Bela krajina

Sustainable development in karst areas should be adapted to its specificities and take into account its vulnerability. The assessment of the development potential and management of karst areas is of great importance in Slovenia. This book presents the analyses of the impact of landscape features on the land use and sustainable development in a marginal Slovenian karst landscape ‒ Bela krajina. In order to draw attention to the combination of social perspectives with natural conditions for an integrative view of the karst landscapes, three approaches were used: 1. assessment of the degree of human disturbance to the karst landscape, 2. analyses of land use dynamics, and 3. quantitative and qualitative analyses of the sustainable development of Bela krajina. Karst landscape features affect sustainable development of the study region both positively and negatively. According to local stakeholders the positive effects are mainly connected with tourism, and the negative effects are mainly connected with hampered agriculture. The main message is that karst landscape features should not only be seen as limiting factors, but also for their development potential.Trajnostni razvoj na kraških območjih bi moral biti prilagojen posebnostim te pokrajine, posebej njeni ranljivosti. Pravilno vrednotenje razvojnega potenciala in upravljanje s kraškimi območji je zelo pomembno za Slovenijo. V tej knjigi so predstavljene analize vpliva pokrajinskih značilnosti na rabo zemljišč in trajnostni razvoj obmejne slovenske kraške pokrajine ‒ Bele krajine. Da bi opozorili na kombinacijo družbenih dejavnikov in naravnih razmer za celostni pogled na kraško pokrajino, smo uporabili tri pristope: 1. oceno obremenjenosti kraške pokrajine zaradi človeka, 2. analizo sprememb rabe zemljišč in 3. kvantitativno in kvalitativno analizo trajnostnega razvoja Bele krajine. Kraški pojavi na trajnostni razvoj lahko vplivajo tako pozitivno kot negativno. Po mnenju lokalnih deležnikov so pozitivni učinki povezani večinoma s turizmom, negativni učinki pa predvsem v omejenem kmetijstvu. Sklenimo z mislijo, da so kraški pokrajinski pojavi lahko več kot le omejevalni dejavniki, saj imajo tudi razvojni potencial

Mining legacy of Douro Coal Basin: remains of a system under process

A identificação do legado mineiro da Bacia Carbonífera do Douro levanta questões face aos paradigmas de reconhecimento patrimonial e da intervenção sobre as marcas de uma actividade económica estritamente ligada ao território que transforma e que hoje nos chegam. No território transformado pelo Sistema Carbonífero do Douro (SCD) o actual enquadramento patrimonial identifica “fragmentos” que não permitem a sua leitura como integrante de um sistema sociotécnico. Como alternativa, é apresentado um entendimento de como o Sistema se vai territorializando, sedimentando uma “paisagem antropogeográfica”. De um exclusivo reconhecimento das “construções resistentes” hoje existentes, propomos a identificação do conjunto de relações temporal e espacialmente transversais que, assumindo formas distintas em função do contexto integrado, foram determinando as morfologias do SCD, imputando-lhes a dimensão identitária inerente a um sistema produtivo motor de aculturação, possibilitando ler o Sistema como tal. Face à capacidade de representação dos valores que estabelecem vínculos entre o presente e o passado sobre a qual deverá incidir a condição patrimonial, identifica-se neste conjunto de relações as regras para a transformação futura do território que o SCD moldou, procurando-se veicular um entendimento prospectivo de património, então potenciador de uma requalificação territorial integrada e dotada de significado.The recognition of the mining legacy of Douro Coal Basin raises issues regarding the paradigms of heritage recognition and of intervention on the present marks of an economic activity strictly linked to the territory, so transformed by it. In the territory transformed by the Carboniferous System of Douro (SCD) the current heritage context identifies only “fragments”, which don’t allow their understanding as an integrated part of a sociotechnical system. As an alternative, it is proposed the understanding of how the SCD was being territorialized and settling an “anthropogeographic landscape”. This paper identifies temporal and spatial correlations that supported SCD territorialisation. These correlations assumed distinct forms according to the surrounding context and determined SCD morphologies, attributing to them the identity inherent to a productive system that enhanced acculturation and allowing its perception as a system. The evaluation of heritage condition and future interventions should consider values and needs that result from the links between present and past. Under this assumption, a group of intervention principles for the transformation of the SCD territory are identified. The goal is to move closer to a prospective understanding of heritage, enabling an integrated and meaningful territory requalification.Peer Reviewe

The Impact of (Opportunity and Necessity) Entrepreneurship on Economic Growth: Does Human Capital Matter?

