Genetic engineering approaches for enhanced lignocellulosic-based bioprocesses

Book of Abstracts of CEB Annual Meeting 2017Lignocellulosic biomass is the most abundant, low-cost, bio-renewable resource. It has a recognised potential as a sustainable platform for the production of biofuels and other bio-chemicals. To improve the accessibility of the cellulose component from complex lignocellulosic structures to the enzymes, a pretreatment step is necessary. Enzymatic saccharification of resulting whole slurry is highly desirable as it avoids the solid-liquid separation step, the need for detoxification and related waste disposal problem, and increases final sugar concentration. However, lignin residues and other inhibitory compounds resulting from pretreatment negatively affect the digestibility of the whole slurry and compromise fermentation efficiency. To tackle these pitfalls, genetic engineering strategies have been developed and integrated in the process to improve both stages. For improving the fermentation efficiency, our approach has been to intensify the process by using high solid loadings and both pentose and hexoses fractions, enriching sugar concentration available for fermentation. To work under such demanding conditions robust yeast strains are crucial. We have selected natural robust yeast isolates and identified key genes necessary for yeast growth and maximal fermentation rate in hydrolysates. Selected robust yeast chassis have been metabolic engineered for cofermentation of glucose and xylose from hemicellulose fraction using a novel metabolic assembly tool and key tolerance genes expression has been simultaneously evaluated for the valorization of biomass of different origins. Results obtained pointed to the importance of designing from the very beginning a tailor-made yeast considering the specific raw material and process [1]. The flexibility of the metabolic assembly tool developed and the selected robust yeast backgrounds envisioned the developing of effective yeast platforms for biomass processing into different products. For improving the saccharification of whole slurry, our strategy has been to use the efficient recombinant protein production system from Escherichia coli to produce hydrolysis enhancers, namely a family 3 carbohydrate-binding module (CBM3). The purified CBM3 was used as an additive in the enzymatic hydrolysis of the whole slurry from hydrothermally-pretreated Eucalyptus globulus wood among other biotechnological applications [2]. The results obtained show an increase in glucose yield when CBM3 was added, compensating the negative effect of inhibitors on the enzymatic efficiency of whole slurry saccharification. Thus, CBM3 is a valid additive for enhanced lignocellulosics saccharification and a valuable alternative to costly additives (e.g. BSA) as it can be affordably obtained from heterologous bacterium or integrated in the developed yeast platforms, thus contributing to more cost-efficient and environmental-friendly biomass conversion bioprocesses.info:eu-repo/semantics/publishedVersio

A Guarda Nacional Republicana e as Redes Sociais: Usos, Potencialidades e Riscos

O presente Trabalho de Investigação Aplicada está subordinado ao tema: "A Guarda Nacional Republicana e as redes sociais: usos, potencialidades e riscos". A forma como exploramos as novas tecnologias de informação e comunicação tem vindo a evoluir, desde a livre circulação de informação até ao seu fácil acesso, características da época em que vivemos. Pretende-se, por isso, caraterizar o uso que a Guarda Nacional Republicana faz das redes sociais e, ao mesmo tempo, averiguar aspetos sensíveis quanto à dinâmica que esse uso implica. Este relatório encontra-se estruturado em três partes distintas, sendo elas a parte teórica, que consiste num levantamento bibliográfico e documental, efetuada em fontes fidedignas sobre o assunto, e a parte prática, que está relacionada com o trabalho de campo, especificamente a recolha e tratamento de dados, e respetiva apresentação, análise e discussão dos mesmos. É na última parte, a conclusiva, que se verificam as hipóteses e se responde às perguntas levantadas. Para tal utilizam-se os dados dos inquéritos por entrevista aplicados a interlocutores privilegiados e ainda outros documentos, designadamente estatísticas, fornecidos pela Divisão de Comunicações e Relações Públicas da GNR. Os resultados permitem apurar que a Guarda Nacional Republicana utiliza o facebook de uma forma significativa, distinguindo-o das restantes redes, enquanto programa de comunicação global. Concluiu-se que as redes sociais funcionam como democratizadoras da comunicação, permitindo através do seu bom uso projetar a Guarda Nacional Republicana para um campo de uma força de segurança moderna e em sintonia com a mudança da sociedade atual.Abstract This Applied Research Work is subordinated to the theme: "The Guarda Nacional Republicana and its use of social networks: opportunities and risks". The way we explore the new technologies of information and communication has been evolving since the free flow of information to its easy access, which characterizes the time in which we live. Thus, it is our intention to analyze how the Guarda Nacional Republicana uses the social networks and, at the same time, to look sensitive aspects regarding the dynamics that such use entails. This report is structured into three distinct parts, being the theoretical part, which consists of a bibliographical and documentary survey, conducted on reliable sources on the subject, and the empirical part, which is related to field work, specifically the collection and data processing, and respective presentation, analysis and discussion of the results. It is in the last part, the conclusive one, which we verify the hypotheses and answer the questions raised. To achieve that purpose the data was obtained by interviewing the privileged interlocutors on this issue and also by consulting the statistical analysis by the Divisão de Comunicação e Relações Públicas. The results allow to establish the Guarda Nacional Republicana uses Facebook in a meaningful way, making it stand out from the other networks, in the overall communications program. It was concluded that social networks act as democratizing communication, allowing through its good use, to project the strenght of Guarda Nacional Republicana for field strength of a modern security and in line with the change of the current society

