Integrated strategies for the production, maturation and storage of functional cardiomyocytes derived from human pluripotent stem cells

The production of cardiomyocytes (CM) from human pluripotent stem cells (hPSC) holds great promise for cardiotoxicity drug testing, disease modeling and cardiac regeneration [1]. However, the complex nets of signalling pathways involved in cardiomyogenesis compromises the effectiveness of the existing differentiation protocols to reproducibly produce high-quality CM from hPSC (hPSC-CM). Produced hPSC-CM are immature compared with adult CMs, express typical fetal cardiac genes, have immature electrophysiological properties and use glucose as major energy source [2]. The applicability of hPSC-CM in the clinic/industry is also dependent on the development of efficient methods for worldwide shipment of these cells. In this study we aim to overcome these hurdles by devising an integrated strategy for scalable production, maturation and storage of functional hPSC-CM. hPSC (hiPSC and hESC lines) were cultured as aggregates in environmentally controlled bioreactors, where the necessary conditions to control stem cell fate are tightly tuned [3]. Dissolved oxygen and hydrodynamic forces were manipulated in order to improve CM differentiation yields [4]. CM differentiation was monitored using flow cytometry and qRT-PCR. Novel feeding strategies were tested aiming at improving hPSC-CM enrichment and maturation. We also evaluated the feasibility to cold store monolayers and aggregates of hPSC-CM using a clinical compatible preservation formulation. After storage the ultrastructure and functionality of the hPSC-CM were assessed by TEM and electrophysiology analyses. Our results showed that hypoxia and hydrodynamic forces affect cell differentiation towards functional CM. The bioreactor protocol herein described (i.e. the controlled hypoxic and specific hydrodynamic environment) improved PSC differentiation by enhancing culture homogeneity, process reproducibility, and CM productivities. Moreover, enhanced CM maturation was attained when hPSC-CM were cultured in glucose depleted media supplemented with fatty acids; hPSC-CM showed a more elongated structure with organized sarcomeric pattern and displayed higher expression of genes responsible for contraction, calcium handling and electrophysiology. Noteworthy, using gas chromatography-mass spectrometry (GC-MS) analysis and 13C labeled subtracts we showed that hPSC-CM use fatty acid β-oxidation as energy source, a typical feature of adult CMs. At the end, we showed that monolayers of hPSC-CM and cardiospheres can be stored up to 7 days at hypothermic conditions without compromising cell viability, morphology and electrophysiological properties. This work describes significant advances towards mass production of mature hPSC-CM and their short-term storage, meeting some of the needs of the cardiac regenerative medicine market and industrial field

BUBBLE-NET : system to catch fish and the respective method of use

Fishing is a socio-economic activity of great relevance worldwide. However the environmental sustainability of the fishing gears is far from being effective. It is necessary to find more selective types of fishing to ensure sustainable catches of target species and also to protect other organisms accidentally caught with no commercial value. The answer to these problems arises, often, from the nature itself, as in the case of bubble net feeding, the feeding behavior of the humpback whale (Megaptera novaengliae). These whales trap the school of fish surrounding them with a wall of bubbles they send by the blowhole (Leighton et al, 2004) (Johson et al, 1984), (fig. 1). Thus, we are developing a device that mimics the whale behavior. This device presents large environmental benefits such as the reduction of bycatch, the absence of ghost fishing, clean capture, that is the absence of human handling, and sustainable yield by allowing the capture of the legal quantities and sizes. There are also economic advantages, such as the reduction of fishing gear, increased device lifetime and reduction of maintenance cost associated

IL-10 underlies distinct susceptibility of BALB/c and C57BL/6 mice to Mycobacterium avium infection and influences efficacy of antibiotic therapy

Increased production of IL-10 has been frequently associated with augmented susceptibility to infection. However, the correlation between IL-10 activity and susceptibility to mycobacterial infection is still uncertain. Although studies using transgenic mice overexpressing IL-10 consistently showed an increased susceptibility to mycobacterial infection, experimental approaches in which IL-10 activity was reduced or abrogated originated inconclusive data. We show here that this controversy might be due to the mouse strains used in the various experimental procedures. Our results show that BALB/c mice are more susceptible than C57BL/6 to Mycobacterium avium infection. This increased susceptibility of BALB/c mice is, to a great extent, due to distinct activity of IL-10 between the two mouse strains. In accordance, reduction of IL-10 activity through the administration of anti-IL-10R mAb, or the absence of IL-10 as studied in IL-10 knockout mice, clearly decreased the susceptibility of BALB/c mice to M. avium but had a less obvious effect in C57BL/6 mice. Moreover, abrogation of IL-10 activity in infected BALB/c mice increased the efficacy of antimycobacterial therapy, whereas for the C57BL/6 mice it produced no effect. These observations show that the activity of IL-10 in response to the same mycobacterial stimulus influences not only the susceptibility to infection but also the efficacy of antimycobacterial therapy. This should now be considered in the context of human response to mycobacterial infection, particularly as a possible strategy to improve treatment against infections by mycobacteria