O empreendedorismo é geralmente definido como a criação de novas empresas e, de acordo com a literatura, é o processo pelo qual novas empresas são criadas e se tornam sustentáveis. Um considerável número de pesquisas estudou o impacto do empreendedorismo no crescimento económico. Contudo, poucos desses estudos analisaram o impacto dos tipos (oportunidade versus necessidade) de empreendedorismo no crescimento económico. Além disso, o último conjunto de estudos ignorou a relevância do capital humano como fator intermediário na relação entre o (tipo de) empreendedorismo e crescimento económico. O presente estudo tem como objetivo ultrapassar a lacuna acima mencionada, avaliando até que ponto o impacto direto e indireto do (tipo de) empreendedorismo, via capital humano, é importante para o crescimento económico dos países. Em termos metodológicos, recorremos a estimações de dados em painel envolvendo um conjunto alargado de (OCDE e não-OCDE) países num extenso período de tempo (1990-2016). Os resultados sugerem que o empreendedorismo global apresenta um impacto positivo no crescimento económico. Distinguindo os tipos de empreendedorismo, há claras evidências de que o empreendedorismo de oportunidade apresenta um impacto positivo no crescimento económico enquanto que o empreendedorismo de necessidade inibe o crescimento económico. De forma interessante, o capital humano tende a mitigar o impacto negativo do empreendedorismo de necessidade sobre o crescimento económico. No caso do empreendedorismo de oportunidade, o impacto direto positivo fica reduzido em contextos caracterizados por elevados níveis de capital humano, o que pode ser influenciado pelo aumento dos custos de oportunidade.Entrepreneurship is generally defined as the creation of new firms and according to literature, it is the process by which new enterprises are founded and become viable. Although considerable research has been devoted to the study of the impact of entrepreneurship on economic growth, fewer studies have analyzed the impact of the types (opportunity vs necessity) of entrepreneurship on economic growth. Moreover, the latter set of studies overlooked the relevance of human capital as mediating factor in the relation between (types of) entrepreneurship and economic growth. The aim of the present study was to fill in the above mentioned gap, by assessing the extent to which the direct and indirect impact of (the types of) entrepreneurship, via human capital, matters for countries' economic growth. In methodological terms, we resort to fixed effects panel data estimations, involving a large set of (OECD and non-OECD) countries, over a relatively long time span (1990-2016). The results suggest that total entrepreneurship have a positive impact on economic growth. Distinguishing between types of entrepreneurship, there is clear evidence that opportunity entrepreneurship fosters economic growth, whereas necessity entrepreneurship inhibits it. Interestingtly, human capital tends to mitigate the negative impact of necessity entrepreneurship on economic growth. In the case of opportunity entrepreneurship, the direct positive impact observed is reduced in contexts characterized by high levels of human capital, which might reflect increased opportunity costs

Mechanisms and Variability of Glyphosate Resistance in Amaranthus Palmeri and Ipomoea Lacunosa

The resistance of Palmer amaranth (PA) and the tolerance (natural resistance) of pitted morningglory (PM) to glyphosate have made these species among the most common and troublesome weeds in the southeastern U.S. since the adoption of glyphosate-resistant (GR) crops. Populations of GR PA (R1 and R2) were identified in Mississippi. The inheritance of glyphosate resistance was examined in reciprocal crosses (RC) between glyphosate-resistant (R) and -susceptible (S) parents (Female-S × Male-R, S/R, and Female-R × Male-S, R/S), and second reciprocal crosses (2RC) (Female-S/R × Male-S/R, S/R//S/R, and Female-R/S × Male-R/S, R/S//R/S). Dose-response assays resulted in 17- to 4old resistance to glyphosate compared with S. Population S accumulated 325- and 8-times more shikimate at the highest glyphosate dose than in R1 and R2, respectively. cDNA sequence analysis of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene indicated no target site mutation. Genomes of R1, R2, RC, and 2RC contained from 1- to 59old more copies of EPSPS gene than S; EPSPS was highly expressed in R1 and R/S, but was poorly expressed in S, S/R, and R2. EPSPS activity was lower in S and S/R than in R and R/S, glyphosate absent; all were inhibited by glyphosate. Western Blot analysis confirmed an increased EPSPS protein level to EPSPS copy number correlation. Thus, the level of resistance was decidedly influenced by the direction of the cross. R and S female plants were reproductively isolated and seed were still produced, suggesting that PA can produce seed both apomictically and sexually (facultative apomixis). This mode of reproduction determined the low copy number inheritance, as well as guaranteeing the GR trait stability in the R populations. Dose-response assays resulted in 2.6old variability in tolerance to glyphosate between the most tolerant (MT) and the least tolerant (LT) PM populations. The level of tolerance positively correlated with the time of exposure to GR-crop system. Less shikimate was recovered in MT as compared to LT. Levels of aminomethylphosphonic acid (AMPA) were not different between populations and sarcosine was not present in either populations. Consequently, metabolism of glyphosate to AMPA or sarcosine is not a common factor in explaining natural resistance levels

Explaining mortality rates from COVID-19 : an application of business analytics

The COVID-19 pandemic has generated a lot of demand for responses to prevention, treatment, how to control, how to predict evolutions, among others. This thesis aims to answer the question about what affects mortality. Thus, through the use of Analytics, 26 different variables were studied for 37 duly selected countries. The results showed that the country's economic structure has no impact on mortality, while vaccination policy for BCG, changes in mobility within the country, such as “stay at home”, and the prevalence of diabetes have an impact on mortality.A pandemia COVID-19 tem gerado muita procura por respostas para prevenção, tratamento, como controlar, como prever evoluções, entre outras. Esta tese pretende responder à pergunta sobre o que afeta a mortalidade. Assim, através do uso do Analytics foram estudadas 26 diferentes variáveis para 37 países devidamente selecionados. Os resultados permitiram concluir que a estrutura económica do país não tem impacto na mortalidade, enquanto que a política de vacinação para a BCG, as alterações da mobilidade dentro do país, tais como o “stay at home”, e a prevalência de diabetes têm impacto para a mortalidade

