subjects (18-60 years old) of either gender. Studies were excluded when the intervention could not be assessed independently. The methodological quality of RCTs was independently assessed with the PEDro Scale by three reviewers. Results: Thirty-three studies were included in the systematic review; eight analyzed the effects of the massage, ten analyzed the effects of the cryotherapy, eight the effect of stretching and seventeen focused low-intensity exercise intervention. The results suggest that massage is the most effective intervention and that there is inconclusive evidence to support the use of cryotherapy; whereas the other conventional, namely stretching and low-intensity exercise, there is no evidence to prove their efficacy. Conclusion: The results allow the conclusion that massage is the physiotherapeutic intervention that demonstrated to be the most effective in the relief of symptoms and signs of exercise-induced muscle damage, as a result, massage should still be used in the muscular recovery after sports activities.

Identification of neuronal alterations induced by Shank3 mutations using iPS cells from Phelan-McDermid Patients' Fibroblasts

A família de proteínas Shank é o principal conjunto de proteinas de suporte e está localizada na densidade pós-sináptica das sinapses excitatórias. Existem 3 genes na família Shank, Shank1, Shank2 e Shank3 e são caracterizados por múltiplos domínios repetidos de anquirina próximo ao N-terminal seguido pelos domínios Src homologo 3 e PDZ, uma região longa rica em prolina e um domínio de motivo α estéril próximo ao C-terminal. Shank proteínas conectam duas subunidades de receptors glutamatérgicos, recetores NMDA e recetores metabotrópicos de glutamato do tipo-I (mGluRs). O domínio PDZ da Shank conecta-se ao C-terminal do GKAP e este, liga-se, ao complexo recetor PSD-95-NMDA. Por outro lado, a proteína Homer interage com o domínio rico em prolina para confirmar a associação entre a proteína Shank com o mGluR tipo-I. A proteína específica em estudo, Shank3, é haploinsuficiente em pacientes com sindrome Phelan-McDermid devido à deleções no braço comprido do cromossoma 22 levando à danos intelectuais, ausência ou atraso no discurso, comportamentos semelhantes ao autismo, hipotonia e características dismórficas. Neste trabalho, investigamos o papel da Shank3 na função sináptica para compreender a relação entre alterações nesta proteína e as características neurológicas presente em Pacientes com síndrome Phelan-McDermid. Foram utilizados dois modelos diferentes, ratinhos knockout Shank3 e hiPSC de pacientes com PMS. Ratinhos geneticamente modificados são ferramentas uteis no estudo de genes e na compreensão dos mecanismos que experiências in vitro não são capazes de reproduzir, mas de maneira a compreender melhor as patologias humanas, decidimos trabalhar também com células humanas. Os fibroblastos dos pacientes com síndrome Phelan-McDermid fora reprogramados em hiPS cells, diferenciados em neurónios e comparados com os neurónios obtidos a partir de doadores saudavéis e da mesma idade. A reprogramação em iPSC foi realizada por infecção de lentivirus com quatro genes de reprogramação OCT4, c-MYC, SOX2 e KFL4 para posteriormente serem diferenciados em neurónios, com cada passo sendo positivamente confirmado através de marcadores neuronais. Através dos neurónios diferenciados, analisamos a expressão de proteínas sinápticas. Pacientes com haploinsuficiencia na proteína Shank3 apresentam níveis elevados de proteína mGluR5 e decrescidos de proteína Homer sugerindo que a haploinsuficiencia leva a desregulação do complexo mGluR5-Homer-Shank3 conduzindo também, a defeitos na maturação sináptica. Assim, a expressão da proteína mGluR5 está alterada nos pacientes com PMS podendo estar relacionada com defeitos encontrados na diferenciação neuronal e maturação sináptica observados nos neurónios de pacientes. Conclusivamente, iPS cells representam um modelo fundamental no estudo da proteína Shank3 e a sua influência no sindrome de Phelan-McDermid.