Modelling Strain Penetration Effects in Rc Walls with Smooth Steel Bars

Reinforced concrete (RC) framed and dual wall-frame structures represent an important fraction of the building stock in some European cities. Many of these were constructed without seismic provisions. Therefore, it is important to assess their ability to withstand seismic events and to define strategies to reduce their potential vulnerability. This work focuses on the numerical simulation of the seismic behaviour of RC walls making use of fibre-based nonlinear beam-column elements. Even though it is common to assume a perfect bond between the reinforcing bars and concrete, the relative bond-slip deformations between the two materials can contribute up to 40% of the total lateral deformation of columns when ribbed rebars are used and up to 90% when in the presence of smooth rebars. This paper intends to be a contribution to understand the importance of these effects in old RC walls, namely through the consideration of both ribbed and smooth rebars with different anchorage lengths and provide indications regarding the use of a simplified bond-slip model.info:eu-repo/semantics/publishedVersio

Uma aproximação da permacultura ao design. O caso do sanitário ecológico em contexto rural

Tese de Mestrado em Design de ProdutoEsta Dissertação de Mestrado reflete o estudo do Design de Produto no âmbito da Permacultura, um conceito ainda em formação. Encaramos a Permacultura como uma filosofia de vida sustentável, que através de esforços locais tem vindo a diminuir a nossa pegada ecológica. É, portanto, perti¬nente uma reflexão sobre este conceito visto que atravessamos uma crise ambiental, financeira e de valores. É nosso objetivo averiguar se um objeto/sistema pode ser concebido com base no conceito da Permacultura. No fundo é tentar perceber as potencialidades do conceito e adaptá-lo à linha de pensamento de um Designer de Produto. Propõe-se também um enfoque num objecto/sistema em particular, o sanitário ecológico, um sistema utilizado em Permacultura, como exemplo perfeito de um ciclo completo que tem como objetivo combater a problemática da escassez de água e a utilização de fertilizantes químicos. Deste modo, é feita uma análise teórica cruzando vários autores e fazendo ressaltar pontos comuns entre o Design de Produto e a Permacultura. É utilizada uma metodologia mista que passa pela realização de um questionário a Designers e Permacultores, e pela análise de casos de estudo. É de ressaltar que para se realizar a análise dos casos de estudo recorreu-se ao tra¬balho de campo – voluntariado - num local onde é praticado este conceito - a Herdade do Freixo do Meio. Foi a partir das carências e potencialidades analisadas neste local que foi escolhido um obje¬to/sistema, que melhor representasse um ciclo completo – o sanitário ecológico. Posteriormente foi realizada uma análise intensiva aos sanitários ecológicos através de casos de estudo e literatura referente ao tema.Concluímos que um objeto/sistema pode ser inspirado com base no conceito de Permacultura e que o mesmo é uma mais valia para a linha de pensamento e prática de um Designer.ABSTRACT - This Master dissertation proposes the study of Product Design integrated in Permaculture’s envi¬ronment. We identify Permaculture as a philosophy based on a sustainable life, which several people until now have been following thus improving our ecological footprint. Then, it is pertinent to pon¬der about this concept since we are dealing with an ambiental, economic and values crises. Our main goal is to understand if a product can be developed inspired by Permaculture’s principles. Basically we’ll try to understand the concept’s potentialities and adapt it to a Designer’s thinking process. We, also, propose a study about ecological sanitation as a perfect example of a Permacultural’s pro¬duct/system. This system aim to supress the need of potable water and chemists fertilizers used in agriculture. To develop this study, we will analyze theoretically this concept, make a qualitative/quantitative study based on the objects used in this type of communities and make a survey. We also present an esquematic model of a ecological sanitation, as a propose to be considered in fur¬thers investigations. We highlight the fact that, this research begun with a volunteer experience in a place where Permaculture’s practices are done - Freixo do Meio’s fields. In fact, our case stu¬dies depart from these fields. As a result we concluded that an object or system can be inspired in a Permaculture’s point of view, elevating even more a Designer’s process thinkin

