Chronic PEGylated obestatin treatment prevents the onset of high fat diet-induced hypertension and endothelial dysfunction in the absence of metabolic actions

Background: Obestatin is a gastrointestinal peptide with established metabolic actions and emerging vascular effects which involve activation of NO signalling. The aim of this study was to investigate effects of a recently-characterised stable analogue, PEGylated obestatin (PEG-OB), in the setting of diet-induced obesity which is associated with both metabolic and vascular dysfunction. Methods: Male Sprague Dawley rats (6 weeks; n=8) were maintained on standard (SD) or high fat (HF) diet (60% fat) for 8 weeks with once-daily injection of either PEG-OB (50nmol/kg/day) or saline from 2 weeks. Results: HF feeding for 8 weeks resulted in marked body weight gain which was not affected by chronic PEG-OB treatment (HF saline, 175.0±12.2; HF PEG-OB, 190.4±6.4g; P=NS). Similarly, blood glucose, as indicated by HbA1c (HF saline, 6.30±0.15; HF PEG-OB, 6.13±0.36%; P=NS) and insulin tolerance (HF saline, 105.2±52.5; HF PEG-OB, 90.3±45.4mmol/L.min; P=NS), were unaltered by PEG-OB. Despite the apparent lack of metabolic effects, chronic PEG-OB treatment markedly attenuated development of HF-induced hypertension (HF saline, 146.5±4.9mmHg; HF PEG-OB, 123.0±9.7mmHg; P<0.01), assessed by tail-cuff plethysmography. Furthermore, organ bath pharmacology in isolated aortic rings, indicated that HF diet-induced endothelial dysfunction was completely prevented by PEG-OB (acetylcholine, EC50: SD saline, 335±113; HF saline, 758±164; HF PEG-OB, 277±85nmol/L; P<0.05). However, contraction to phenylephrine and relaxation to the NO donor, sodium nitroprusside, were unaltered between groups. Conclusions: PEG-OB exerts beneficial effects on hypertension and endothelial function in diabetes independently of metabolic actions suggesting that obestatin signalling may represent a novel therapeutic target to reduce the risk of associated cardiovascular complications.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog, Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana-BI. The cDNA sequencing was obtained by 'shotgun' cloning. The amino acid sequence of the mature peptide was identified as Gly-Leu-Leu-Ser-Gly-Lys-Ser-Val-Lys-Gly-Ser-Ile-OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI-TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana-BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana-BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide

Mitochondrial plasticity in pathophysiological conditions

Both skeletal and cardiac muscles daily burn tremendous amounts of ATP to meet the energy requirements for contraction. So, it is not surprising that the maintenance of mitochondrial morphology, number, distribution and functionality in striated muscle are important for muscle homeostasis. In these tissues mitochondria present the added dimension of two populations, the intermyofibrillar (IMF) and the subsarcolemmal (SS) mitochondria, being IMF the most abundant one. In the present thesis, the molecular mechanisms harboured in mitochondria of striated muscles were studied using animal models, to better comprehend the role of mitochondrial plasticity in several pathophysiological conditions such as aging, diabetes mellitus and bladder cancer. The comparative analysis of IMF and SS populations isolated from heart evidenced a higher respiratory chain activity of mitochondria interspersed in the contractile apparatus. The higher susceptible of SS respiratory chain complexes subunits to carbonylation, but not to nitration, seems to justify the lower respiratory chain activity observed in this mitochondrial population. Our results showed that in heart from aged mice there is an accumulation of dysfunctional mitochondria. The age-related decrease of oxidative phosphorylation activity seems to be justified, at least partially, by the increased proneness of mitochondrial proteins as OXPHOS subunits and MnSOD to oxidative modifications. Moreover, a sedentary lifestyle seems to worsen the functional consequences of aging in heart by increasing mitochondrial proteins susceptibility to nitration. In skeletal muscle from rats with type 1 diabetes mellitus induced by streptozotocin administration, we verified the accumulation of dysfunctional mitochondria due, at least in part, to the impairment of PQC system. Indeed, the decreased activity of AAA proteases was accompanied by the accumulation of oxidatively modified mitochondrial proteins with impact in respiratory chain activity. The diminishing of mitochondria activity also underlies cancer-induced muscle wasting. Indeed, using a rat model of chemically induced urothelial carcinoma we verified that the loss of gastrocnemius mass was related to mitochondrial dysfunction due to, at least partially, the down-regulation of PQC system involving the mitochondrial proteases paraplegin and Lon. PQC impairment resulted in the accumulation of oxidatively modified mitochondrial proteins. In overall, regardless the pathophysiological stimuli that promote mitochondrial alterations, there are similarities in the pattern of disease-related mitochondrial plasticity. The diminished capacity for ATP production in striated muscle seems to be due to increased oxidative damage of mitochondrial proteins, namely subunits of respiratory chain complexes, metabolic proteins and MnSOD. Our data highlighted, for the first time, the impact of mitochondrial PQC system impairment in the accumulation of oxidized proteins, exacerbating the dysfunction of this organelle in striated muscle in several pathophysiological conditions.