Improvement of project management practices: a car industry case study

Dissertação de mestrado integrado em Industrial Engineering and ManagementThe present master dissertation project was developed on a competence center (MFT3 section) at Bosch Car Multimedia in Braga. It has as main objective the improvement of project management practices in order to enhance the performance of this particular section in the organization, translated into a set of four sub-objectives: 1) redefinition of an integrated project management process; 2) definition of a template for project planning supported with Work Breakdown Structure (WBS) tool; 3) customization of a software tool to support the project management process; and 4) development of a performance measurement system. Firstly, a literature review was performed in order to understand which project management improvements could be proposed to MFT3 section at Bosch Car Multimedia in Braga. Important subjects were studied, such as organizational project management, including project, program and portfolio management; project planning and WBS; tools to support project and portfolio management; and project management success through performance measurement systems. Secondly, having a better theoretical knowledge about project management approaches, some improvements were proposed to accomplish the project’s dissertation objectives. The software customized to support the project management process was the software Track&Release which integrates the WBS template and improves time tracking, the visualization of every activity with its respective status, planning, prioritization of work and communication as well as decreases unproductive time and misunderstandings through organization of all team work. The Track&Release also allows data extraction in order to have relevant information to create interesting performance measurement indicators graphics. These graphics were created through an EXCEL file developed to transform the data from Track&Release to well-established key performance indicators graphics. Besides some difficulties on this research, namely the totally new environment for the researcher and the lack of time of Bosch collaborators, the established objectives were fulfilled and there was a significant contribution to the improvement of project management practice to MFT3 section at Bosch Car Multimedia in Braga, which makes this master dissertation project a successful one.A presente dissertação de mestrado foi desenvolvida no Centro de Competências de montagem de PCBs (secção MFT3) da Bosch Car Multimedia em Braga. A fim de melhorar o desempenho e organização desta secção, foi proposto como principal objetivo a melhoria das práticas de gestão de projetos, subdividindo este em quatro objetivos mais específicos: 1) redefinição do processo de gestão de projetos integrado; 2) definição de um template de planeamento de projetos apoiado pela ferramenta Work Breakdown Structure (WBS); 3) customização de um software para suporte da gestão de projetos; e 4) desenvolvimento de um sistema de medição de desempenho. Inicialmente foi realizada uma revisão da literatura de modo a apurar quais as melhorias que poderiam ser propostas à secção MFT3 da Bosch Car Multimedia em Braga. Vários assuntos foram estudados, tais como, organização da gestão de projetos, incluindo gestão de projeto, programa e portefólio; planeamento de projetos e WBS; ferramentas de apoio à gestão de projetos e portefólio; e sucesso aplicado à gestão de projetos por meio de sistemas de medição de desempenho. Após um conhecimento teórico mais sólido, foram propostas algumas melhorias para alcançar os objetivos do projeto de dissertação. Deste modo, o software personalizado para suportar o processo de gestão de projetos foi o Track&Release que integra o WBS template e acrescenta melhorias a nível do controlo de tempo, visualização de todas as atividades com seu respetivo status, planeamento, priorização de trabalho e comunicação, assim como diminui o tempo improdutivo e equívocos através da organização de todo o trabalho da equipa. O Track&Release também permite a extração de dados a fim de ter informações relevantes para criar gráficos de medição de indicadores de desempenho. Estes gráficos são criados através de um ficheiro de EXCEL desenvolvido para transformar os dados do Track&Release nos gráficos predefinidos. Apesar das dificuldades encontradas, nomeadamente a pouca experiência do investigador e a falta de tempo dos colaboradores da Bosch, os objetivos estabelecidos foram cumpridos e houve uma contribuição significativa para a melhoria da prática de gestão de projetos para a seção MFT3 na Bosch Car Multimedia em Braga

Targeting miR-124 in motor neurons as a therapeutic strategy to prevent neurodegeneration and glial activations in ALS