The control of inflammation in airway epithelial cells

COPD is a severe chronic and complex airway disease that represents a major financial burden on the healthcare and economic system. Environmental risk factors such as cigarette smoke have been associated with the predisposition to COPD. Other factors such as exposure to viral pathogens can exacerbate airway inflammation and tissue destruction generated by recruited neutrophils, culminating in altered epithelial cell responses in COPD. This thesis investigated the physiological role of Pellino-1, an E3-ubiquitin ligase, and its regulation in human primary bronchial epithelial cells (HBEpCs) in response to viral infection. The viral mimic poly(I:C) increased Pellino-1 protein and gene expression in HBEpCs. In addition, Pellino-1 gene expression was significantly increased by RV-16 and RV-1B infection in primary bronchial epithelial cells from COPD patients. Pellino-1 knockdown in HBEpCs led to a reduction in NF-κB regulated cytokines CXCL8, IL-1α and β gene expression and release of CXCL8 in response to poly(I:C) while having no measurable effect on IFNβ mRNA expression. \ud Furthermore, the transient knockdown of Pellino-1 resulted in the decrease in IKKα/β phosphorylation. The role for Pellino-1 in the non-canonical NF-κB pathway was also investigated and while Pellino-1 knockdown did not alter the expression of the non-canonical NF-κB precursor protein NFKB1 following poly(I:C) stimulation, NFKB2 protein expression was suppressed. In contrast to Pellino-1 knockdown, the transient knockdown of NFKB2 resulted in significant increase in CXCL8 mRNA and protein expression and in turn did not regulate Pellino-1 mRNA expression to poly(I:C). These data suggest that following viral infection in airway epithelial cells, TLR3 activation culminates in the up-regulation of Pellino-1 leading to an increase in NFKB2 expression, resulting in the suppression of NF-κB specific gene transcription. In addition to activating non-canonical NF-κB, NFKB1 regulates the activation of ERK signalling via MEK1. Treatment of HBEpCs with MEK1 inhibitors, PD98059 and U0126, resulted in a significant reduction in Pellino-1 protein and gene expression which led to the suggestion of ERK as a potential Pellino-1 regulator. Proteomics analysis of primary epithelial cells obtained from COPD patient airways further identified a potential novel mechanism of action for Pellino-1 in the NF-κB signalling pathway, wherein it Pellino-1 may inhibit A20’s negative regulatory role or its adaptor proteins TNIP1 or TAX1BP. Taken in combination these data support Pellino-1 as a potential target to down-regulate neutrophilic inflammation whilst retaining antiviral immunity by selectively mediating the TLR3 TRIF-dependent NF-κB/MAPK pathway and not TLR3-mediated IRF3 and IFNβ activation

Indirect taxes on e-commerce

In questo lavoro si analizza l’imposizione indiretta nel commercio elettronico; l’analisi si è basata sullo studio della normativa Comunitaria (Direttive Europee ) e la legislazione e Italiana, esponendo anche le differenze con il profilo legislativo brasiliano (softwares e libri).Esposti i contributi delle istituzione internazionali (conferenze ed/o proposte della Unione Europea) per l’inquadramento tipologico e fiscale del commercio elettronico, sono stati analizzati i profili generali dell’istituto della stabile organizzazione ai fini dell’imposizione dell’IVA e al commercio elettronico, distinguendo anche tra le transazioni elettroniche considerabili come cessione di beni e quelle considerabili prestazioni di servizi, in base alla materialità o alla dematerializzazione del bene scambiato. Anche il principio di territorialità nelle prestazioni di servizi è stato analizzato tramite analisi dei regimi ordinario e speciale riguardanti gli operatori extracomunitari.In this work we analyze the indirect taxation in e-commerce, the analysis was based on the study of EC regulations (EU Directives) and the Italian legislation and, exposing the differences with the legislative Brazilian (softwares and books). Exposed the contributions of the international institution (conferences and / or proposals by the European Union) for the classification of types of e-commerce and taxation, we have analyzed the general aspects of the institute of the permanent establishment for tax purposes of VAT and e-commerce , distinguishing between electronic transactions also consider them as sale of goods and the considerable services, according to the material or the dematerialization of the exchanged item. The principle of territoriality in the provision of services was analyzed by analysis of ordinary and special regimes relating to non-EU operator

Aprendizagem Cooperativa e Inclusão Educacional: Projeto de Extensão Comunidades de aprendizagem no Pampa