Caracterização não-invasiva da morfologia e fisiologia cardíaca de Halobatrachus didactylus: aplicação da ecocardiografia

Tese de Doutoramento, Biologia, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2001

Resiliência, sustentabilidade e qualidade de vida em sistemas urbanos : efeitos da crise (pós-2008) em Portugal e no sistema urbano do oeste

Tese de doutoramento, Geografia (Planeamento Regional e Urbano), Universidade de Lisboa, Instituto de Geografia e Ordenamento do Território, 2014

Children's television: the soft underbelly of public service broadcasting

The provision of children's content should be a key constituent of the public service brand, but has often been viewed as a programme category at risk. Certainly in many countries children's television has moved from the 'scarcity' associated with terrestrial provision, to the 'plenty' of digital (see Ellis 2000). However in spite of a range of dedicated public service children's channels in Europe (CBeebies, Kika, Z@ppelin), domestically produced children's television in Europe is notoriously under-resourced if not marginalised. There is a pronounced reliance on imports (particularly on commercial television) notwithstanding the launch by US-owned multinationals (Disney, Nickelodeon, Cartoon Network) of localised versions of their children's television channels in many European countries. Within the broader context of global developments in children's media, this paper starts by outlining the recent and rapid crisis in British children's television and the factors that caused it. This was a crisis, which caught broadcasters and producers by surprise in the middle of 2006, but reflects many of the challenges faced by the children's television sector in other countries. It clearly demonstrated how a combination of the lack of regulatory protection, a change in commercial priorities among broadcasters, advertising restrictions, budgetary pressures and the competitive environment at home and abroad all combined to reinforce the trend towards a contraction of domestic production. The crisis also served to underline the dominance of the BBC - both as a representative of public service principles, and as the dominant producer and commissioner in the market. With the reasons underpinning the crisis explained, the paper will then analyse how the children's television community responded to the crisis and with what effect. Based on interviews, contemporary accounts and documentary evidence the paper will chart the converging and diverging views of broadcasters, producers, regulatory authority Ofcom, and a range of advocacy groups which represent children's interests and the industry. What arguments were elaborated in favour of protecting children's television as an integral part of the public service media brand? Can lessons be learned about how best to ensure the origination of children's media within a public service environment? Can developments in the UK be used to provide insight into how children's media might develop further?

Acute hypoxia improves insulin sensitivity (SI2*) and β cell function in individuals with Type 2 Diabetes

