DoE to improve supercoiled p53-pDNA purification by O-phospho-L-tyrosine chromatography

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O phospho-L-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chro matographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-en coding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was ap plied. The dynamic binding capacity of the O-phospho-L-tyrosine agarose column was 0.35 ± 0.02 mg pDNA/ mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of en dotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agenciesinfo:eu-repo/semantics/publishedVersio

Mobilidade de microserviços em centros de dados suportada por Software Defined Networking

The growing adoption of software architectures based on microservices, and consequently the proliferation of the use of containers in environments with hybrid infrastructures (local data centers and public cloud providers), poses several challenges in terms of integrating this new paradigm into already existing solutions. In this context, this article initially focuses on the challenges of integrating a container orchestration platform (Kubernetes) with existing solutions in the data center, namely: Network infrastructure (Cisco ACI - Application Centric Infrastructure), load balancing (F5) and storage (NFS). In a second stage, this paper also addresses the issue of integration with orchestration platforms hosted in a public cloud (Microsoft Azure). This integration will allow the movement of containers from the local data center to the cloud without having to reconFigure the network infrastructure or the current security policy.A crescente adoção de arquiteturas de software baseadas em microserviços, e consequentemente a proliferação da utilização de containers em ambientes com infraestruturas híbridas (centros de dados locais e operadores de clouds públicas), lança vários desafios do ponto de vista da integração deste novo paradigma nas soluções existentes. Neste contexto, este artigo centra-se inicialmente nos desafios de integrar uma plataforma de orquestração de containers (Kubernetes) com soluções já existentes no centro de dados, nomeadamente com a componente de rede (Cisco ACI – Application Centric Infrastructure), com o balanceador de carga (F5) e armazenamento (NFS). Num segundo momento é também abordada a integração com plataformas de orquestração alojadas numa cloud pública (Microsoft Azure). Esta integração vai permitir a movimentação de containers do centro de dados local para a cloud sem necessitar de reconfigurar a infraestrutura de rede ou as políticas de segurança em vigor.This work has been supported by FCT – Fundação para a Ciência e Tecnologia within the R&D Units Project Scope: UIDB/00319/202

Explaining the social acceptance of renewables through location-related factors: an application to the Portuguese case

The public perception of renewable energy sources is generally positive, due to their role in air pollution and CO2 emission mitigation policies. However, there are local environmental detrimental effects, and empirical evidence is not consistent as to the support of local communities. In the present paper, we analyse the antecedents of public generic perceptions of renewables grounded on objective location-related factors. Personal location-related factors can originate in the involvement of individuals with renewable energy sources. Regional location-related factors concern the importance of the renewable energy source in the district of residence and in relation to other renewables. We implement a questionnaire on public perceptions of renewable energy sources by the general population in mainland Portugal and complement respondent-level responses with renewable energy district information. Regression analysis shows that these objective location-related factors, both personal and regional, help explain public perceptions of renewables and thus we find empirical support for the proposed approach. These results can inform and guide policymakers in tackling future social acceptance issues of renewable energy policies towards lower carbon emissions and less polluting energy production.L.M.C.P. and M.V. acknowledge the financial support of NIPE (National Funds of the FCT-Portuguese Foundation for Science and Technology within the project UID/ECO/03182/2019)

Additive Manufacturing Tools to Improve the Performance of Chromatographic Approaches

Chromatography is widely applied industrially. However, some limitations are associated with its common supports, and the impossibility to fully control their structural features is particularly restrictive. Additive manufacturing (AM) is emerging as a fast, highly precise, and reproducible technology for producing chromatographic supports that can improve its performance.info:eu-repo/semantics/publishedVersio

Composite Central Face Design—An Approach to Achieve Efficient Alginate Microcarriers