Anais do 35º Seminário de Extensão Universitária da Região Sul - Área Temática: EducaçãoEste artigo compartilha experiência referente ao Projeto Comunidades de Aprendizagem no Pampa, aprovado pelo Edital PDA UNIPAMPA no 375/2016, em execução desde maio de 2017, que tem por objetivo contribuir com a inclusão educacional de pessoas com deficiência na Educação Básica e com a promoção de uma cultura de paz no cotidiano escolar por meio da aprendizagem cooperativa e da acessibilidade ao currículo. O público-alvo da proposta é formado por alunos e educadores da Escola Estadual de Ensino Fundamental João Goulart, localizada na zona urbana de São Borja/RS, que atende, em tempo integral, 254 alunos de uma realidade socioeconômica bastante vulnerável, dos quais, 42 alunos apresentam diagnóstico de deficiência. O grupo executor vinculado à UNIPAMPA é formado por docentes, técnicos administrativos e discentes do Campus São Borja. A contribuição para a formação dos discentes da graduação está vinculada à produção de registros audiovisuais das atividades realizadas na escola, articulando conhecimentos técnicos específicos da sua área de formação com a ressignificação de conceitos relativos à deficiência, aprendizagem, trabalho colaborativo, acessibilidade. A metodologia utilizada é a pesquisa-ação colaborativa. É possível observar, como resultados parciais do trabalho, maior autoconfiança e participação efetiva dos estudantes com deficiência nas atividades propostas, bem como mudança de expectativa dos demais envolvidos no cotidiano escolar em relação àqueles diante da vivência de experiências comuns. Conclui-se, por ora, que a metodologia colaborativa é um instrumento potente para promover a reciprocidade entre universidade e escola com vistas a fomentar práticas educacionais democratizadoras do conheciment

Impact of the hydrodynamic environment on cardiomyocyte differentiation of iPSC

Ventricular-like cardiomyocytes have the potential to be used for cardiac repair in cases of cardiovascular diseases, most commonly myocardial infarction, which can ultimately lead to heart failure. The regenerative capacity of the adult myocardium is approximately 1% per annum, therefore, novel solutions are being explored where cardiomyocytes are derived from pluripotent stem cells (PSCs). From the initial promise of PSCs (including embryonic and induced pluripotent stem cells-ESC and iPSC, respectively) for clinical application, their use in therapies has already covered heart, eye diseases and diabetes (www.clinicaltrials.gov/) Furthermore, iPSC have been revolutionizing pre-clinical research as a tool for disease modelling and drug discovery. Biochemical and physical cues have been demonstrated to have a significant impact on the PSC differentiation yield, i.e. the number and quality of the resulting cardiomyocytes. In particular electrical and mechanical stimulation emulates the in vivo heart environment and has proven to have significant effects on morphology, cell density, cardiomyogenesis, maturity and functionality of differentiated cardiomyocytes. This is dependent on the type of mechanical cue applied, commonly cyclic strain/stretch or fluid shear stress (hydrodynamic forces), in addition to magnitude, duration and time of application (Acimovic et al., 2014; Geuss and Suggs, 2013; Savla et al., 2014). Correia et al. (2014) developed an iPSC-based differentiation protocol in a stirred tank bioreactor and mechanically varied the environment by using different agitation modes. It was found that combining hypoxic culture conditions with intermittent agitation resulted in 1000-fold increase in the cardiomyocytes yield compared to normoxic and continuous agitation mode. This work combines rigorous fluid dynamics investigation and flow frequency analysis with iPSC differentiation experiments to identify and quantify the flow characteristics leading to a significant increase of the differentiation yield in the aforementioned study, towards a better understanding of the physical relationship between frequency modulation and embryoid bodies suspension, and the development of dimensionless correlations applicable at larger scales. Laser Doppler Anemometry (LDA) and Fast Fourier Transform analysis were used to identify characteristic flow frequencies under different agitation modes. The use of intermittent agitation resulted in a pattern of low intensity frequencies at reactor scale. It was found that the observed frequencies and their intensity can be controlled by varying rotation time, i.e. the impeller stirrer speed (rpm), the dwell time, during which the motor is stopped and the interval time between in-motion and stationary phases. It can be inferred that the presence of such low frequencies is responsible for the observed improvement in differentiation yield and that this can be further optimised by tuning three significant parameters identified in this study. The results of the engineering studies have informed the biological verification experiments using iPSC. This work introduces the concept of fine-tuning the physical hydrodynamic cues within a three-dimensional flow system to improve cardiomyocyte differentiation of PSC. Acimovic I, Vilotic A, Pesl M, Lacampagne A, Dvorak P, Rotrekl V, Meli A (2014). Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools. BioMed Research Int. Vol. 2014. Geuss LR and Suggs LJ (2013). Making cardiomyocytes: how mechanical stimulation can influence differentiation of pluripotent stem cells. Biotech. Progress, 29(5):1089-1096. Savla JJ, Nelson BC, Perry CN, Adler ED (2014). Induced pluripotent stem cells for the study of cardiovascular disease. J. Am. Coll Cardiol. 64(5):512-519. Correia C, Serra M, Espinha N, Sousa M, Brito C, Burkert K, Zheng Y, Hescheler J, Carrondo M, Saric , Alves PM (2014). Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes. Stem Cell Reviews, 10(6):786-801