Type 2 diabetes is a multifactorial metabolic disease characterized by defects in β-cells function, insulin sensitivity, glucose effectiveness and endogenous glucose production (1). It is widely accepted that insulin and exercise are potent stimuli for glucose transport (2). Acute exercise is known to promote glucose uptake in skeletal muscle via an intact contraction stimulated mechanism (3), while post-exercise improvements in glucose control are due to insulin-dependant mechanisms (2). Hypoxia is also known to promote glucose uptake in skeletal muscle using the contraction stimulated pathway. This has been shown to occur in vitro via an increase in β-cell function, however data in vivo is lacking. The aim of this study was to examine the effects of acute hypoxia with and without exercise on insulin sensitivity (SI2*), glucose effectiveness (SG2*) and β-cell function in individuals with type 2 diabetes. Following an overnight fast, six type 2 diabetics, afer giving informed written consent, completed 60 min of the following: 1) normoxic rest (Nor Rest); 2) hypoxic rest [Hy Rest; O2 = 14.6 (0.4)%]; 3) normoxic exercise (Nor Ex); 4) hypoxic exercise [Hy Ex; O2 = 14.6 (0.4)%]. Exercise trails were set at 90% of lactate threshold. Each condition was followed by a labelled intravenous glucose tolerance test (IVGTT) to provide estimations of SI2*, SG2* and β-cell function. Values are presented as means (SEM). Two-compartmental minimal model analysis showed SI2* to be higher following Hy Rest when comparisons were made with Nor Rest (P = 0.047). SI2* was also higher following Hy Ex [4.37 (0.48) x10-4 . min-1 (μU/ml)] compared to Nor Ex [3.24 (0.51) x10-4 . min-1 (μU/ml)] (P = 0.048). Acute insulin response to glucose (AIRg) was reduced following Hy Rest vs. Nor Rest (P = 0.014 - Table 1). This study demonstrated that 1) hypoxia has the ability to increase glucose disposal; 2) hypoxic-induced improvements in glucose tolerance in the 4 hr following exposure can be attributed to improvements in peripheral SI2*; 3) resting hypoxic exposure improves β-cell function and 4) exercise and hypoxia have an additive effect on SG2* in type 2 diabetics. These findings suggest a possible use for hypoxia both with and without exercise in the clinical treatment of type 2 diabetes.

Akt/PKB activation and insulin signaling: a novel insulin signaling pathway in the treatment of type 2 diabetes

Type 2 diabetes is a metabolic disease categorized primarily by reduced insulin sensitivity, β-cell dysfunction, and elevated hepatic glucose production. Treatments reducing hyperglycemia and the secondary complications that result from these dysfunctions are being sought after. Two distinct pathways encourage glucose transport activity in skeletal muscle, ie, the contraction-stimulated pathway reliant on Ca2+/5′-monophosphate-activated protein kinase (AMPK)-dependent mechanisms and an insulin-dependent pathway activated via upregulation of serine/threonine protein kinase Akt/PKB. Metformin is an established treatment for type 2 diabetes due to its ability to increase peripheral glucose uptake while reducing hepatic glucose production in an AMPK-dependent manner. Peripheral insulin action is reduced in type 2 diabetics whereas AMPK signaling remains largely intact. This paper firstly reviews AMPK and its role in glucose uptake and then focuses on a novel mechanism known to operate via an insulin-dependent pathway. Inositol hexakisphosphate (IP6) kinase 1 (IP6K1) produces a pyrophosphate group at the position of IP6 to generate a further inositol pyrophosphate, ie, diphosphoinositol pentakisphosphate (IP7). IP7 binds with Akt/PKB at its pleckstrin homology domain, preventing interaction with phosphatidylinositol 3,4,5-trisphosphate, and therefore reducing Akt/PKB membrane translocation and insulin-stimulated glucose uptake. Novel evidence suggesting a reduction in IP7 production via IP6K1 inhibition represents an exciting therapeutic avenue in the treatment of insulin resistance. Metformin-induced activation of AMPK is a key current intervention in the management of type 2 diabetes. However, this treatment does not seem to improve peripheral insulin resistance. In light of this evidence, we suggest that inhibition of IP6K1 may increase insulin sensitivity and provide a novel research direction in the treatment of insulin resistance.

A loucura e o cinema: representações cinematográficas de personalidade limite

Dissertação apresentada à Escola Superior de Comunicação Social como parte dos requisitos para obtenção de grau de mestre em Audiovisual e Multimédia.

Efeitos imediatos da mobilização com movimento na dor, Amplitude de movimento e actividade electromiográfica dos músculos da cintura escapular em indivíduos com síndrome do conflito subacromial