Eficácia do Constraint-Induced Movement Therapy, em utentes com sequelas de Acidente Vascular Encefálico, até 9 meses

O Acidente Vascular Encefálico é uma das principais causas de morte, tornando-se cada vez mais iminente processos de reabilitação que minimizem as sequelas, nomeadamente as limitações do membro superior que dificultam o envolvimento em atividades da vida diária. O Constraint-Induced Movement Therapy, surge como uma abordagem que incrementa o uso do membro superior mais afetado. A presente investigação trata-se de um estudo de casos múltiplos. Pretende-se verificar se existem melhorias na funcionalidade do membro superior mais afetado, analisar em que atividades da vida diária são visíveis melhorias funcionais e compreender se o maior envolvimento nas atividades diárias está diretamente relacionado com a melhoria na capacidade funcional. Pretende-se ainda que os valores obtidos no Wolf Motor Function Test sejam um contributo para a sua validação para a população portuguesa. Utilizou-se um questionário para recolha de dados pessoais e clínicos (amplitudes de movimento, dor e espasticidade); o Wolf Motor Function Test e o Action Research Arm Test para verificar a funcionalidade do membro superior mais afetado; e a Motor Activity Log que avalia o envolvimento em atividades da vida diária. O grupo é constituído por 3 utentes que sofreram um primeiro Acidente Vascular Encefálico até 9 meses de evolução, internados na Santa Casa da Misericórdia de Monção e que cumpriam os critérios de inclusão. O programa foi implementado três horas/dia, durante 10 dias, mantendo a restrição no membro superior menos afetado durante 90% do dia acordado. Como se trata de um estudo de casos múltiplos, analisou-se cada participante individualmente e verificou-se a diferença entre os resultados finais e iniciais para cada uma das variáveis. Os resultados obtidos revelam ganhos na amplitude de movimento, velocidade de execução e capacidade funcional do membro superior mais afetado, nomeadamente nas funções de preensão e pinça da mão, bem como se testemunhou minimização do fenómeno learned nonuse. Verificaram-se ganhos funcionais em todos os participantes nas atividades da vida diária apesar de serem diferentes de participante para participante. Dois participantes afirmaram que voltariam a participar no programa.Conclui-se, assim que a técnica resulta em ganhos funcionais nestes utentes, indicando um caminho alternativo a outras abordagens de reabilitação.

Proposta de um modelo de avaliação de websites de turismo de habitação

actividade de turismo de habitação tem crescido de forma sustentada ao longo dos últimos anos em Portugal e o recurso à internet como canal de comercialização deste serviço tem substituído gradualmente os restantes canais mais tradicionais. No entanto, esta generalização do recurso a websites, nesta e noutras áreas da actividade económica, não tem sido sempre sinónimo de qualidade, justificando assim a procura de sistemas de avaliação deste tipo de produto de software, que possam ser aplicados sistemática e eficazmente. Nesse sentido, foi desenvolvido este trabalho, no propósito de conseguir um modelo de avaliação de websites, específico para o turismo de habitação, na convicção de que para áreas específicas se exigem abordagens específicas. A pesquisa por modelos orientados para esta actividade não produziu qualquer resultado, ao contrário de outras áreas onde já existem modelos adequados, como sejam a área académica e outras. Daí a necessidade de conjugar ideias e conceitos mais genéricos, de fontes diversas, com elementos específicos de turismo de habitação, no intuito de os combinar e adequar aos objectivos deste trabalho. Assim, a partir de elementos da Norma ISO 9126; de conceitos de usabilidade, funcionalidade, credibilidade, e outros; da opção por uma abordagem de desempenho empresarial; de modelos já existentes, embora direccionados para outras áreas; e de um modelo exploratório de carácter empírico entretanto desenvolvido, foi concebido e implementado um modelo que se caracteriza no essencial, por ser estruturado em três níveis, contemplar um conjunto de trinta e oito atributos em avaliação, com maior incidência naqueles relacionados com aspectos que se considera terem maior influência no desempenho da actividade económica subjacente, e que tem uma ponderação variável do seu impacto no resultado final. No intuito de dar flexibilidade ao modelo e de contrariar a carga subjectiva resultante do processo de ponderação do impacto de cada atributo avaliado, foram implementados no modelo três cenários alternativos com ponderações distintas, cada um valorizando determinado tipo de atributos e desvalorizando outros. Naturalmente que, aquando da implementação do modelo exploratório foram já retiradas algumas conclusões sobre o panorama geral, todavia o modelo definitivo veio dar-lhes maior consistência e pormenor.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

