The role of the restriction-modification system of Clostridium pasteurianum on its electro-transformation

Dissertação de mestrado em BioengenhariaClostridium pasteurianum is a Gram-positive and anaerobic bacterium with a great biotechnological potential. It is one of the few microorganisms capable of hydrolyzing glycerol to produce solvents as ethanol and butanol, which have a wide applicability in the market as biofuels. The development of a genetic system for this microorganism would increase its application opportunities since gene overexpression or inactivation could improve their solventogenic characteristics. Its genetic information is already known but this organism has a particular resistance to transformation. This resistance can be explained by a very efficient restriction system that does not allow the entrance of non-methylated DNA or DNA with a methylation pattern different from it. Therefore, foreign DNA must be correctly methylated prior to transformation. For this purpose, a specific methyltransferase is needed to transfer methyl groups to a certain nucleotide of a specific sequence. The goal of this thesis was to create a genetic system in C. pasteurianum that allows genome modification and foreign protein expression, ultimately improving C. pasteurianum DSM 525 transformation. Preliminary simple electro-transformations in which the parameters to make competent cells and the electroporation conditions were altered, did not result in positive results. Being aware of the possibility of a restriction system presence in this organism, experiments with M.MspI methylated DNA were performed, however they demonstrated the inability of this methyltransferase to improve the microorganism transformation. The presence of restriction enzymes was confirmed when a characterization of the restriction system of C. pasteurianum was performed using MspI methylated and non-methylated DNA. The presence of a discrete digestion pattern was detected, and M.MspI methylation could not protect the foreign DNA from C. pasteurianum restriction action. The polyamine spermidine, with known affinity for negatively charged DNA, showed to be efficient against C. pasteurianum crude extract digestion action, however not sufficiently to facilitate this microorganism electrotransformation. By accessing the genome information, the Restriction/Modification (R/M) systems of this microorganism were analyzed. The GATC type IIP R/M system was chosen in order to verify the restriction and methylation enzymes activity with the same target sequence. Three genes, one REase (DpnII) and two MTases (Dam and MdpnII) were cloned in pETduet-1, followed by overproduction in BL21 (DE3). The codon usage of the host and original organism were not compatible, and the protein production in tRNAs provider strains was tested. Protein production was detected, however was not possible to re-confirm their presence. The common protein folding problems were analyzed using a disulfide bond enhancer strain. Nevertheless, the production problem may not be related to this, since no different protein over-production was detected. Restriction reactions with the REase BstUI and C. pasteurianum crude extract, using DNA methylated by M.SssI (m5CG), were developed and showed that the REase responsible for hindering foreign DNA entering C. pasteurianum recognizes the sequence 5'-CGCG- 3'. In a second analysis of the C. pasteurianum genome a methyltransferase-encoding gene was identified that may be involved in methylating the sequence 5'-CGCG- 3'. The in silico analysis was performed and its codon usage was also improved to be compatible with E. coli. In this work, the reasons for C. pasteurianum’s recalcitrance to transformation were identified, the knowledge about its R/M systems was extended, and a proposal to efficiently transform this bacterium was provided.Clostridium pasteurianum DSM 525 é uma bactéria Gram-positiva anaeróbia com um elevado potencial biotecnológico. Este é um dos poucos microrganismos capaz de hidrolisar glicerol para produzir solventes como etanol e butanol, que têm uma grande aplicabilidade no mercado. O desenvolvimento de um sistema genético para este organismo permitiria aumentar as suas oportunidades de aplicação sendo que a sobre-expressão ou inativação de um determinado gene pode melhorar as suas características solventogénicas. A sua informação genética já é conhecida, mas este microrganismo apresenta uma particular resistência à transformação. Esta resistência pode ser explicada pela presença de um eficiente sistema de restrição que não permite a entrada de DNA não metilado ou DNA metilado de forma diferente da própria bactéria. Desta forma, o DNA estranho deve ser corretamente metilado antes da transformação. Para que isto seja possível é necessária a presença de uma metilase específica para transferir grupos metilo para um determinado nucleótido de uma sequência específica. O objetivo desta tese foi criar um sistema genético em C. pasteurianum que permitisse modificações no genoma e a expressão de proteínas heterólogas, ou seja, que permitisse melhorar a transformação de C. pasteurianum DSM 525. Foram realizadas transformações preliminares simples com parâmetros que diferem na forma de obter células competentes e nas condições de eletroporação, contudo os resultados obtidos não foram positivos. Tendo conhecimento da possibilidade da presença de um sistema de restrição neste organismo, foram realizadas experiências com DNA metilado pela enzima M.MspI, sendo que estas demonstraram a incapacidade da metiltransferase para melhorar a transformação deste microrganismo. Foi confirmada a presença de enzimas de restrição aquando da caracterização do sistema de restrição de C. pasteurianum usando DNA não metilado ou metilado pela enzima M.MspI. Foi detetada a presença de um padrão de digestão distinto, verificando-se que a enzima M.MspI não consegue proteger o DNA estranho da ação de restrição de C. pasteurianum. A poliamina espermidina, com conhecida afinidade por DNA negativamente carregado, mostrou ser eficiente contra a ação de digestão do extrato cru de C. pasteurianum, contudo não o suficiente para facilitar a electrotransformação deste microrganismo. Tendo acesso ao genoma, foi então analisado o sistema de Restrição e Modificação (R/M) deste microrganismo. Foi escolhido o sistema R/M tipo IIP GATC para verificar a atividade de enzimas de restrição e metilação com a mesma sequência de reconhecimento. Foram clonados três genes no vetor pETduet-1, uma enzima de restrição (REase – DpnII) e duas metiltransferases (MTases – Dam and Mdpn), seguindo-se a produção em BL21 (DE3). O conjunto de codões usados pelo hospedeiro e pelo organismo de origem não eram compatíveis, foi então testada a produção proteica em estirpes fornecedoras de tRNAs. Foi observada produção proteica contudo não foi possível re-avaliar a sua presença. Foram analisados problemas de enrolamento (do inglês folding), usando uma estirpe que facilita a formação de pontes dissulfito. No entanto, o problema na produção não deve estar associado ao enrolamento proteico sendo que não foi detectada produção proteica nestas condições. Foram desenvolvidas reações de restrição com a REase BstUI e extrato cru de C. pasteurianum usando DNA metilado pela enzima M.SssI (m5CG) e foi mostrado que a REase responsável pelo impedimento da entrada de DNA em C. pasteurianum reconhece a sequência 5'-CGCG- 3'. Numa segunda análise ao genoma de C. pasteurianum DSM 525 foi identificado um gene que codifica uma metiltransferase que pode estar envolvida na metilação da sequência 5'-CGCG- 3'. Foi feita a análise in silico e o tipo de codões usados foi melhorado para ser compatível com E. coli. Neste trabalho, foram identificadas as razões para a resistência deste microrganismo à transformação, foi consolidado o conhecimento sobre o seu sistema de R/M e foi proposta uma metodologia para transformar de forma eficiente esta bactéria

