Correction to the pathogenic alternative splicing, caused by the common GNB3 c.825C>T allele, using a novel, antisense morpholino

The very common GNB3 c.825C>T polymorphism (rs5443), is present in approximately half of all human chromosomes. Significantly the presence of the GNB3 825T allele has been strongly associated, with predisposition to essential hypertension. Paradoxically the presence of the GNB3 825T allele, in exon 10, introduces a pathogenic alternative RNA splice site into the middle of exon 9. To attempt to correct this pathogenic aberrant splicing, we therefore bioinformatically designed, using a Gene Tools® algorithm, a GNB3 specific, antisense morpholino. It was hoped that this morpholino would behave in vitro as either a potential “ splice blocker and/or exon skipper, to both bind and inhibit/reduce the aberrant splicing of the GNB3, 825T allele. On transfecting a human lymphoblast cell line homozygous for the 825T allele, with this antisense morpholino, we encouragingly observed both a significant reduction (from ~58% to ~5%) in the production of the aberrant smaller GNB3 transcript, and a subsequent increase in the normal GNB3 transcript (from ~42% to ~95%). Our results demonstrate the potential use of a GNB3 specific antisense morpholino, as a pharmacogenetic therapy for essential hypertension.

Tecnologias CAC e Inquéritos de Percepção da Opinião Pública sobre “O papel da Captação e do Armazenamento/Sequestração de Dióxido de Carbono no Futuro da Energia na Europa”:O caso-de-estudo da Comunidade Fernando Pessoa