Introdução: A síndrome do conflito subacromial (SCSA) é a causa mais frequente de dor no ombro. Alterações na cinemática escapuloumeral e na activação dos músculos escapulares têm sido identificadas em pessoas com SCSA. A mobilização com movimento (MWM) é uma técnica de terapia manual, desenvolvida por Mulligan, que visa normalizar a cinemática articular. Objectivos: Determinar os efeitos imediatos da MWM na dor, na amplitude de movimento (ADM) de abdução no plano da escápula (APE), e na amplitude do sinal electromiográfico (EMG) do trapézio e grande dentado (GD), em pessoas com SCSA. Métodos: Foram incluídas no estudo 24 pessoas com SCSA, divididas de forma aleatória em 2 grupos de 12, MWM e Placebo. As medidas de resultados avaliadas foram: a dor nos testes de Neer e Hawkins-Kennedy; o limiar de dor à pressão; a ADM de APE até ao início da dor; e a percentagem da contracção isométrica voluntária máxima dos músculos trapézio (superior, médio e inferior) e GD. Resultados: A aplicação da MWM resultou numa significativa diferença, com redução da dor, no teste de Hawkins-Kennedy (p=0,028), num aumento do limiar de dor à pressão (p=0,002) e da ADM de APE até ao início da dor (p=0,010), e numa diminuição da actividade EMG do trapézio superior (TS), na fase concêntrica, abaixo dos 90˚ (p=0,028), comparativamente ao grupo Placebo. Foi, ainda, identificada uma diminuição estatisticamente significativa da actividade EMG do TS, nas restantes fases do movimento (p<0,05), um aumento do limiar de dor à pressão (p<0,001) e da ADM até ao início da dor (p=0,006) entre, antes e após a intervenção com MWM. Conclusão: A MWM poderá ser uma técnica efectiva em indivíduos com SCSA, pelos seus efeitos na redução de dor, aumento de ADM até ao início da dor e diminuição da actividade EMG do TS.

Contraste e alteridade na viagem contemporânea

No mundo contemporâneo globalizado, definido pela sua qualidade essencialmente fluida e instável, o carácter distintivo da viagem parece dissolver-se face à contracção do planeta, à economia das ―trocas simbólicas‖ e a um alegado processo de diluição das diferenças e de homogeneização cultural. Com efeito, a mediatização da sociedade e a proliferação icónica contemporâneas produzem um aparente estado de saturação da geografia real e de multiplicação de lugares enquanto representações e imagens, permitindo pôr em causa a própria necessidade e urgência de deslocação, bem como admitir a abolição do ―estatuto de privilégio‖ de certos lugares e a derradeira quebra no conceito aurático das férias e das viagens, tradicionalmente assente em antinomias cruciais entre o quotidiano/familiar e o diferente/extraordinário. O presente artigo propõe-se abordar o paradigma da mobilidade contemporânea, nomeadamente no que diz respeito à traumática aniquilação do espaço e do tempo e ao seu impacto fortemente disruptivo sobre a dimensão ontológica de uma prática cultural cujo poder aurático se encontra tradicionalmente relacionado com a conquista de distâncias, a percepção de diferenças e a experiência de alteridade. O artigo pretende, por outro lado, refutar a declaração pós-moderna de que a familiarização com o outro conduz a uma diminuição do potencial de choque cultural no turismo contemporâneo, discutindo a relevância e a prevalência da busca de contraste e formas de vivência de alteridade no complexo novelo de motivações da viagem turística contemporânea.

Principais dificuldades que os alunos de viola de arco apresentam quando ingressam no ensino superior português: relatório de estágio

A maioria dos instrumentistas de cordas friccionadas apresentam problemas ou dificuldades ao longo de toda a sua aprendizagem musical. Como professora e violetista, realizei este relatório de estágio com o intuito de pesquisar onde se focam a maioria desses problemas. Este relatório de estágio está dividido em duas diferentes secções, a primeira apresenta todo o trabalho pedagógico efectuado com três diferentes alunos, de diferentes níveis ao longo do ano lectivo de 2012/13 na Fundação Musical dos Amigos das Crianças (FMAC), e a segunda apresenta toda a investigação que realizei para concluir quais as principais dificuldades que os alunos de viola de arco apresentam quando ingressam no Ensino Superior. A investigação apresenta uma vasta pesquisa literária e o testemunho dos professores de várias instituições, como a Universidade do Minho, Escola Superior de Música e Artes de Espectáculo, Universidade de Aveiro, Escola Superior das Artes Aplicadas de Castelo Branco e Escola Superior de Música de Lisboa que ajudaram à profunda análise deste tema. Os resultados revelaram que os principais problemas apresentados nos estudantes de viola de arco são de origem técnica, ou seja, postura (posição do corpo, distribuição do peso correcto pelas pernas e escolha adequada do instrumento de acordo com a fisionomia do aluno); mão direita (pega correcta dos dedos no arco, distribuição de arco e contracção muscular); mão esquerda (posição do polegar, ombro e braço que afectam a afinação, flexibilidade, mudanças de posição e vibrato); influência da escolha do repertório por parte de um professor e autonomia do aluno.