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2018Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by the loss of motor neurons (MNs) from the motor cortex, brainstem and spinal cord (SC). Despite being studied for several years, there are only two available therapies, each one playing a modest impact on disease outcome. Initially, it was thought initially that the disease was only caused by motor neuron degeneration, but today it is known that ALS is a non-cell-autonomous disease involving glial cells, which show several dysfunctionalities considered to be aberrant. Glial cells were shown to lose their neuro-supportive function and to contribute to neurodegeneration and disease progression. However, it is not known which dysregulated mechanisms are affecting cell-to-cell communication, and the molecular mediators of neuronal injury, either existing as soluble factors or mediated by extracellular microvesicles. MicroRNAs (miRNAs) are non-coding RNAs whose function is to suppress gene expression through their binding to mRNAs. Lately, miRNAs were discovered as diagnostic biomarkers and potential therapeutic targets in ALS. Differentially expressed miRNAs were identified, some with recognized association with known or suggested pathways in ALS pathogenesis. miRNAs can act in a tissue-/cell-type-specific manner and are involved in cellular communication, where they may affect gene expression, either on the cells of origin or in recipient ones. MiRNA(miR)-124 is one of the most expressed in the central nervous system (CNS) and described as usually associated with neuronal differentiation. It has been found elevated in the cerebrospinal fluid of ALS patients, as well as in the ALS mice with the G93A mutation in the SOD1 gene (mSOD1), namely at active neurodegeneration sites, and identified in our studies as being upregulated in MN NSC-34 cell line with the same mutation. Interestingly, we noticed that miR-124 was upregulated in exosomes derived from such MNs, which showed to induce several activated subtypes once collected by microglia. In physiological conditions, miR-124 is involved in neuronal maturation, as well as in cell cytoskeleton organization, neurite outgrowth and autophagic regulation. Lately, dysregulation of miR-124 has been found in several CNS disorders associated with neurodegeneration, stress, neuro-immune dysregulation, and brain tumors, among others. Here, we aimed to explore the consequences of miR-124 inhibition and overexpression on the MN function and structure, as well as on microglia and astrocyte immunoregulation. For that, we used the NSC-34-MN-like cell line, either non-mutated (wild type, WT), or expressing the G93A mutation (mSOD1), which is a model that mimics the SOD1 accumulation and the neuronal degeneration observed in ALS. We transfected WT MNs with miR-124 mimic (pre-miR-124), and mSOD1 MNs with the miR-124 antagonist (anti-miR-124), to assess whether the effects produced by their secretome differently affected glia reactivity. In this sense, the collected secretome was incubated with mice microglial N9 cells for 4 h and with SC astrocytes from WT and mSDO1 8-days-old mice for 24 h. MN viability was not changed by both miR-124 modulations and that using anti-miR-124 prevented SOD1 accumulation. This one also prevented the increase in gene and protein expression of High Mobility Group Box 1 (HMGB1), as well as of the inducible Nitric Oxide Synthase (iNOS) protein, and of S100 Calcium-binding Protein B (S100B) mRNA. Overexpression of miR-124 in WT MNs was reflected only in S100B mRNA increase. At miRNAs level, anti-miR-124 in mSOD1 MNs decreased the overexpression of miR-125b and increased that of miR-21 and miR-146a, which were diminished in the disease model. Interestingly, pre-miR-124 in WT MNs led to the profile observed in mutated cells, namely to upregulation of miR-125b and to downregulation of miR-21 and miR-146a, validating miR-124 as a driver of such miRNA dysregulation. Importantly, anti-miR-124 was also capable of diminishing the expression of the pre-synaptic marker synaptophysin and of the retrograde transport marker dynein, both elevated in mSOD1 MNs and in WT-treated pre-miR-124 MNs, toward WT levels, highlighting miR-124 as a major contributor to such increases. In opposite, this miRNA revealed to be associated with the decrease of the post-synaptic marker PSD-95 and the anterograde transport marker kinesin, since anti-miR-124 led to a remarkable increase in the expression of their genes. Thus, we can conclude that anti-miR-124 favors anterograde transport (from soma to the end of the axon), deficient in the mutated cells, in which dynein elevation favors retrograde transport (back to the soma) described to be associated with miR-124 upregulation. Overexpressed miR-124 was also shown to relate with fewer ramifications and number of primary neurites, as well as with their increased length, which were counteracted to values close to control levels with anti-miR-124 in mSOD1 MNs. This indicates the influence of this miRNA in cytoskeleton regulation, cellular morphology, and MN function. In fact, anti-miR-124 was able to recover mitochondrial viability in mSOD1 MNs, whose decrease seems to be related with miR-124 upregulation. It also favored Mitofusin expression (an inducer of mitochondrial fusion), found diminished in mutated cells, and decreased DRP1 (associated with mitochondrial fission), increased in mutated cells. Once again, the overexpression of miR-124 showed to be involved in DRP1 upregulation in the mutated cells. Such alterations suggest the existence of oxidative stress associated with miR-124 overexpression and that anti-miR-124 may have a beneficial action by favoring anti-oxidant mechanisms. Relatively to the immunoregulatory action exerted by the MN secretome, we verified that the increased area, perimeter and Ferret’s diameter, as well the circularity reduction in microglia treated with secretome derived from mutated MNs, was not manifested upon incubation with the secretome from mSOD1 MNs treated with anti-miR-124. These parameters showed a direct correlation with the miR-124 overexpression. Microglia phagocytic ability was also affected by the secretome from WT MNs treated with pre-miR-124, as well as with that from mSOD1 modulated or not with anti-miR-124, indicating that miR-124 inhibition is not enough to recover such microglial property. However, anti-miR-124 modulation effectiveness was again manifested when the secretome from mSOD1 MNs acquired the capability to decrease the gene expression of iNOS, arginase1, Tumor Necrosis Factor alpha (TNF-α) and Interleukin 1 beta (IL-1β). All these inflammatory genes were found upregulated in microglia treated with both mSOD1 and pre-miR-124 WT MNs. Additionally, it led to a decrease of HMGB1, highly enhanced after incubation with the secretome from mSOD1 MNs, and to