Guidelines to reach high-quality purified recombinant proteins

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity, and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization - denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS), and circular dichroism (CD) - are revisited with focus on the protein, and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.This study was funded by the Fundação para a Ciência e a Tecnologia (FCT), Portugal, under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01- 0145-FEDER-006684) and the Post-Doctoral grant SFRH/BPD/ 110640/2015, and by the BioTecNorte operation (NORTE-01-0145- FEDER-000004) supported by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Aborto: da demonização da mulher na Idade Média à criminalização no ordenamento jurídico contemporâneo

O presente trabalho pretende demonstrar o papel da mulher no contexto da acumulação primitiva, com ênfase na transição do sistema feudal para o sistema capitalista, situando a vida sexual e reprodutiva da mulher como objetos do mecanismo de controle do Estado e da Igreja para alcance de seus interesses e perpetuação de seu poder. Analisamos o fenômeno da caça às bruxas dentro da obra Calibã e a Bruxa (2004), de Silvia Federici, pautando a questão da criminalização do aborto na legislação brasileira como resultante da biopolítica e do biopoder.PALAVRAS-CHAVE: Criminalização; Aborto; Inquisição; Biopolítica; Biopoder

A comparative study of recombinant and native frutalin binding to human prostate tissues

<p>Abstract</p> <p>Background</p> <p>Numerous studies indicate that cancer cells present an aberrant glycosylation pattern that can be detected by lectin histochemistry. Lectins have shown the ability to recognise these modifications in several carcinomas, namely in the prostate carcinoma, one of the most lethal diseases in man. Thus, the aim of this work was to investigate if the α-<smcaps>D</smcaps>-galactose-binding plant lectin frutalin is able to detect such changes in the referred carcinoma. Frutalin was obtained from different sources namely, its natural source (plant origin) and a recombinant source (<it>Pichia </it>expression system). Finally, the results obtained with the two lectins were compared and their potential use as prostate tumour biomarkers was discussed.</p> <p>Results</p> <p>The binding of recombinant and native frutalin to specific glycoconjugates expressed in human prostate tissues was assessed by using an immuhistochemical technique. A total of 20 cases of prostate carcinoma and 25 cases of benign prostate hyperplasia were studied. Lectins bound directly to the tissues and anti-frutalin polyclonal antibody was used as the bridge to react with the complex biotinilated anti-rabbit IgG plus streptavidin-conjugated peroxidase. DAB was used as visual indicator to specifically localise the binding of the lectins to the tissues. Both lectins bound to the cells cytoplasm of the prostate carcinoma glands. The binding intensity of native frutalin was stronger in the neoplasic cells than in hyperplasic cells; however no significant statistical correlation could be found (<it>P </it>= 0.051). On the other hand, recombinant frutalin bound exclusively to the neoplasic cells and a significant positive statistical correlation was obtained (<it>P </it>< 0.00001). However, recombinant frutalin did not recognise all malignant prostate cases and, when positive, the binding to those tissues was heterogeneous.</p> <p>Conclusion</p> <p>Native and recombinant frutalin yielded different binding responses in the prostate tissues due to their differences in carbohydrate-binding affinities. Also, this study shows that both lectins may be used as histochemical biomarkers for the prostate cancer. Moreover, the successful use of a recombinant lectin in immunohistochemical studies of prostate cancer was for the first time demonstrated, highlighting the advantages of using recombinant systems in the preparation of pure lectin samples for diagnostic purpose.</p

