Conservation of Lactuca watsoniana Trelease, an Azorean priority species : phylogenetics, population genetics and propagation

Tese de Doutoramento, Biologia, 28 de maio de 2018, Universidade dos Açores.Lactuca watsoniana Trel., é uma herbácea perene, endémica do Arquipélago dos Açores. Localmente denominada de “Alfacinha”, pode ser encontrada em quatro das nove ilhas do Arquipélago dos Açores (Faial, Pico, Terceira e São Miguel), estando possivelmente extinta da ilha de São Jorge. O tamanho total estimado das populações é de 500 a 2000 indivíduos, mas observações recentes no campo indicam que poderão ser menos de 500 indivíduos. Considerada como taxon prioritário na Directiva Habitats (Annex B-II) bem como na Convenção de Berna em termos de conservação e está listada como ameaçada (EN B2ab (i, ii, iii); C2a(i)) na Lista Vermelha da IUCN 2017. Atualmente, a Lactuca watsoniana encontra-se restrita a zonas húmidas, ravinas profundas e estreitas, crateras zonas declivosas, prados naturais de Festuca, matos de Erica e florestas de Juniperus entre 600-800 m. Está ameaçada por perda e degradação de habitat, resultante de expansão dos pastos, invasão por espécies exóticas, introdução de herbívoros e distúrbio de áreas sensíveis por turistas e locais. Neste trabalho investigaram-se os seguintes tópicos: i) propagação vegetativa por sementes; ii) genética de populações; e iii) filogenética e filogeografia. Foi estabelecido um protocolo eficaz e exequível para propagação por via seminal, germinando as sementes com 0.1 mg l-1 GA3, ethephon, e um filtro de luz vermelha e um regime de temperatura alterna de 15/10°C, com um fotoperíodo de 12 horas. Este protocolo permitiu a produção de centenas de plântulas viáveis que foram replantadas na população de origem. Dada a necessidade de proceder ao reforço das populações naturais em algumas ilhas, pretendeu-se definir se existiam riscos de contaminação genética resultante da troca de material entre as diferentes ilhas, para isso estudou-se a estrutura e variabilidade genética das populações, usando 8 microssatélites nucleares, desenvolvidos especificamente para a L. watsoniana. Os resultados mostraram uma clara separação entre as várias ilhas dos Açores, estando a maior variação genética dentro das populações (55.0%), mas com níveis relativamente altos de variação entre os clusters (27.3%). Evidenciando um grau de diferenciação entre as populações elevado e fluxo genético com valores inferiores a 1, bem como a existência de isolamento e barreiras geográficas. De modo a clarificar a posição taxonómica da L. watsoniana, nomeadamente em relação a espécies próximas da Europa, Norte da América e de outros Arquipélagos da Macaronésia (Canárias), recorremos a marcadores moleculares nucleares (ITS) e quatro do cloroplasto. Na análise das sequências combinadas de ITS e do cloroplasto (Parsimónia, Máxima verosimilhança e Inferência Baeysiana) o taxon dos Açores, mostrou a existência de um padrão filogeográfico através do arquipélago, com a presença de 5 haplótipos distintos para a L. watsoniana, quatro endémicos em apenas uma ilha (Faial, Pico, Terceira e São Miguel) e um haplótipo com informação genética partilhada do Faial e Pico, bem como uma relação filogenética, bem suportada, com as Lactuca Norte Americanas.ABSTRACT: Lactuca watsoniana Trel., is a perennial herb, endemic to the Azores archipelago. Locally called "Alfacinha", the species is restricted to only four of the nine islands of the archipelago (São Miguel, Terceira, Pico and Faial) and is possibly extinct in São Jorge Island. Estimates of its total population size are 500 to 2,000 individuals, but recent field observations indicate that it can be fewer than 500 individuals. It is therefore considered a priority species for conservation and was listed as endangered on the IUCN Red List 2017. Furthermore, it was included in Annex II of the EC Habitats Directive and is protected under the Berne Convention. Lactuca watsoniana is currently restricted to the steep slopes of craters and ravines in temperate juniper rain forest between 600-800 m of altitude. The species is threatened by habitat loss and degradation resulting from changes in land use, namely expansion of pasture, invasion by exotic species, consumption by introduced herbivores and disturbance of sensitive areas by tourists and locals. In this thesis, we investigated the following topics: i) seed propagation; ii) population genetics; and iii) phylogenetics and phylogeography. We established and efficient and feasible seed propagation protocol by germinating the seeds germinate seeds with 0.1 mg l-1 GA3, ethephon, a red-light filter and a temperature regime of 15/10°C, and 12 hours’ photoperiod. This protocol allowed producing hundreds of viable seedlings that were reintroduced in the source population. Given the need to reinforce natural populations in some of the islands, we aimed to find out if any outbreeding risks existed, resulting from material exchanges between the different islands. For that, we studied the genetic structure and variability of the populations, using eight nuclear microsatellites, developed specifically, for Lactuca watsoniana. Our findings showed a clear separation among the islands, with the largest proportion of genetic variation found within populations (55.0%) but with relatively high levels of genetic variation both among clusters (27.3%). Furthermore, a high variability between the populations, a gene flow value below one, and the presence of geographical isolation and barriers were estimated. In order to clarify the taxonomic position of L. watsoniana, namely the phylogeographic and phylogenetic relationship with the morphologically similar species from Europe, North America and the geographically close taxa from the Canary Islands, we used one nuclear region (ITS) and four chloroplast markers. Parsimony, Maximum likelyhood and Bayesian Inference analyses were conducted and revealed a strongly supported monophyletic Lactuca clade, and a clear separation of Macaronesian endemic Lactuca in sister clades. Lactuca watsoniana, endemic to Azores, showed a phylogeographic pattern across the archipelago, with the presence of five different haplotypes for L. watsoniana: four single island endemics (Faial, Pico, Terceira e São Miguel) and one occurring on both Faial and Pico and a strongly supported phylogenetic relationship with North America Lactuca.Fundo Regional da Ciência pelo financiamento da Bolsa de Doutoramento (M3.1.2/F/032/2011 e FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE

Successful isolation and cultivation of Cylindrospermopsis raciborskii strains isolated from finished drinking water samples

This work presents the successful establishment of Cylindrospermosis raciborskii cultures isolated from water samples collected at the exit point of Water Treatment Plant (WTP). An intense bloom dominated by filamentous cyanobacteria (Aphanizomenon spp., Planktothrix spp., Cylindrospermosis raciborskii, Anabaena spp.) occurred in the summer of 2015 in Roxo reservoir (south Portugal). Several cyanotoxins (microcystins, saxitoxins, cylindrospermopsin) were detected in raw and treated water, although at levels below the corresponding regulatory and/or guideline values. Nevertheless, this bloom caused intense unpleasant odour and taste in the water supplied to the populations and cyanobacterial cells (up to 1000 cells.mL-1) were detected in finished water samples collected at the exit point of WTP. Treated water samples collected at the WTP and at the city water deposit were inoculated in Z8 culture medium and cyanobacterial growth was followed by optical microscopy. After 30 days, cyanobacterial growth was observed showing resistance to the treatment processes and maintenance of reprodution capacity. Interestingly, morphometric and molecular analysis revealed the presence of C. raciborskii. Three isolates of this species were obtained and none were cylindrospermopsin- or microcystins-producers, as confirmed by Enzyme Linked Immunosorbent Assays (ELISA) and by amplification of genes (PS, PKS, mcyA-cd, mcyAB, mcyB) involved in those cyanotoxin synthesis. However, the ELISA for saxitoxins was positive for the 3 isolates and confirmation of this toxin production is in progress. To our knowledge, this is the first report on the establishment of successful cultures of C. raciborskii that survived to conventional water treatment processes.N/

Impact of European Structural Funds used on competitiveness and financing of portuguese Small-Medium enterprises

The objective of this paper is to empirically determine the impacts of European Structural Funds used on the competitiveness and financing of Portuguese Small-Medium Enterprises. Some empirical studies have already been carried out regarding the impact of European Structural Funds in several European countries, especially in macroeconomic terms, but the analysis focused on the microeconomic impact of Small and Medium-sized companies has been less studied. This paper fills that gap, particularly in the case of Portugal, focusing on eligible Small-Medium Enterprises for Portugal 2020. Using a panel data period methodology and considering an unbalanced sample of 228 eligible firms for Portugal 2020 for the period 2015-2019, the results indicate that Small-Medium Enterprises financed by European Structural Funds tend to present a better performance in certain parameters and, consequently, these funds seem reasonable financing option for companies eligible for Portugal 2020. This paper gives a contribution to clarify which are the impact of European Union Structural Funds on Small-Medium Enterprises competitiveness, which is an important issue not only for managers but for all the stakeholders.O objetivo deste artigo é analisar empiricamente o impacto dos Fundos Estruturais Europeus utilizados no financiamento e competitividade das pequenas e médias empresas portuguesas. Algum estudo empírico foi já efetuado relativamente ao impacto dos Fundos Estruturais Europeus em vários países europeus, especialmente em termos macroeconómicos, mas a análise centrada no impacto microeconómico nas pequenas e médias empresas tem sido aparentemente menosprezada. Este artigo procura preencher, particularmente para o caso de Portugal, focando-se nas pequenas e médias empresas elegíveis para Portugal 2020. Utilizando uma metodologia de dados em painel e considerando uma amostra não equilibrada de 228 empresas elegíveis para o Portugal 2020 no período 2015-2019, os resultados indicam que as pequenas e médias empresas financiadas pelos Fundos Estruturais Europeus, neste caso o Portugal 2020, tendem a apresentar um desempenho superior em certos parâmetros e, consequentemente, estes fundos aparentam ser uma opção de financiamento que as empresas com projetos elegíveis deverão ativamente considerar. Assim, os resultados deste trabalho contribuem para esclarecer o impacto positivo dos Fundos Estruturais da União Europeia na competitividade das pequenas e médias empresas, o que é uma questão relevante não apenas para os gestores, mas para a própria sociedade em geral