O carvão e outros combustíveis fósseis, continuarão a ser, por décadas, a principal matéria-prima energética para as Centrais Térmicas, não obstante os esforços para, dentro do possível, substituir os combustíveis fósseis por fontes de energia renovável.Tal como está, hoje, bem documentado, a produção de gases com efeito estufa (GEE), designadamente CO2, resulta da combustão dos ditos combustíveis fósseis, sendo que se espera ser possível mitigar substancialmente a emissão de tais gases com a aplicação das chamadas Tecnologias Limpas do Carvão.Há, pois, necessidade de promover o abatimento do CO2 através de Tecnologias de Emissão Zero ou Tecnologias Livres de Carbono, incluindo designadamente a Captura, o Transporte e a Sequestração geológica de CO2 correspondentes ao que é costume designar por Tecnologias CAC (Captação e Armazenamento de Carbono). De facto, tais tecnologias e, designadamente, o armazenamento geológico de CO2 são as únicas que, no estado actual do conhecimento, são capazes de permitir que se cumpram as metas do ambicioso programa da EU para a energia e o ambiente conhecido por “20 20 para 2020” em conjugação com os aspectos económicos das directivas relativas ao Comércio Europeu de Licenças de Emissão – CELE (Directivas 2003/87/EC, 2004/101/EC e 2009/29/EC).A importância do tema está, aliás, bem demonstrada com o facto da Comissão Europeia ter formalmente admitido que as metas supracitadas serão impossíveis de atingir sem Sequestração Geológica de CO2. Esta é, pois, uma das razões de ter sido recentemente publicada a Directiva Europeia 2009/31/EC de 23 de Abril de 2009 expressamente dedicada ao tema do Armazenamento Geológico de CO2.Ora, a questão do armazenamento geológico de CO2 implica, para além das Tecnologias CAC acima mencionadas e da sua viabilização em termos tanto técnicos como económicos, ou seja, neste último aspecto, competitiva com o sistema CELE, também o conhecimento, da percepção pública sobre o assunto. Isto é, a praticabilidade das Tecnologias CAC implica que se conheça a opinião pública sobre o tema e, naturalmente, que face a esta realidade se prestem os esclarecimentos necessários como, aliás, é reconhecido na própria Directiva Europeia 2009/31/EC.Dado que a Fundação Fernando Pessoa / Universidade Fernando Pessoa através do seu Centro de Investigação em Alterações Globais, Energia, Ambiente e Bioengenharia – CIAGEB tem ultimado um Projecto de Engenharia relativo à Sequestração Geológica de CO2 nos Carvões (Metantracites) da Bacia Carbonífera do Douro – o Projecto COSEQ, preocupou-se naturalmente, desde o início, com o lançamento de inquéritos de percepção da opinião pública sobre o assunto.Tal implicou, nesta fase, a tradução para português e o lançamento do inquérito europeu ACCSEPT que não tinha sido ainda formalmente lançado de forma generalizada entre nós. Antes, porém, de lançar publicamente tal inquérito – o que está actualmente já em curso – resolveu-se testar o método de lançamento, a recolha de dados e o seu tratamento com uma amostra correspondente ao que se designou por Comunidade Fernando Pessoa, i.e. o conjunto de docentes, discentes, funcionários e outras pessoas relacionadas com a Universidade Fernando Pessoa (cerca de 5000 individualidades).Este trabalho diz, precisamente, respeito à preparação, lançamento e análise dos resultados do dito inquérito Europeu ACCSEPT a nível da Comunidade Fernando Pessoa. Foram recebidas 525 respostas representando 10,5% da amostra. A análise de resultados foi sistematicamente comparada com os obtidos nos outros países europeus, através do projecto ACCSEPT e, bem assim, com os resultados obtidos num inquérito homólogo lançado no Brasil. The use of coal, and other fossil fuels, will remain for decades as the main source of energy for power generation, despite the important efforts made to replace, as far as possible, fossil fuels with renewable power sources.As is well documented, the production of Greenhouse Gases (GHG), mainly CO2, arises primarily from the combustion of fossil fuels. The increasing application of Clean Coal Technologies-CCTs, is expected to mitigate substantially against the emission of such gases.There is consequently a need to promote the CO2 abatement through Zero Emission (Carbon Free) Technologies - ZETs, which includes CO2 capture, transport and geological storage, i.e. the so-called CCS (Carbon, Capture and Storage) technologies. In fact, these technologies are the only ones that are presently able to conform to the ambitious EU targets set out under the “20 20 by 2020” EU energy and environment programme, jointly with the economic aspects of the EU Directives 2003/87/EC, 2004/101/EC and 2009/29/EC concerned with the Greenhouse Gas Emissions Allowance Trading Scheme – ETS scheme. The European Commission formal admission that the referred targets will be impossible to reach without the implementation and contribution of geological storage clearly demonstrate the importance of this particular issue, and for this reason the EC Directive 2009/31/EC of April 23, 2009 on Geological Storage of CO2 was recently published.In considering the technical and economical viabilities of CCS technologies, the latter in competition with the ETS scheme, it is believed that public perception will dictate the success of the development and implementation of CO2 geological storage at a large industrial level. This means that, in order to successfully implement CCS technologies, not only must public opinion be taken into consideration but objective information must also be provided to the public in order to raise subject awareness, as recognized in the referred Directive 2009/31/EC.In this context, the Fernando Pessoa Foundation / University Fernando Pessoa, through its CIAGEB (Global Change, Energy, Environment and Bioengineering) RDID&D Unit, is the sponsor of an Engineering Project for the Geological Sequestration of CO2 in Douro Coalfield Meta-anthracites - the COSEQ Project, and is therefore also engaged in public perception surveys with regards to CCS technologies.At this stage, the original European ACCSEPT inquiry was translated to Portuguese and submitted only to the “Fernando Pessoa Community” - comprising university lecturers, students, other employees, as well as, former students and persons that have a professional or academic relationship with the university (c. 5000 individuals). The results obtained from this first inquiry will be used to improve the survey informatics system in terms of communication, database, and data treatment prior to resubmission of the inquiry to the Portuguese public at large.The present publication summarizes the process and the results obtained from the ACCSEPT survey distributed to the “Fernando Pessoa Community”. 525 replies, representing 10.5% of the sample, have been received and analysed. The assessment of the results was systematically compared with those obtained from other European Countries, as reported by the ACCSEPT inquiry, as well as with those from an identical inquiry launched in Brazil.