Cytotoxic Effects of Native and Recombinant Frutalin, a Plant Galactose-Binding Lectin, on HeLa Cervical Cancer Cells

Frutalin is the α-D-galactose-binding lectin isolated from breadfruit seeds. Frutalin was obtained from two different sources: native frutalin was purified from its natural origin, and recombinant frutalin was produced and purified from Pichia pastoris. This work aimed to study and compare the effect of native and recombinant frutalin on HeLa cervical cancer cells proliferation and apoptosis. Furthermore, the interaction between frutalin and the HeLa cells was investigated by confocal microscopy. Despite having different carbohydrate-binding affinities, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cells growth (IC50~100 μg/mL) and equally induced cell apoptosis. The interaction studies showed that both lectins were rapidly internalised and targeted to HeLa cell's nucleus. Altogether, these results indicate that frutalin action is not dependent on its sugar-binding properties. This study provides important information about the bioactivity of frutalin and contributes to the understanding of the plant lectins cytotoxic activity

Physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya gossypii

The blockage of the de novo pyrimidine biosynthetic pathway at the orotidine-5-phosphate decarboxylase level was previously demonstrated to affect riboflavin production in the industrial producer fungus Ashbya gossypii. However, the molecular basis for the unusual sensitivity to uracil displayed by the pyrimidine auxotroph A. gossypii Agura3 was unknown. Here, uridine was shown to be the only intermediate of the pyrimidine salvage pathway able to fully restore this mutants growth. Conversely, uracil, which is routinely used to rescue pyrimidine auxotrophs, had a dose-dependent growth-inhibitory effect. Uracil phosphoribosyltransferase (UPRT) is the pyrimidine salvage pathway enzyme responsible for converting uracil to uridine monophosphate in the presence of phosphoribosyl pyrophosphate (PRPP). Characterization of the A. gossypii UPRT, as produced and purified from Escherichia coli, revealed that uracil concentrations above 1mM negatively affected its activity, thus explaining the hypersensitivity of the Agura3 mutant to uracil. Accordingly, overexpression of the AgUPRT encoding-gene in A. gossypii Agura3 led to similar growth on rich medium containing 5mM uracil or uridine. Decreased UPRT activity ultimately favors the preservation of PRPP, which otherwise may be directed to other pathways. In A. gossypii, increased PRPP availability promotes overproduction of riboflavin. Thus, this UPRT modulation mechanism reveals a putative means of saving precursors essential for riboflavin overproduction by this fungus. A similar uracil-mediated regulation mechanism of the UPRT activity is reported only in two protozoan parasites, whose survival depends on the availability of PRPP. Physiological evidence here discussed indicate that it may be extended to other distantly related flavinogenic fungi.This study was supported by the Portuguese Foundation for Science and technology-FCT(strategic funding UID/BIO/04469/2013, Post-Doc grant SFRH/BPD/110640/2015 and PhD grant PD/BD/113812/2015), and European Regional Development Fund under the scope of Compete2020 (POCI-01-0145-FEDER-006684) and Norte2020 Programa Operacional Regional do Norte (BioTecNorte operation NORTE-01-0145-FEDER-000004).info:eu-repo/semantics/publishedVersio