an elevation of the mannose receptor CD206 expression, associated with macrophage endocytic and phagocytic properties. Interestingly, here we found an indirect correlation in that overexpressed miR-124 triggered a CD-206 gene expression decrease. In what concerns the consequences of mSOD1 MN secretome after miR-124 modulation on astrocytes, it was interesting to observe that GLT-1 levels were sustained in this condition, contrasting with the reduction found with the secretome from WT MNs treated with pre-miR-124, as well as with that from mSOD1 MNs. Similar effect was noticed on GFAP levels, whose expression increased relatively to WT and mSOD1 astrocytes treated with the secretome from mSOD1 MNs as well as the WT astrocytes exposed to the secretome from WT MNs modulated with pre-miR-124. Therefore, we can hypothesize that, at least in part, miR-124 elevation in mSOD1 MNs is associated with the production of a secretome determining GFAP and GLT-1 reduced expression, features that relate with the aberrancy of astrocytes in ALS. Overall, our results indicate that miR-124 overexpression in mSOD1 MNs has a key role in the degeneration and dysfunction of these cells, remarkably contributing to ALS pathogenesis and progression by compromising cellular homeostasis, due to a secretome with immunostimulatory and inductive aberrant features in either WT or mSOD1 astrocytes. Anti-miR-124 showed beneficial effects in preventing SOD1 accumulation in mutated MNs, while exerting synaptic transmission regulation (increase of the neuritic tree, kinesin and PSD-95), anti-oxidant effect (increased mitochondrial viability and Mitofusin and diminished DRP1) and anti-inflammatory action (increase of miR-21 and miR-146a, with decrease of miR-125b). Furthermore, secretome from mSOD1 MNs modulated with anti-miR-124 acquire properties that support microglia functionality (normal morphological characteristics and levels of iNOS, Arginase1, TNF-α, IL-1β and HMGB1 similar to those observed for the secretome from non-modulated WT MNs) and that of astrocytes (increase of GLT-1 and GFAP expression), beyond the elevation of microglial receptor CD206, associated with endocytic and phagocytic abilities. Thus, we can conclude that the decrease of miR-124 expression toward basal levels in mSOD1 MNs backup their capacity to sustain their appropriate function, while preventing a reactive glial response triggered to a secretome with immunostimulatory properties, thus counteracting neurodegeneration and disease progression. In conclusion, this work supports that the selective targeting of miR-124 in mSOD1 MNs may turn in a promising and broad therapeutic strategy for ALS treatment, by allowing the recovery of MN function and by preventing secretome-mediated glial reactivity, thus acting in multiple targets as usually intended with a combined therapy.A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa fatal que evolui de forma progressiva e que é caracterizada pela perda de neurónios motores (NMs) do córtex motor, tronco encefálico e medula espinhal (ME). Apesar de ser estudada há vários anos a ELA é uma doença ainda sem cura. Apenas existem duas terapias disponíveis para o seu tratamento, mas com pouco impacto na progressão da doença e na saúde dos doentes. Inicialmente pensava-se ser uma doença unicamente do NM, mas atualmente sabe-se que a ELA envolve também alterações nas células gliais. Estas apresentam diversas disfuncionalidades consideradas aberrantes, perdendo a sua função de suporte aos neurónios e muito contribuindo para a neurodegeneração e progressão da doença. Desconhece-se, contudo, quais os mecanismos de comunicação célula-célula que se encontram alterados e os mediadores envolvidos, quer solúveis, quer os contidos em vesículas extracelulares, como os microRNAs (miRNAs). Os miRNAs não codificantes têm como função suprimir a expressão de genes através da sua ligação a mRNAs. Nos últimos anos, os miRNAs têm sido indicados como possíveis biomarcadores para o diagnóstico da doença e como alvos terapêuticos na ELA. Determinados miRNAs foram já encontrados como estando diferencialmente expressos, entre os quais alguns com reconhecida associação com vias conhecidas ou sugeridas como estando envolvidas na patogénese da ELA. A sua ação pode ser dirigida a um tecido, ou a um tipo de célula, sendo também elementos ativos na comunicação celular, sendo que afetam a expressão génica tanto das células de onde derivam, como das células onde vão atuar. O miRNA (miR)-124, um dos mais abundantes no sistema nervoso central (SNC) e normalmente associado à diferenciação neuronal, foi recentemente identificado como estando elevado no líquido cefalorraquidiano de doentes com ELA, bem como sobre-expresso em ratinhos com a mutação G93A no gene da SOD1 (mSOD1) nos locais de visível neurodegeneração, tendo sido por nós observado na linha celular de NMs NSC-34 com a mesma mutação, uma das mais abundantes nos casos familiares de ELA. Curiosamente, verificámos que a sua expressão estava aumentada em exossomas libertados desses NMs, os quais evidenciaram produzir ativação de diversos subtipos de microglia. Em condições fisiológicas o miR-124 encontra-se envolvido na maturação neuronal, bem como na organização do citoesqueleto celular, comprimento das neurites e processos associados à autofagia da célula. Uma desregulação na expressão de miR-124 tem sido encontrada em diversas doenças do SNC e associada a neurodegeneração, stress, desregulação neuro-imune e tumores cerebrais, entre outras. Neste trabalho o nosso objetivo foi avaliar de que modo é que a inibição ou a sobre-expressão de miR-124 se refletia na estrutura e função dos NMs, bem como na imunorregulação da microglia e dos astrócitos. Para isso, usámos uma linha de NMs expressando SOD1 humana, quer não mutada (wild type, WT), quer com mutação G93A (mSOD1), de modo a mimetizar a acumulação desta proteína e a degeneração neuronal observadas na ALS. Para avaliar de que modo o secretoma de NMs WT, após tratamento com pre-miR-124, e o de NMs mSOD1 modulados com anti-miR-124, influenciava a reatividade da microglia após 4 h de incubação e a dos astrócitos após 24 h de tratamento, usámos a linha de células microgliais de ratinho N9 e os astrócitos isolados da ME de ratinhos transgénicos SOD1G93A, com 8 dias de idade. Qualquer uma das modulações não teve reflexo na viabilidade dos NMs e a acumulação de mSOD1 foi evitada com o anti-miR-124. Este mostrou também prevenir o aumento de expressão génica e proteica da High Mobility Group Box 1 (HMGB1) e da proteína inducible Nitric Oxide Synthase (iNOS), levando ainda a uma diminuição da expressão génica da S100 Calcium-binding Protein B (S100B) nos neurónios mutados. A sobre-expressão do miR-124 nos NMs WT apenas se traduziu num aumento de S100B mRNA. A nível dos miRNAs, o anti-miR-124 nos NMs mSOD1 foi capaz de diminuir a sobre-expressão de miR-125b