Energia eólica

Actualmente, é indiscutível a importância das energias ditas renováveis no contexto da produção mundial de energia, destacando-se, dentro destas, a energia eólica pelas suas inúmeras vantagens. Neste artigo descrevem-se as vantagens e desvantagens deste tipo de energia e apresenta-se uma discussão da evolução da potência instalada de acordo com os dados fornecidos pelos organismos correspondentes. Pretende-se, além disso, explicar a evolução da potência instalada na Europa e, mais especificamente, no caso de Portugal, explicar a baixa potência instalada tendo em conta o elevado potencial eólico disponível. The importance of renewable energies in the context of the world energy production is nowadays unquestionable, namely in the case of the eolic energy, due to its specific advantages. In this paper, the advantages and disadvantages of this type of energy are described, and a discussion of the evolution of the wind power installed is presented according to the data published by the different organisms involved. It is intended to show and explain the evolution of the wind power capacity installed in the European Union. Finally, it is discussed the Portuguese specificity characterized by a low wind power installed despite the high eolic potential available.

Vantagens do ciclo combinado a gás natural face a outras tecnologias de produção de energia. Estudo de caso

Seria impensável concebermos os nossos dias sem a utilização de energia eléctrica. Esta forma de energia é responsável pelo desenvolvimento económico e a sua disponibilidade é indicadora da qualidade de vida dos povos. A procura de formas de obtenção desta energia que minimizem os impactes para o ambiente tem levado à adopção de energias renováveis mas também ao desenvolvimento de novas tecnologias que permitam aumentar a eficiência de conversão de energia entre as suas várias formas. Neste sentido procedeu-se à análise de um estudo de caso da central termoeléctrica a gás natural com tecnologia de ciclo combinado da Tapada do Outeiro, Portugal. Living without electricity is nowadays unconceived. The economic growth and quality of life is strongly dependent on this source of energy. The search for new forms of producing electricity in order to minimise environmental impacts has lead to the adoption of renewable energies and to the improvement of new technologies which allow at the same time to reach high efficiency in the process of energy conversion from the chemical form to the electrical one. This article is about a case study of a natural gas turbine power plant with combined cycle, at “Tapada do Outeiro”, Portugal.

Linking Visual Cortex to Visual Perception: An Alternative to the Gestalt Bubble

Lehar's lively discussion builds on a critique of neural models of vision that is incorrect in its general and specific claims. He espouses a Gestalt perceptual approach, rather than one consistent with the "objective neurophysiological state of the visual system" (p. 1). Contemporary vision models realize his perceptual goals and also quantitatively explain neurophysiological and anatomical data.

Zinc finger nuclease gene repair as a treatment for cystinosis

Cystinosis is a multi-system autosomal recessive disorder caused by mutations and/or deletions in both alleles of CTNS, a gene encoding for the low pH dependent lysosomal cystine exporter cystinosin. Cystinosis occurs in approximately 1:200,000 newborns worldwide and is characterised by an accumulation of cystine in the lysosomes. The most severe form of the disorder is nephropathic cystinosis presenting Fanconi syndrome and leads without treatment to an end-stage renal failure before the age of ten. The only treatment available so far is cysteamine therapy, which delays disease progression by five years, but does not provide a cure for cystinosis patients. Current gene and cell based therapeutic approaches have not yet provided a suitable alternative. A potentially approach for a long-term treatment could be to generate autologous gene–modified stem cells by repairing the gene. Zinc Finger Nucleases (ZFNs) serve as a tool to increase HDR up to a 200,000-fold by introducing a double-stranded break (DSB). Thus, simple mutations in the CTNS gene could be corrected by introduction of a double-stranded break using ZFNs to boost the process of HDR with a suitable donor DNA sequence. A permanent repair of the most common lesion CTNS, a 57 kb deletion, could be achieved by ZFN-mediated HDR using a minigene CTNS promoter/cDNA construct. The thesis describes the design and testing of seven zinc finger nuclease pairs for their cleavage activity in vitro and in cellulo.. A highly sensitive assay to detect even low levels of ZFN-mediated HDR was also developed. Finally, to further investigate the role of autophagy in tissue injury in cystinotic cells an assay to monitor autophagy levels in the cells was successfully developed. This assay provides the opportunity to demonstrate functional restoration of CTNS after successful ZFN-HDR in cystinotic cells.