Via de sinalização mTOR no carcinoma urotelial da bexiga

Trabalho de Projeto apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Análises Laboratoriais Especializadas, área de especialização em Análise BiomédicaMundialmente, o cancro da bexiga (CB) é o 7º tipo de cancro mais frequente em homens e o 17 º mais frequente em mulheres. É uma doença com etiologia multifatorial associada a vários agentes ambientais e genéticos, dos quais o tabagismo é o fator de risco mais importante. Os carcinomas uroteliais da bexiga (CUBs) são geralmente superficiais em 70% a 80% dos pacientes e invasores em 20% a 30%. Os CUBs não-invasores têm uma alta taxa de recorrência e progressão e os invasores e metastáticos representam a principal causa de morbidade e mortalidade entre os pacientes com CB. A via fosfatidilinositol-3cinase (PI3K) /AKT (Proteína cinase b) -alvo da rapamicina em mamíferos (m-TOR) é uma importante via envolvida no crescimento celular, tumorogénese, invasão celular e resposta a drogas. Esta via é frequentemente ativada em muitas neoplasias e o descontrolo da sinalização PI3K-AKT-mTOR no CB pode contribuir para o crescimento do tumor, angiogénese e metastização. Neste trabalho, foram estudados 96 casos de tumores diagnosticados como CUBs de vários graus e estádios e foi avaliada a imunoexpressão do phospho-mTOR (Ser2448) e do phospho-S6 (Ser235/236). Em relação à expressão do p-mTOR verificou-se que mais de 50% dos casos de CUB não apresentavam ou apresentavam uma baixa expressão desta proteína sendo que não foi encontrada associação deste com estádio ou grau de diferenciação tumoral. Em relação à proteína p-S6 a sua expressão nos CUB foi igualmente nula ou baixa, no entanto encontrou-se uma associação estatisticamente significativa com o estádio e grau de diferenciação, em duas formas de avaliação qualitativa. Estes dois marcadores imunohistoquímicos, quando analisados em conjunto apresentaram uma correlação positiva moderada nos CUB, no entanto, estudos futuros são necessários para avaliar a sua validade como marcadores biológicos e, eventualmente, alvos terapêuticos. Worldwide, bladder cancer (BC) is the 7th most frequent type of cancer in men and the 17th most common in women. It is a disease with multifactorial etiology associated with multiple genetic and environmental agents, of which smoking is the most important risk factor. The urothelial carcinomas of the bladder (CUBs) are generally superficial in 70% to 80% of patients and invasive in 20% to 30%. The non-invasive CUBs have a high rate of recurrence and progression, and the metastatic and invaders ones are the leading cause of morbidity and mortality among patients with CB. The 3 cinase via phosphatidylinositol PI3K/AKT (cinase protein b) target of rapamycin in mammals (m-TOR) is an important pathway involved in cell growth, tumorigenesis, cell invasion and drug response. This pathway is frequently activated in many tumors and uncontrollable signaling PI3K/AKT/mTOR pathway in BC may contribute to tumor growth, angiogenesis and metastasis. In this study, 96 cases of tumors diagnosed as CUBs of various grades and stages were studied, and it was evaluated the immunoreactivity of phospho-mTOR (Ser2448) and phospho-S6 (Ser235/236). Concerning the p-mTOR expression, it was observed that more than 50% of CUB didn`t express or had a low expression of this protein, and it wasn`t found any association between this and the state or grade of the tumor. In what concerns the p-S6 protein, it was equally low or absent its expression in the CUB, although it was found a statistical association with the state and grade of the tumor, in two forms of qualitative evaluation. When analyzed, together, these two markers presented a positive moderated correlation in the CUB, however future studies are necessary to access its validity as biological markers and, eventually, therapeutic targets

Modernização administrativa e casos de gabinetes de apoio ao Munícipe

Mestrado em Administração e Gestão PúblicaO presente relatório de estágio incide sobre as temáticas da modernização administrativa e a gestão da qualidade na administração pública, mais concretamente na administração local. A qualidade é vista como uma realidade na Administração Pública reflexo da Reforma da Administração Pública. A mesma é regulada pelas normas de certificação que são vistas como um factor de confiança na sociedade, daí que a implementação dos sistemas de gestão da qualidade na Administração Publica tem vindo a aumentar. Com o nosso estudo pretendemos perceber com que instrumentos se criaram os gabinetes de atendimento ao munícipe e o impacto nos mesmos, da modernização administrativa e a gestão da qualidade.The present report of stage training reflects the themes of the administrative modernization and the quality management in public administration, specifically local administration. Quality is looked upon as a reality that reflects the reform of the Public Administration. Quality is regulated by the norms of certification that are seen as a trust factor in the society, for that reason the implementation of the systems of management of the quality in the Public Administration is increasing. With our study we intend to understand what instruments were created in the offices of city hall and the impact of the administrative modernization and the quality management

Perspectivas analíticas e interpretativas sobre Suplício Tantálico: uma década de imersão na obra de Frederico Richter

O presente artigo traz algumas reflexões acerca de conclusões teórico-analíticas e decisões interpretativas sobre a canção Suplício Tantálico, de Frederico Richter, tendo como ponto de partida estudos realizados sobre a obra em 2010 e 2019, dentro do Mestrado em Música e, posteriormente, do Doutorado, levando em consideração questões de relação entre texto e música