Esteroisómeros na terapêutica com bloqueadores beta-adrenérgicos

A maioria dos fármacos comercializados atualmente são estereoisómeros, podendo estes apresentarse na forma de enantiómeros ou diastereómeros. Desde que a talidomida causou inúmeros casos de malformações fetais, as indústrias química e farmacêutica aperceberam-se da importância do estudo da quiralidade dos fármacos e das suas propriedades estereoquímicas. A análise da correlação entre a quiralidade e as propriedades toxicológicas e farmacológicas dos compostos levou, não só à eliminação de efeitos adversos, como também a benefícios terapêuticos. Tem surgido uma tendência para a comercialização de novos fármacos sob a forma de enantiómeros puros, pois apresentam vantagens em relação às misturas racémicas. A escolha entre um enantiómero ou uma mistura racémica depende das vantagens terapêuticas, dos efeitos adversos e dos custos de desenvolvimento. Este trabalho evidência as diferenças a nível da atividade biológica e dos parâmetros farmacocinéticos entre os racematos e os estereoisómeros dos bloqueadores β-adrenérgicos. À semelhança do que acontece com outros fármacos, na sua maioria, os bloqueadores β são prescritos como racematos.Contudo, em humanos, os seus S-enantiómeros são muito mais eficazes que os seus antípodas, por exemplo, no tratamento de patologias cardíacas, podendo a purificação e administração do eutómero traduzir-se em benefício clínico para o doente.info:eu-repo/semantics/publishedVersio