Background: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2- octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Mechanisms for reflection-based monitoring of real- time systems

Monitoring is a very important aspect to consider when developing real-time systems. However, it is also important to consider the impact of the monitoring mechanisms in the actual application. The use of Reflection can provide a clear separation between the real-time application and the implemented monitoring mechanisms, which can be introduced (reflected) into the underlying system without changing the actual application part of the code. Nevertheless, controlling the monitoring system itself is still a topic of research. The monitoring mechanisms must contain knowledge about “how to get the information out”. Therefore, this paper presents the ongoing work to define a suitable strategy for monitoring real-time systems through the use of Reflection.

Ligand-Induced structural changes in TEM‑1 probed by molecular dynamics and relative binding free energy calculations

The TEM family of enzymes has had a crucial impact on the pharmaceutical industry due to their important role in antibiotic resistance. Even with the latest technologies in structural biology and genomics, no 3D structure of a TEM- 1/antibiotic complex is known previous to acylation. Therefore, the comprehension of their capability in acylate antibiotics is based on the protein macromolecular structure uncomplexed. In this work, molecular docking, molecular dynamic simulations, and relative free energy calculations were applied in order to get a comprehensive and thorough analysis of TEM-1/ampicillin and TEM-1/amoxicillin complexes. We described the complexes and analyzed the effect of ligand binding on the overall structure. We clearly demonstrate that the key residues involved in the stability of the ligand (hot-spots) vary with the nature of the ligand. Structural effects such as (i) the distances between interfacial residues (Ser70−Oγ and Lys73−Nζ, Lys73−Nζ and Ser130−Oγ, and Ser70−Oγ−Ser130−Oγ), (ii) side chain rotamer variation (Tyr105 and Glu240), and (iii) the presence of conserved waters can be also influenced by ligand binding. This study supports the hypothesis that TEM-1 suffers structural modifications upon ligand binding.

POSIX trace based behavioural reflection

Traditional Real-Time Operating Systems (RTOS) are not designed to accommodate application specific requirements. They address a general case and the application must co-exist with any limitations imposed by such design. For modern real-time applications this limits the quality of services offered to the end-user. Research in this field has shown that it is possible to develop dynamic systems where adaptation is the key for success. However, adaptation requires full knowledge of the system state. To overcome this we propose a framework to gather data, and interact with the operating system, extending the traditional POSIX trace model with a partial reflective model. Such combination still preserves the trace mechanism semantics while creating a powerful platform to develop new dynamic systems, with little impact in the system and avoiding complex changes in the kernel source code.

Evaluation of the role of glutathione in the lead-induced toxicity in saccharomyces cerevisiae

The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3 h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (l-glutamic acid, l-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability.

Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 μmol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 .−) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative ρ0 strain, lacking mitochondrial DNA, was used. The ρ0 strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 .− precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.

Predicting Xerostomia induced by IMRT treatments: A logistic regression approach

Radiotherapy is one of the main treatments used against cancer. Radiotherapy uses radiation to destroy cancerous cells trying, at the same time, to minimize the damages in healthy tissues. The planning of a radiotherapy treatment is patient dependent, resulting in a lengthy trial and error procedure until a treatment complying as most as possible with the medical prescription is found. Intensity Modulated Radiation Therapy (IMRT) is one technique of radiation treatment that allows the achievement of a high degree of conformity between the area to be treated and the dose absorbed by healthy tissues. Nevertheless, it is still not possible to eliminate completely the potential treatments’ side-effects. In this retrospective study we use the clinical data from patients with head-and-neck cancer treated at the Portuguese Institute of Oncology of Coimbra and explore the possibility of classifying new and untreated patients according to the probability of xerostomia 12 months after the beginning of IMRT treatments by using a logistic regression approach. The results obtained show that the classifier presents a high discriminative ability in predicting the binary response “at risk for xerostomia at 12 months”