e aumentar o miR-21 e o miR-146a, que se encontravam reduzidos. Curiosamente, o pre-miR-124 nos NMs WT conduziu ao perfil evidenciado pelas células mutadas, ou seja, a um aumento de miR-125b e a uma diminuição de miR-21 e de miR-146a, comprovando a sua contribuição em tal desregulação. Importante foi ainda observar que o anti-miR-124 reduziu a expressão do marcador pré-sináptico sinaptofisina e da proteína motora dineína, ambas elevadas nos NMs mSOD1 e WT tratados com pré-miR124, para valores próximos das células WT, ficando claro que o pre-miR-124 está por detrás de tais aumentos. Pelo contrário, este mostrou estar ligado à diminuição do marcador pós-sináptico Postsynaptic Density Protein 95 (PSD-95) e da proteína motora cinesina, já que o anti-miR-124 levou a um marcado aumento da expressão génica destas proteínas. Podemos então concluir que o anti-miR-124 favorece o transporte anterógrado (do soma para a extremidade do axónio), deficiente nas células mutadas, nas quais a elevação da dineína privilegia o transporte retrógado (de volta ao soma) associado à elevação da expressão de miR-124. Esta, mostrou estar também interligada com o menor número de ramificações e de neurites primárias, bem como do seu comprimento, as quais foram corrigidas para valores próximos dos do controlo pelo anti-miR-124 nos NMs mutados, assim indicando a sua influência no citoesqueleto celular, morfologia e funcionalidade dos NMs. De facto, o anti-miR-124 foi capaz de recuperar a viabilidade mitocondrial nos NMs mSOD1, cuja diminuição poderá estar relacionada com o aumento de expressão de miR-124, bem como ainda de favorecer a expressão de Mitofusina (que induz fusão da mitocôndria), a qual se encontrava diminuída nas células mutadas, bem como diminuir a de DRP1 (associada à fissão da mitocôndria) que estava aumentada nas mesmas. Mais uma vez a sobre-expressão de miR-124 mostrou ser responsável pelo aumento de DRP1 observado nas células mutadas, se bem que não evidenciou associação com a Mitofusina. Tais modificações são sugestivas da existência de stress oxidativo associado à sobre-expressão de miR-124 e que o anti-miR-124 poderá atuar de forma benéfica por favorecer mecanismos anti-oxidantes e protetores da mitocôndria. Relativamente à ação imunoreguladora, verificámos que tanto o aumento da área, perímetro e diâmetro de Ferrett’s, como a diminuição de circularidade da microglia após tratamento com o secretoma dos NMs mutados, não se detetavam quando a célula era tratada com o secretoma proveniente dos modulados com o anti-miR-124, sendo que tais parâmetros mostraram correlação direta com a sobre-expressão deste miRNA. De realçar, que a capacidade fagocítica da microglia revelou ser afetada quando exposta ao secretoma dos NMs WT tratados com pré-miR-124, ou ao proveniente dos mSOD1 não modulados ou modulados com anti-miR-124, indicando que este não é suficiente para repor a capacidade fagocítica da microglia. Contudo, foi de novo muito evidente a eficácia da utilização do anti-miR-124 nos NMs mutados quando o seu secretoma se mostrou eficaz na diminuição da expressão génica de iNOS, arginase, Tumor Necrosis Factor alpha (TNF-α) e Interleukin 1 beta (IL-1β), todas elas aumentadas tanto na microglia tratada com o secretoma proveniente dos NMs mSOD1, como na tratada com o de NMs WT modulados com pre-miR-124. Ainda, conseguiu diminuir tanto a expressão de HMGB1, muito aumentada pelo secretoma de NMs mSOD1, como elevar a expressão do recetor da manose, o CD206, associado à endocitose e fagocitose nos macrófagos. Curiosamente, encontrámos aqui também uma correlação, desta vez indirecta, com a sobre-expressão neuronal do miR-124, cujo secretoma decresce a expressão de CD206. Quanto ao efeito do secretoma de NMs mSOD1 tratados com anti-miR-124 nos astrócitos WT e mSOD1, verificou-se que ele foi capaz de preservar a expressão do glutamate transporter 1 (GLT-1), encontrado diminuído nos astrócitos WT e mSOD1 após incubação com o secretoma dos NMs WT tratados com pre-miR-124 e com o dos mutados não tratados. Efeito semelhante foi obtido para a Glial Fibrillary Acidic Protein (GFAP), cuja expressão aumentou relativamente aos astrócitos WT e mSOD1 tratados com o secretoma dos NMs mSOD1, bem como aos WT expostos ao secretoma dos NMs WT tratados com pre-miR-124. Pode-se então pensar que a expressão elevada do miR-124 no NM mutado é, pelo menos em parte, responsável pela produção de um secretoma que determina a diminuição da expressão de GFAP e de GLT-1, uma característica dos astrócitos aberrantes na ALS. Em suma, os resultados obtidos sugerem que a sobre-expressão do miR-124 nos NMs mSOD1 se encontra envolvido na disfuncionalidade e degeneração destas células e contribui marcadamente para a patogénese e progressão da ALS ao comprometer a homeostasia celular em resultado de um secretoma com propriedades imunoestimuladoras e indutor de características aberrantes nos astrócitos, quer em células saudáveis como em mutadas. O anti-miR-124 revelou ter efeitos benéficos na prevenção da acumulação de mSOD1 nos NMs, exercendo ação reguladora da transmissão sinática (aumento da árvore neurítica, da cinesina e da PSD-95), anti-oxidante (maior viabilidade mitocondrial, aumento de Mitofusina e diminuição de DRP1) e anti-inflamatória (subida de miR-21 e miR-146a, com diminuição de miR-125b). Para além disso, os NMs mSOD1 modulados com anti-miR-124 revelaram produzir um secretoma que suporta a funcionalidade da microglia (manutenção de uma morfologia normal e expressão de iNOS, Arginase, TNF-α e IL-1β e HMGB1 semelhante à encontrada após incubação com o secretoma de NMs WT), bem como a dos astrócitos (aumenta a expressão de GLT-1 e de GFAP), para além de elevar a expressão do recetor microglial CD206, associado à endocitose e à fagocitose. Desta forma, podemos inferir que a diminuição da expressão de miR-124 para níveis basais nos NMs mutados sustenta a sua funcionalidade e previne a reatividade das células gliais mediada por um secretoma com propriedades imunoestimuladoras, contrariando a neurodegeneração e a progressão da doença. Em conclusão, este trabalho sugere que o targeting do miR-124 especificamente nos NMs mSOD1 poderá ser uma estratégia terapêutica muito promissora e abrangente para o tratamento da ELA, ao permitir não só a recuperação da funcionalidade neuronal, mas também a prevenção da ativação das células gliais, significando a sua atuação em diversos alvos à semelhança do pretendido com a terapia combinada por associação de fármacos.This work was funded by Research Grant of the Santa Casa Scientific Research Program on ALS (SCML-Project ELA-2015-002 to DB), by Fundação para a Ciência e a Tecnologia (FCT), (UID/DTP/04138/2013 to iMed.ULisboa and PTDC/MED-NEU/31395/2017 to DB) and by Programa Operacional Regional de Lisboa and Programa Operacional Competitividade e Internacionalização (LISBOA-01-0145-FEDER-031395 to DB)