Ensaio sobre cegueiras: cruzamentos intersecionais e (in)visibilidades nos media

A atualidade não se pode dissociar de vivências multideterminadas e sempre presentes. Quer sejamos atravessadas/os por privilégios ou opressões, e sobretudo por ambas, nos mais variados contextos, é impossível minimizar o impacto que estes têm na sedimentação ou desconstrução das relações de poder, na nossa subjetividade e experiências diárias.info:eu-repo/semantics/publishedVersio

How to Predict the Innovation to SMEs? Applying the Data Mining Process to the Spinner Innovation Model

Funding: This paper is financed by National Funding awarded by the FCT—Portuguese Foundation for Science and Technology to the project «UIDB/04928/2020» and NECE-UBI, R&D unit funded by the FCT —Portuguese Foundation for the Development of Science and Technology, Ministry of Education and Science, University of Beira Interior, Management and Economics Department, Estrada do Sineiro, 6200-209 Covilhã, Portugal.Despite the importance of small and medium-sized enterprises (SMEs) for the growth and development of companies, the high failure rate of these companies persists, and this correspondingly demands the attention of managers. Thus, to boost the company success rate, we may deploy certain approaches, for example predictive models, specifically for the SME innovation. This study aims to examine the variables that positively shape and contribute towards innovation of SMEs. Based on the Spinner innovation model, we explore how to predict the innovation of SMEs by applying the variables, namely knowledge creation, knowledge transfer, public knowledge management, private knowledge management and innovation. This study applied the data mining technique according to the cross industry standard process for data mining (CRISP-DM) method while the Statistical Package for the Social Sciences (SPSS_Version28) served to analyze the data collected from 208 SME employees in Oporto, Portugal. The results demonstrate how the Spinner innovation model positively influences the contributions of the SMEs. This SME-dedicated model fosters the creation of knowledge between internal and external interactions and increases the capacity to predict the SME innovation by 56%.info:eu-repo/semantics/publishedVersio

Children and youths at risk/danger: contributions to the characterization of a CPCJ from north of Portugal

A promoção e proteção das crianças em risco funda-se na dignidade da pessoa humana que, no nosso ordenamento jurídico, tem consagração constitucional (art.º 1.º CRP) e na lei ordinária, em especial, na Lei 147/99, de 1/9. Na comunidade internacional esta problemática está vazada na Convenção sobre os Direitos da Criança. É objetivo deste trabalho descrever/compreender as situações de perigo da criança/jovem, sinalizadas numa Comissão de Proteção de Crianças e Jovens (CPCJ) da região Norte do país (2011-2013). A amostra foi constituída por 62 processos, já arquivados, de crianças/jovens em risco, de ambos os géneros, com idades entre os 0-17 anos e aos quais foi aplicada, pelo menos, uma medida de promoção e proteção pela CPCJ. As situações de risco/perigo ocorreram, predominantemente, em famílias nucleares com filhos (56,6%) e monoparentais femininas (26,4%), com crianças entre 0-2 anos (14,5%,), 2-7 anos (17,7%), 7-11 anos (20,9%), 11-15 anos (37,2%) e 16-17 anos (9,7%). Das 62 crianças, 35,4% apresentavam problemas de cognição/saúde. As sinalizações foram realizadas, sobretudo, pela escola (19,4%), anónimos (17,7%) e autoridades policiais (11,3%) e fundaram-se, essencialmente, em motivos como a negligência da saúde da criança (22,6%) e o absentismo escolar (19,4%). As medidas aplicadas foram, na sua maioria, em meio natural de vida, designadamente apoio junto dos pais (87,1%) e junto de familiar (9,7%). A duração dos processos situou-se entre 3 e 40 meses, com a moda nos 12 e 34 meses. Importa realizar mais estudos que nos permitam conhecer sincrónica e diacronicamente a realidade portuguesa das CPCJ.The promotion and protection of children at risk is based on the dignity of the human person that in our legal system has constitutional defense (Article 1 CRP) and ordinary law (Law 147/99, 1/9). In the international community this issue is assured in the Convention on the Rights of the Child. The aim of this study is to describe/understand the risk/danger situations of the children/youths signalized on a Child and Youth Protection Commission (CPJC) from the North region of the country (2011-2013). The sample consisted of 62 cases, already filed, of children/youths at risk, of both genders, 0-17 years and to which was applied by the CPCJ at least one promotion and protection measure. Situations of risk/danger occurred predominantly in nuclear families with children (56.6%) and female single-parent families (26.4%), with children aged 0-2 years (14.5%), 2-7 years (17,7%), 7-11 years (20.9%), 11-15 years (37.2%), and 16-17 years old (9.7%). Of the 62 children, 35.4% had cognitive/health problems. Referrals were made mainly by the school (19.4%), anonymous (17.7%) and the police (11.3%) and there were founded essentially situations such as health neglect (22.6%) and school absence (19.4%). The measures were applied, mostly in natural living environment, including support to parents (87.1%) and other family member (9.7%). The duration of processes ranged between 3 and 40 months, with mode in 12 and 34 months. Further studies that allow us to know synchronic and diachronic Portuguese reality of CPCJ are essential.info:eu-repo/semantics/acceptedVersio