Funding: This work was supported by Portuguese Agency for Innovation (PT2020) through the projects (CENTRO-01-0145-FEDER-000014) and (POCI-01-0247-FEDER-017771). It was also supported by the project PAMI – Portuguese Additive Manufacturing Initiative (project nº22158 – SAICT- AAC nº 01/SAICT/2016) and also, by the CDRSP– ID/Multi/04044/2019, funded by the Portuguese Government through FCT/MCTES and co-funded by the European Regional Development Fund through the Partnership Agreement PT2020. This work was also supported by funds from the Health Sciences Research Center (CICS-UBI) through National Funds by FCT - Foundation for Science and Technology (UID / Multi / 00709/2019).Microparticulated drug delivery systems have been used as promising encapsulation systems for protecting drugs for in vitro and in vivo applications, enhancing its stability, providing an increased surface to volume ratio, reducing adverse effects, and hence an improvement in bioavailability. Among the studied microparticles, there is a rising interest in the research of alginate microparticles for pharmaceutical and biomedical fields confirming its potential to be used as an effective matrix for drug and cell delivery. Moreover, calcium alginate has been one of the most extensively forming microparticles in the presence of divalent cations providing prolonged drug release and suitable mucoadhesive properties. Regarding the above mentioned, in this research work, we intended to produce Ca-alginate micro-vehicles through electrospraying, presenting high encapsulation efficiency (EE%), reduced protein release across the time, reduced swelling effect, and high sphericity coefficient. To quickly achieve these characteristics and to perform an optimal combination among the percentage of alginate and CaCl2, design of Experiments was applied. The obtained model presented to be statistically significant (p-value < 0.05), with a coefficient of determination of 0.9207, 0.9197, 0.9499, and 0.9637 for each output (EE%, release, swelling, and sphericity, respectively). Moreover, the optimal point (4% of alginate and 6.6% of CaCl2) was successfully validated.info:eu-repo/semantics/publishedVersio

Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

Funding: This work was supported by FEDER funds through the POCI—COMPETE 2020 Operational Programme Competitiveness and Internationalization in Axis I—Strengthening research, technological development, and innovation (Project POCI-01-0145-FEDER-007491), and National Funds by FCT Foundation for Science and Technology (Project UID/Multi /00709/2013). This work was also developed within the scope of the project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. G.M. Azevedo acknowledges the support and fellowship of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/203482/2014-0). J.F.A. Valente acknowledges the PhD fellowship (Ref SFRH/BD/96809/2013) from FCT.Acknowledgments: The authors would like to thank Thomas Roberts for providing the pcDNA3–FLAG–p53 construct through Addgene, ref: 10838.The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.info:eu-repo/semantics/publishedVersio

O PROGRAMA DE AGROINDÚSTRIA DO BANCO DO NORDESTE – AVALIAÇÃO DE RESULTADOS

O presente estudo apresenta os resultados da avaliação do programa de financiamento à agroindústria desenhado e implementado pelo Banco do Nordeste a partir de 1990 – o FNE AGRIN. O conceito de agroindústria utilizado compreende os processos de estocagem, manejo, beneficiamento, preservação e transformação industrial de matérias-primas de origem agrícola, pecuária, florestal e pesqueira. Isto implica na integração entre a produção, abastecimento de matérias-primas e transformação, além da instalação de unidades de processamento próximas aos locais de produção. O FNE-AGRIN tem contribuído para implantar e expandir diferentes agroindústrias na Região, contudo, o desenvolvimento da agroindústria no Nordeste depende do fornecimento de insumos e matérias-primas com qualidade e regularidade, além do estabelecimento, disseminação e fiscalização de controles de qualidade e de normas e padrões sanitários e agroindustriais. --------------------------------------------The concept of agroindustry here utilized refers to the processes of stocking, handling, producing, preserving and transforming agricultural as well as livestock, forestry and fishing raw materials. This process represents an integration of production, raw material supply and transformation, as well as constructing agroindustry plants near the original places of agricultural production. Evaluation consists on an important phase of a project cycle. The present study shows the results of an agroindustry financing program that was designed and implemented by Banco do Nordeste. The mentioned program has helped to introduce and expand different agroindustries plants in the Brazilian Northeast. Nevertheless, the development of a sustainable agroindustry sector in this Region depends on quality and regularity of raw materials, as well as on the introduction of inspections services aimed to improve the quality control of food products and (agro)sanitary measures.Agroindústria, Avaliação, Fundo Constitucional, Nordeste, Agroindustry, Evaluation, Constitutional Fund, Northeast, Agribusiness, Livestock Production/Industries,

Ways and means to comfort people at the end of life: how is the nurse a privileged player in this process?