The expression and regulation of pregnancy-specific glycoproteins in the mouse

Pregnancy-Specific Glycoproteins (PSG) are the most abundant fetally expressed proteins in the maternal bloodstream at term. This multigene family are immunoglobulin superfamily members and are predominantly expressed in the syncytiotrophoblast of human placenta and in giant cells and spongiotrophoblast of rodent placenta. PSGs are encoded by seventeen genes in the mouse and ten genes in the human. Little is known about the function of this gene family, although they have been implicated in immune modulation and angiogenesis through the induction of cytokines such as IL-10 and TGFβ1 in monocytes, and more recently, have been shown to inhibit the platelet-fibrinogen interaction. I provide new information concerning the evolution of the murine Psg genomic locus structure and organisation, through the discovery of a recent gene inversion event of Psg22 within the major murine Psg cluster. In addition to this, I have performed an examination of the expression patterns of individual Psg genes in placental and non-placental tissues. This study centres on Psg22, which is the most abundant murine Psg transcript detected in the first half of pregnancy. A novel alternative splice variant transcript of Psg22 lacking the protein N1-domain was discovered, and similar to the full length isoform induces TGFβ1 in macrophage and monocytic cell lines. The identification of a bidirectional antisense long non-coding RNA transcript directly adjacent to Psg22 and its associated active local chromatin conformation, suggests an interesting epigenetic gene-specific regulatory mechanism that may be responsible for the high level of Psg22 expression relative to the other Psg family members upon trophoblast giant cell differentiation

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.

Isolation, characterization, and functional assessment of novel smooth muscle like stem progenitors

Vascular smooth muscle cells (VSMC) are one of the key players in the pathogenesis of cardiovascular diseases. The origin of neointimal VSMC has thus become a prime focus of research. VSMC originate from multiple progenitors cell types. In embryo the well-defined sources of VSMC include; neural crest cells, proepicardial cells and EPC. In adults, though progenitor cells from bone marrow (BM), circulation and tissues giving rise to SMC have been identified, no progress has been made in terms of isolating highly proliferative clonal population of adult stem cells with potential to differentiate into SMC. Smooth muscle like stem progenitor cells (SMSPC) were isolated from cardiopulmonary bypass filters of adult patients undergoing CABG. Rat SMSPC have previously been isolated by our group from the bone marrow of Fischer rats and also from the peripheral blood of monocrotaline induced pulmonary hypertension (MCT-PHTN) animal model. Characterization of novel SMSPC exhibited stem cell characteristics and machinery for differentiation into SMC. The expression of Isl-1 on SMSPC provided unique molecular identity to these circulating stem progenitor cells. The functional potential of SMSPC was determined by monitoring adoptive transfer of GFP+ SMSPC in rodent models of vascular injury; carotid injury and MCT-PHTN. The participation of SMSPC in vascular pathology was confirmed by quantifying the peripheral blood, and engrafted levels of SMSPC using RT-PCR. In terms of translating into clinical practice, SMSPC could be a good tool for detecting the atherosclerotic plaque burden. The current study demonstrates the existence of novel adult stem progenitor cells in circulation, with the potential role in vascular pathology.