Experimental Study of a Single Droplet Impinging upon Liquid Films: Jet Fuel and Biofuel Mixtures

The present work is focused on a single droplet impinging upon a liquid film of the same fluid. This particular study is a matter of interest for several research areas and has a wide variety of applications such as fuel injection in internal combustion engines and processes involving spray paints, coatings and systems cooling. The human being started searching for new alternatives to reduce pollution, and since transports contribute with a significant portion, it is extremely necessary to bet on alternatives to fossil fuels. The introduction of biofuels in the aviation sector could be an example. The huge challenge is to modify and optimize piston engines to operate efficiently with alternative fuels. In order to achieve that, in these experiments, Jet Fuel and Biofuel mixtures were used. The main goal of this dissertation is to visualize the dynamic behavior of single droplets impinging upon liquid films with different relative thicknesses, several outcomes are possible. To accomplish that, four fluids were used: water (as reference), 100% Jet A-1, 75%/25% and 50%/50% mixtures of Jet A-1 and NEXBTL, respectively, since civil aviation only accept mixtures with at least 50% Jet Fuel in volume. To assure the accuracy of the calculations, the fluids physical properties were measured. An experimental facility was designed and built, and the setup includes a high-speed digital camera that was manually triggered with a specific exposure time. The impact site was illuminated by a led lamp through a diffusion glass to provide uniform back lighting. A syringe pump connected to the needle released the droplets with a specific pumping rate. The liquid film is held by a perspex container. Five needles were used with different inner diameters to yield five distinct droplet sizes for each fluid. Additionally, three impact heights were established to provide three impact velocities and Weber numbers for each needle. The liquid films depths considered were 10%, 50% and 100% of the droplet diameter. The existence of splash was reported as well as its characteristics. Some conclusions about the influence of the impact conditions and the fluids physical properties were indicated. Using the obtained data comparisons were made with some splashing thresholds available in the literature.O presente trabalho foca-se no impacto de uma única gota com um filme de líquido do mesmo fluido. Este estudo particular tem interesse para várias áreas de pesquisa e tem uma grande variedade de aplicações tais como injeção de combustível em motores de combustão interna e processos que envolvem pintura a spray, revestimentos e arrefecimento de sistemas. O Ser Humano começou a procurar novas alternativas para reduzir a poluição e visto que os transportes contribuem com uma porção significativa é extremamente necessário apostar em alternativas aos combustíveis fósseis. A introdução de biocombustíveis no sector da aviação poderia ser um exemplo. O grande desafio passa então por modificar e otimizar motores a pistão de forma a operarem eficientemente com combustíveis alternativos. De forma a alcançar isso, nestes ensaios experimentais foram usadas misturas de Jet Fuel e Biocombustível. O principal objetivo desta dissertação é visualizar o comportamento dinâmico do impacto de gotas únicas com filmes de líquido com diferentes espessuras relativas, vários resultados são possíveis. Para o obter foram usados quatro fluidos: água (como referência), 100% Jet A-1 e misturas de 75%/25% e 50%/50% de Jet A-1 e NEXBTL, respetivamente, visto que na aviação civil só são aceites misturas com no mínimo 50% de Jet Fuel em volume. Para garantir cálculos precisos as propriedades físicas dos fluidos foram medidas. Uma montagem experimental foi idealizada e construída. A instalação inclui uma câmara digital de alta velocidade que foi manualmente acionada com um tempo de exposição específico. O local de impacto foi iluminado por uma lâmpada led através de um vidro difusor de forma a fornecer uma luz uniforme de frente para a câmara. Uma bomba infusora foi conectada à agulha e libertava as gotas a uma taxa de bombeamento específica. Um recipiente de perspex conteve o filme de líquido. Foram utilizadas cinco agulhas com diâmetros internos diferentes para produzir cinco diâmetros de gota diferentes para cada fluido. Adicionalmente, foram estabelecidas três alturas de impacto para proporcionar três velocidades de impacto e três números de Weber para cada agulha. Foram consideradas espessuras do filme de líquido de 10%, 50% e 100% do diâmetro da gota. A existência de splash foi reportada, assim como as suas características. Algumas conclusões sobre a influência das condições de impacto e das propriedades físicas das substâncias foram indicadas. Usando os dados obtidos foram feitas comparações com os limites de splashing disponíveis na literatura