Comparação do efeito da técnica de facilitação neuromuscular proprioceptiva com a técnica de músculo energia no alongamento muscular dos isquiotibiais a curto prazo

Introdução: A aplicação das técnicas de Contrair-Relaxar com Contracção do Antagonista (CRCA) e de Músculo Energia (TME) promovem um aumento da flexibilidade muscular, contudo poucos estudos comparam a eficácia de ambas. Apresentam aspectos comuns como a contracção prévia do músculo a alongar sendo esta máxima na CRCA e uma percentagem da máxima na TME. Contudo, alguma evidência sugere que não existe correspondência entre a força produzida e a desejada pelo que este aspecto da TME carece de explicação. Objectivos: Confirmar se a técnica CRCA e a TME são efectivas no alongamento muscular dos isquiotibiais a curto prazo, caso sejam determinar qual a mais efectiva. Pretende-se ainda avaliar se a percepção ao esforço durante a aplicação da TME corresponde à força efectivamente realizada. Métodos: Efectuou-se um estudo experimental com 45 voluntários distribuídos aleatoriamente pelos grupos CRCA, TME e Controlo. Avaliou-se a amplitude articular passiva de extensão do joelho antes e depois de aplicar as técnicas, utilizando um goniómetro. Nos participantes submetidos à TME avaliou-se a percepção ao esforço, solicitando uma contracção submáxima isométrica de 40% medida através do dinamómetro de mão. Resultados: Verificou-se um efeito das técnicas entre as avaliações (Teste ANOVA medidas repetidas factor tempo: p<0,001) e entre os grupos (tempo*grupo: p<0,001). Comparando os grupos dois a dois, verificaram-se diferenças entre o grupo CRCA e o grupo Controlo (Teste Post Hoc Games-Howell: p=0,001) e entre o grupo TME e o grupo Controlo (p=0,009), não existindo diferenças entre os grupos CRCA e TME (p=0,376). Os grupos CRCA e TME obtiveram um ganho de 10,7º e de 11,4º respectivamente, não havendo diferenças significativas entre os ganhos (Teste T-Student Independente: p=0,599). Existiram diferenças significativas entre os 40% CMVI produzida e desejada (Teste Wilcoxon: p=0,018). Conclusão: Ambas foram efectivas no aumento da flexibilidade muscular dos isquiotibiais a curto prazo. Os efeitos foram comparáveis, mas dada a menor complexidade e menor solicitação a TME foi considerada mais eficiente. A percepção ao esforço durante a aplicação da TME não correspondeu ao esforço desejado, existindo uma tendência para a produção de intensidades de contracções maiores.

Anti-vibration gloves and the forearm efforts during tools operations

The objective of this paper is to analyze the forearm muscular contraction levels associated to the use of anti-vibration gloves, by comparing the contraction levels with gloves and without gloves. Two different vibration tools were used in a simulated work environment: (1) A compact Duty Multi-Cutter Bosch and (2) and a Percussion Drill with a drill bit Ø20 mm. Standard operations were performed by each subject in the following materials: (1) Performing cross- sectional cuts in 80x40 mm pine section and (2) performing 20 mm diameter holes in a concrete slab 2 x 2 m, 70 mm thick. The forearm contraction level were measured by surface electromyography in four different muscles: Flexor Digitorum Superficialis (FDS), Flexor Carpi Ulnaris (FCU), Extensor Carpi Radialis Longus (ECRL) e Extensor Carpi Ulnaris (ECU). For the flexor muscles (FDS, FCU), an increase tendency in muscular contraction was observed when the operations are performed without gloves (2-5% MVE increase in the FDS and 3-9% MVE increase in the FCU). For the extensor muscles ECU a decrease tendency in muscular contraction was observed when the operations are performed without gloves (1-10% MVE decrease). Any tendency was found in the ECRL muscle. ECU was the muscle with the highest contraction level for 79% and 71% of the operators, during the operations respectively with the multi-cutter (P50= 27-30%MVE) and with the percussion drill (P50=46-55%MVE). As a final conclusion from this study, anti- vibration gloves may increase the forearm fatigue in the posterior region of the forearm (ECU muscle) during operations with the mentioned tools