Mesenchymal stem cell therapy for the treatment of myocardial infarction

Mesenchymal stem cells (MSCs) are currently under investigation as repair agents in the preservation of cardiac function following myocardial infarction (MI). However concerns have emerged regarding the safety of acute intracoronary (IC) MSC delivery specifically related to mortality, micro-infarction and microvascular flow restriction post cell therapy in animal models. This thesis aimed to firstly identify an optimal dose of MSC that could be tolerated when delivered via the coronary artery in a porcine model of acute MI (AMI). Initial dosing studies identified 25x106 MSC to be a safe MSC cell dose, however, angiographic observations from these studies recognised that on delivery of MSC there was a significant adverse decrease in distal blood flow within the artery. This observation along with additional supportive data in the literature (published during the course of this thesis) suggested MSC may be contributing to such adverse events through the propagation of thrombosis. Therefore further studies aimed to investigate the innate prothrombotic activity of MSC. Expression of the initiator of the coagulation cascade initiator tissue factor (TF) on MSC was detected in high levels on the surface of these cells. MSC-derived TF antigen was catalytically active, capable of supporting thrombin generation in vitro and enhancing platelet-driven thrombus deposition on collagen under flow. Infusion of MSC via IC route was associated with a decreased coronary flow reserve when delivered but not when coadministered with an antithrombin agent heparin. Heparin also reduced MSC-associated in situ thrombosis incorporating platelets and VWF in the microvasculature. Heparin-assisted MSC delivery reduced acute apoptosis and significantly improved infarct size, left ventricular ejection fraction, LV volumes, wall motion and scar formation at 6 weeks post AMI. In addition, this thesis investigated the paracrine factors secreted by MSC, in particular focusing on the effect on cardiac repair of a novel MSC-paracrine factor SPARCL1. In summary this work provides new insight into the mechanism by which MSC may be deleterious when delivered by an IC route and a means of abrogating this effect. Moreover we present new data on the MSC secretome with elucidation of the challenges encountered using a single paracrine factor cardiac repair strategy.

From respect for nature to agency as realisation in response to the ecological emergency

'The ecological emergency’ describes both our emergence into, and the way we relate within, a set of globally urgent circumstances, brought about through anthropogenic impact. I identify two phases to this emergency. Firstly, there is the anthropogenic impact itself, interpreted through various conceptual models. Secondly, however, is the increasingly entrenched commitment to divergent conceptual positions, that leads to a growing disparateness in attitudes, and a concurrent difficulty with finding any grounds for convergence in response. I begin by reviewing the environmental ethics literature in order to clarify which components of the implicit narratives and beliefs of different positions create the foundations for such disparateness of views. I identify the conceptual frameworks through which moral agency and human responsibility are viewed, and that justify an ethical response to the ecological emergency. In particular, I focus on Paul Taylor's thesis of 'respect for nature' as a framework for revising both the idea that we are ‘moral’ and the idea that we are ‘agents’ in this unique way, and I open to question the idea that any response to the ecological emergency need be couched in ethical terms. This revision leads me to formulate an alternative conceptual model that makes use of Timothy Morton’s idea of enmeshment. I propose that we dramatically revise our idea of moral agency using the idea of enmeshment as a starting point. I develop an alternative framework that locates our capacity for responsibility within our capacity for realisation, both in the sense of understanding, and of making real, sets of conditions within our enmeshment. I draw parallels between this idea of ‘realisation as agency’ and the work of Dōgen and other non-dualists. I then propose a revised understanding of ‘the good’ of systems from a biophysical perspective, and compare this with certain features of Asian traditions of thought. I consider the practical implications of these revisions, and I conclude that the act of paying close attention, or realising, contains our agency, as does the attitude, or manner, with which we focus. This gives us the basis for a convergent response to the ecological emergency: the way of our engagement that is the key to responding to the ecological emergency

The defining characteristics of alternative food initiatives in Ireland: a social movement battling for an alternative food future?