How to promote healthy eating habits in children

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and EconomicsChildhood’s overweight and obesity are a worrying issue in the world nowadays. The purpose of this study was to provide contributions to the promotion of healthy food by analyzing the impact of physical activity, parents’ influence and home meals frequency on children’s food choices. Structured questionnaires were used and were answered by 172 children between 10 and 14 years old and by their respective parents. Children and parents preferred healthy food vs. non-healthy food presenting the children’s healthy food choices a mean of 4.26 and the parent’s healthy food choices a mean of 4.47 in a scale ranging from 0 to 6. Our results also show that physical activity, parent’s education and home meals frequency did not have an impact on children’s food choices, contrasting to the sedentary behavior and parents’ choices which had a negative and positive correlation, respectively, with children’s food choices. Taking these results into account and using them to advise parents and companies, we underline that parents must guarantee an adequate children’s nutrition after doing physical efforts and control the time children watch TV and play computer games and companies may create marketing campaigns and educational programs in order to promote healthy food, improve children’s eating habits and reduce the childhood obesity prevalence

CHEMICAL COMPOSITION OF SELECT SASKATOON BERRY VARIETIES WITH AN EMPHASIS ON PHENOLICS

In this study, three saskatoon berry varieties (Martin, Northline and Pembina) grown in Saskatchewan, Canada were analyzed for their physicochemical properties (berry size, colour, pH and % seeds), proximate composition, amino acids, major carbohydrates/polyols/galacturonic acid, major minerals, oligosaccharides, organic acids and phenolics. In addition, the phenolic subclass composition and antioxidant activities of whole fruit and pomace from commercial and laboratory scale juice production, and aqueous alcohol fractions were determined. Fruit varieties were found to differ in colour and size but showed similar pH and °Brix values. Proximate analysis results ranged from 80.18-82.79% for moisture, 7.39-10.82% for carbohydrate, 1.13-1.79% for protein, 0.28-0.48% for lipid, 4.23-9.42% for total dietary fibre, and 0.53-0.74% for ash. Major carbohydrates and polyol identified were fructose, glucose, and sorbitol. This work represents only the second report of the detection and quantitation of sorbitol in this fruit. Oligosaccharide profiles were determined by high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary gas chromatography with flame ionization detection (CGC-FID) and showed the presence of a number of dextrose (DP2-5) and pectin polymers. Oligosaccharide profiles have not been reported previously. Amino acid contents ranged from 0.83-1.22 g/100 g fresh weight (FW), with arginine, aspartic acid/asparagine, glutamic acid/glutamine and leucine predominating. Major minerals quantified were calcium, magnesium, potassium and sodium, with potassium having the highest concentration that ranged from 219-248 mg/100 g FW. The major organic acids identified were malic (304.7-393.9 mg/100 g FW) and succinic (120.4-316.3 mg/100 g FW). Phenolics from the three fruit varieties were extracted employing water, ethanol:formic acid:water, and methanol:formic acid:water (70:2:28 v:v) mixtures. The ethanol:formic acid:water (EFW) extracts from all samples were found to have the highest phenolic concentrations as determined by total phenolic content analysis. Based on total phenolic chromatographic index (TPCI) results as determined by high performance liquid chromatography with photodiode array detection (HPLC-PDA), the Northline variety had the highest TPCI at 504.2 mg/100 g FW. This variety was also shown to have the highest antioxidant activities by both the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2´-azinobis-3-ethylbenzthiazoline-sulphonic acid (ABTS) radical scavenging assays, of 23.1 1/IC50/100 mg FW and 327.5 mM TEAC/100 mg FW, respectively. Solid phase extraction (SPE) using Amberlite® XAD16N resin and aqueous ethanol (40, 70 and 100%) was employed to produce phenolic fractions from the three fruit varieties. It was found that hydroxybenzoic acids eluted in the 40% ethanol fraction; hydroxycinnamic acids and anthocyanins eluted in the 70% ethanol fraction; and anthocyanins, flavanols and flavonols eluted in the 100% ethanol fraction. The 70% ethanol fraction had the highest TPCI and DPPH/ABTS radical scavenging abilities for all saskatoon berry varieties. Wet and dry pomace from commercial saskatoon berry juice production had TPCI values of 404.2 mg/100 g and 250.0 mg/100 g, respectively. The ABTS values of wet and dry pomace were found to be 304.8 and 327.8 mM TEAC/100 mg, while the DPPH results were 19.4 and 16.8 1/IC50/100 mg FW, respectively. These results show that pomace from commercial juice production was a good source of phenolics with high antioxidant capacities. Results from laboratory scale juice production of the Northline variety employing commercial conditions (i.e. time, temperature, and enzymes and dosages) showed that 29% of the phenolics remained in the pomace after juice production as determined by TPCI analysis