Media and the (im)permeability of public sphere to gender

We cannot continue to look at public sphere as a space of privileged, following an Habermasian logic. In this sense, an idealized definition of public sphere is created drawings upon equality opportunities and upon its intervenient rationalism, where the author clearly tries to retrieve Kantian sense of freedom. However, he focuses on male power holders and citizens’ participation. Moreover, public sphere is nowadays marked by multiple positions and actors that magnify its interactions. We are particularly interested on the uses of public sphere on media by its transformative capacity and the swell of a democratic space for every individuals. The breach of this sphere and its consequent fragmentation has functioned almost paradoxically: on one hand it allows subjects to position themselves and to take a stand but, on the other hand, it reinforces status quo, depriving other subjects from public sphere agency. This discursive (im)permeability carried out by the media against certain groups and individuals has raised debate in many areas. Feminist critique’s legacy has been extremely relevant to this public sphere conceptual evolution and reformulation, contributing to a more plural and equality conceptualization; one that is truly close to citizens and its idiosyncrasies. Feminist critique to Habermas’ bourgeois public sphere comes, first of all, from the recognition of women’s exclusion, as well as of other specific social groups, from figuring in society and from having an active part, therefore excluding them from democratic citizenship. The role of media, however, does not seem easy, as it is one of the privileged vehicles for information dissemination playing an important contribution to citizenship. Yet, in this junction there are diverse social actors that intend to enroll public decision-making. We know that public opinion is shaped mainly by parameters that prioritize some discourses over others, giving them more prominence and power. By highlighting these discourses, assigning greater emphasis to certain themes and people, media equally contribute to the formation and delimitation of public sphere boundaries. In this complex relationship established, and based on a critical feminist outlook, we intend with this paper to question the existence of a single public sphere or of several ones, and to question how far are mainstream media (im)permeable to emancipatory and resistance discourses. Finally, we will question, as well, how does public sphere and democracy notions articulate themselves with the increasing individualism promotion taking place in Western societies.This paper was developed under the scope of the research project “Gender in focus: social representations in Portuguese generalist newsmagazines [Género em foco: representações sociais nas revistas portuguesas de informação generalista]” (PTDC/CCI-COM/114182/2009) funded by FCT - Fundação para a Ciência e Tecnologia (Portuguese Science and Technology Foundation). Project website: http://www.lasics.uminho.pt/genfo

Information mining in patent filings on injectable antineoplastics as a contribution to Health Policy

Introduction: According to data from the United Nations, cancer is the second leading cause of death in the world. Currently, information management has been increasingly difficult due to the large amount of data to be managed. In general, the databases that store patent documents make it possible to read them in full, but do not allow the extraction and treatment of large amounts of data. In this sense, it is necessary to use management software. Objective: To identify, extract, process the data, organize,and make available, in the form of graphical interfaces, the technological information on injectable oncology described in the current patents. Methodology: Patents deposited between January 2002 and July 2022 were analyzed using the ORBIT Intelligence® platform. In the “Advanced Search” field, the “Title, Abstract” filters were applied and the search terms: “injectable AND cancer” were used. Results and Discussion: 115 patent families were identified. The USA stands out in the number of patent documents filed, presenting a total of 56 documents. Inventors Ivan Edward Hofman, Farber Michael, Franco Rodriguez Guillermo and Gutierro Aduriz Ibon were the most productive, each with 3 documents deposited. The institutions Bespoke Bioscience (USA), Immunocore Holdings (United Kingdom) and Mountain Valley MD Holding (Canada) stood out, each holding 3 documents. In the documents analyzed, the most recurrent technological domain went beyond the "pharmaceutical" technological domain, which obtained 109 documents and others such as chemical, biological, electrical, micro and nanotechnology. Final Considerations: The results obtained by mining the data extracted from patent documents proved to be efficient and, can be useful as an effective tool to analyze, compare and monitor research and innovation activities in injectable oncology.publishersversionpublishe