Background: Comfort is a necessity throughout life, and it is a key element in the practice of nursing care for the patient at the end of life. A particular human need and a state related to the experience and culture of the person at the end of life constitute the target of attention and nursing intervention, being a very relevant indicator of the quality of health care. This article is part of a doctoral study in the field of comfort in a palliative care unit, and these are some of the partial results that emerged. Objectives: To understand the ways and means of comfort perceived by the person at the end-of-life hospitalized in a palliative care unit, their family, and health staff as well as the value of the nurse in this process. Design: Qualitative study using an ethnographic approach. Methods: We conducted semistructured interviews with 18 patients at the end of life and their matched significant family members (18) and 21 health professionals. We also conducted participant observation of care situations. Results/Discussion: The ways and means of providing comfort are centered on strategies developed by the entire multidisciplinary team. During this whole process, one of the categories that emerged from the ethnography was the nurse as a privileged player, representing an absolutely essential role in all phases. The results revealed that nurses play a very important role in end-of-life comfort, which is based on a predisposition for end-of-life care (active listening, empathy, congruence, and biographical narrative) and focused attention (global care, attention to detail, family support, and opposition to therapeutic obstinacy). Conclusions: The different ways and means of providing comfort aim to increase care, relieve, and invest in potential different forms of comfort and nurses are recognized by all those involved in this process as someone essential to providing comfort care.info:eu-repo/semantics/publishedVersio

The context of care as a supporting axis for comfort in a palliative care unit

Background: The context of care determines and organizes practices through its structures and guiding principles. It is sometimes a space that generates tension and multiple choices, variable in the provision of different care and uncertain in its duration. We can consider that the construction of the comfort process does not only depend on the will of its actors and the situation itself, but is also conditioned by the professional, cultural, and social context in which it is inserted. This article is part of a doctoral study in the field of comfort in a palliative care unit, and these are some of the partial results that emerged. Design: Qualitative study using ethnographic approach. Methods: We conducted semistructured interviews with 18 patients at the end of life and their matched significant family members (18) and 21 health professionals. We also conducted a participant observation of care situations. Results/discussion: The context of action, where meanings and practices are learned, is linked to a certain identity that is related to practical, contextual knowledge, linked to a collective and to a feeling of belonging. The relationship between the various factors that shape the Care Context in the palliative care unit studied, constitute the three domains of this topic, specifically: the integrative and inclusive environment, the conceptions of care, and the inclusive factors of organizational culture. Conclusion: The specific context was determinant as a supporting axis for comfort in this palliative care unit. The context of care, where objects and provisions support the construction of the comfort process as an entity that integrates culture, established conceptions of care, allowing the deepening of knowledge.info:eu-repo/semantics/publishedVersio

Inteligência emocional dos professores e vulnerabilidade ao stress em contexto escolar

Investigações recentes revelam que o stress é considerado um dos indicadores de mau-estar docente (Jesus, 2005). Para lidar com estas novas realidades, os professores necessitam de controlar/lidar com o stress e as suas emoções nas circunstâncias mais adversas. Desta forma, o principal objectivo deste estudo foi perceber de que forma a percepção emocional e a inteligência emocional são importantes para gerir problemas de stress ocupacional. Para tal, utilizaram-se quatro instrumentos diferentes: O teste de regulação emocional de Berkeley (Gross & John, 2003), o teste de expressividade Emocional de Berkeley (Gross & John, 2003), a escala de meta-conhecimento dos estados emocionais a Trait Meta-Mood Scale” (TMMS 24) (Salovey & Mayer 1990) e a escala de avaliação do grau de vulnerabilidade ao stress a 23QVS (Serra, 2000). Estes testes foram aplicados numa sessão única a 292 professores dos 2.º e 3.º Ciclos do Ensino Básico, do distrito do Funchal, sendo 64,7% do sexo feminino e 35,3% do sexo masculino de diferentes grupos disciplinares. Analisou-se a influência de algumas variáveis como o género, a idade, estado civil, número de filhos, habilitações, anos de serviço, nível de ensino que lecciona e o grupo disciplinar na explicação da percepção emocional, da inteligência emocional e da vulnerabilidade ao stress. Realizaram-se igualmente os estudos correlacionais das diferentes medidas utilizadas. Os resultados mostraram que os professores que apresentam maiores capacidades para clarificar e regular as suas próprias emoções são aqueles que apresentam menor vulnerabilidade ao stress. Para finalizar destacaremos as implicações que este estudo traz para a intervenção em contextos educativos.Universidade da Madeir