Alternative food initiatives (AFIs) have been described as an attempt to change and improve aspects of how the food system operates. They are focused around more traditional, local and sustainable food production and circulation. AFIs such as farmers’ markets, allotments and community gardens, share a desire to reduce the number of steps food goes through from production to plate. The role of these initiatives in the food system, and their potential to impact real change, has however been questioned. Working to better understand this issue is a central concern of this research. To do this a two tier analysis has been deployed. The first tier involves identifying the characteristics and general dynamics of AFIs. Bourdieu’s theory of practice, and the theories of field and capital, are the concepts applied. The research then uses these findings in the second tier of analysis concerned with relating AFI characteristics and dynamics back to their key traits, positive and negative, as well as arguments made about AFI’s role identified from previous research. Another part of this second tier of analysis is exploring if AFIs, the producers, consumers, organisations and groups that make up this phenomenon, can be considered a social movement. AFIs can be referred to collectively as a social movement, but are not often explored theoretically from this perspective. AFIs in Ireland provide the empirical context for this research. A series of qualitative interviews in four areas of Ireland, as well as evidence from primary and secondary sources are analysed. The research finds that AFIs can be understood as the potential beginnings of a lifestyle social movement. Leaders are of central importance to its development. It is also found that an important role of AFIs is revitalising, supporting and contributing to food culture.

The ranking of alternative tariff and quota policies in the presence of domestic monopoly

Bhagwati demonstrated the nonequivalence between tariffs and quotas in the presence of monopoly. This paper also assumes domestic production to be monopolized and shows that giving import licenses or tariff revenues to the domestic producer may raise or lower the welfare cost of protection and the price paid by consumers from the price under other tariff and quota arrangements which maintain the same market share for the domestic producer. However, if the monopolist realizes that commercial policy is an instrument used to maximize the policymaker's welfare function, instead of being a goal in itself, the equivalence of tariffs and quotas re-emerges. © 1977.

Genetic engineering of mesenchymal stem cells and its application in human disease therapy.

The use of stem cells for tissue regeneration and repair is advancing both at the bench and bedside. Stem cells isolated from bone marrow are currently being tested for their therapeutic potential in a variety of clinical conditions including cardiovascular injury, kidney failure, cancer, and neurological and bone disorders. Despite the advantages, stem cell therapy is still limited by low survival, engraftment, and homing to damage area as well as inefficiencies in differentiating into fully functional tissues. Genetic engineering of mesenchymal stem cells is being explored as a means to circumvent some of these problems. This review presents the current understanding of the use of genetically engineered mesenchymal stem cells in human disease therapy with emphasis on genetic modifications aimed to improve survival, homing, angiogenesis, and heart function after myocardial infarction. Advancements in other disease areas are also discussed.

Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix.

OBJECTIVES: Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells with potential for use in cartilage tissue engineering. We hypothesized that these cells show distinct responses to different chondrogenic culture conditions and extracellular matrices, illustrating important differences between cell types. METHODS: Human ASCs and MSCs were chondrogenically differentiated in alginate beads or a novel scaffold of reconstituted native cartilage-derived matrix with a range of growth factors, including dexamethasone, transforming growth factor beta3, and bone morphogenetic protein 6. Constructs were analyzed for gene expression and matrix synthesis. RESULTS: Chondrogenic growth factors induced a chondrocytic phenotype in both ASCs and MSCs in alginate beads or cartilage-derived matrix. MSCs demonstrated enhanced type II collagen gene expression and matrix synthesis as well as a greater propensity for the hypertrophic chondrocyte phenotype. ASCs had higher upregulation of aggrecan gene expression in response to bone morphogenetic protein 6 (857-fold), while MSCs responded more favorably to transforming growth factor beta3 (573-fold increase). CONCLUSIONS: ASCs and MSCs are distinct cell types as illustrated by their unique responses to growth factor-based chondrogenic induction. This chondrogenic induction is affected by the composition of the scaffold and the presence of serum.