Tiempo de sueño, televisión/videojuegos y consumo de bocadillos en niños en edad preescolar: un estudio transversal

This study aims to evaluate the sedentary behavior, caloric content of snacks and sleep time of preschool children in two different regions of Portugal (Bragança and Gondomar). Participated in this study 230 healthy preschoolers (103 girls; 127 boys), of which 176 (76.5%) were from Bragança (mean age of 4.5±0.7 years) and 54 (23.5%) were from Gondomar (mean age of 4.3±1.0 years). A questionnaire about sedentary behavior, sleep time and caloric content of snacks (morning and afternoon) were answer by parents of this preschool children. The principal results of our study indicated that the preschool children slept an average of 9.9 ± 0.9 hours, spent time 1.5 ± 0.8 hours in front of television (TV) during the week, and 2.5 ± 1.5 hours during weekend. Regarding the time spent playing video games, they did 0.6 ± 0.6 hours during the week and 0.9 ± 0.9 hours during the weekend. The children morning snack had an average of 232.7 ± 100.8 kcal and the afternoon snack had an average of 350.4 ± 122.4 kcal. According to the WHO guidelines, the results of this study revealed that preschool children spent less hours sleeping, had more caloric content in the morning and afternoon snacks and spent more time in sedentary behaviors, of what is in the recommendations. These results also suggest that the children’s slept time is affected by the time they spent watching TV and playing video games.This work has been supported by FCT – Fundação para a Ciência e Tecnologia within the Project Scope: UIDB/05777/2020info:eu-repo/semantics/publishedVersio

Gender issues in Portuguese generalist newsmagazines: thematic intersections at Sábado and Visão

As revistas de informação generalista configuram uma interseção entre as representações socioeconómicas, políticas e culturais e os quotidianos da população. Neste artigo apresentamos uma análise temática das revistas de informação generalista Sábado e Visão, as mais lidas do segmento em Portugal. O corpus de análise foi composto por 440 peças jornalísticas de 104 edições que correspondem a todos os números editados em 2011 por estas duas publicações. O estudo empírico, realizado com recurso ao software NVivo 10, revela uma transversalidade da abordagem aos temas político-económicos, espelho da conjuntura nacional e internacional no ano de 2011. As diferenças mais visíveis entre as duas revistas revelam uma maior opção pelos temas Sexualidades e Relações de Intimidade e Jet set na Sábado e Saúde, Lazer e bem-estar na Visão. Além disso, no que concerne às representações de género, presentes em vários temas, surgem na generalidade assentes num binarismo que transpõe o olhar da dimensão de género para o sexo biológico e que opõe as representações tradicionais da feminilidade e masculinidade, condicionando o surgimento de representações alternativas.Generalist newsmagazines constitute an intersection among socioeconomic and political representations and the everyday life of individuals. This paper we introduce a thematic analysis of the two most read Portuguese newsmagazines: Sábado and Visão. We retrieved 440 news articles from 104 newsmagazines that match all the issues of the two newsmagazines in 2011. The empirical study was carried out with the support of the software Nvivo 10 and shows a transversality of both political and economic themes, which refl ects the Portuguese and international conjuncture in the year of 2011. The most visible differences between the two newsmagazines indicate that there is a more frequent option for the themes Sexualities and Intimacies and Jet set in Sábado and for Health, leisure and well being at Visão. Moreover, what concerns the gender representations, that emerge in several themes, they generally arise in a binary way that transpose the look of gender to biological sex and that opposes the traditional representations of femininity and masculinity, conditioning the emergence of alternative representations.(undefined