Shared e-scooters and the promotion of equity across urban public spaces - a case study in Braga, Portugal

Shared e-scooters were introduced in urban public spaces as a way to promote a modal shift from cars in short-distance trips, as well as to improve sustainability, resilience, and equity in urban transport. However, the expansion of shared e-scooter services in 2019 proved that this mode of transport, without integrated planning strategies, can bring some problems to cities, which are related to the illegal parking of e-scooters, an increase in head injuries, and the lack of population diversity among users. Regarding the latest, this research work aims at conducting a case study in the city of Braga, Portugal to reveal who the actual and potential users of shared e-scooters are and how their socioeconomic profile (e.g., gender, age, income range, literacy, occupation) and usage patterns related to the ones found in other cities. For this, a revealed preference survey was deployed on the case study site, and the respondents’ profiles were statistically correlated with the socioeconomic characteristics of the city’s general population in order to assess if this mode of transport provides an equitable service. Results show that shared e-scooters are not equally used by people of different genders, ages, and income ranges. Information on e-scooter usage inequalities across the population is useful for the city to proceed with more socially equitable mobility policies.This research was funded by Fundação para a Ciência e a Tecnologia, grant number 2020.05041.BD

The role of shared E-Scooter systems in urban sustainability and resilience during the COVID-19 mobility restrictions

Shared e-scooter systems were first introduced in 2017 and have since been spreading around the world as a sustainable mode of transport. The success of this mode is also due to new urban mobility strategies and plans, such as the European Sustainable and Smart Mobility Strategy, which relies on non-pollutant modes. To display the range of effects that can be achieved in urban mobility through the proper implementation of shared e-scooter systems, a systematic literature review and a case study were performed. It was found that this shared system can help cities with environmental issues, such as reducing air pollution, reducing inequality in access to transport, promoting money-saving, and improving mobility resilience. During the Covid-19 pandemic, shared e-scooters became a great asset in many cities worldwide, because they promote social distancing and help cities not to rely only on private cars to replace public transport rides, especially for short-distance trips. In the case study of Braga, it was found that the city still relies on shared e-scooter modes as a mobility option after the pandemic, also promoting special fares for people to start using the service.This research was funded by Fundação para a Ciência e a Tecnologia, grant number 2020.05041.BD

The Kidney Vero-E6 Cell Line: A Suitable Model to Study the Toxicity of Microcystins

Microcystins (MCs) are toxins produced by cyanobacteria from water environments that can induce acute and chronic effects on humans and animals, after ingestion/contact with contaminated water. This group of cyclic heptapeptides comprises approximately 80 variants, being microcystin-LR (MCLR) the most frequent and toxic variant. The studies of MCs effects on cell lines often lead to contradictory results, given the fact that distinct MC toxicity endpoints (mainly cytotoxicity and genotoxicity) have been studied in diverse cell lines (and cell clones) under distinct exposure conditions (different doses-ranges, time of exposure, MCs variants, etc). In our work with Vero-E6 cells we tested MCLR (both pure toxin and from cyanobacterial extracts of M. aeruginosa) within a wide range of concentrations (1 nM- 200 μM), using several endpoints and methodologies (cytotoxicity, morphology, genotoxicity, protein expression). In this chapter we will summarize our results and discuss the utility of Vero-E6 cell line to evaluate the toxicological properties of MCLR

In vitro germination of four herbaceous species endemic to the Azores archipelago

Current projects aimed at the conservation of endemic species and restorations of native habitats require information about seed viability, held in a germplasm bank, and ex situ seed and plant production. In this study, we investigated: a) the viability of Spergularia azorica Lebel seeds after 18 years of storage, and the ability of the developed plants to produce viable seeds in ex situ conditions; b) the viability of Leontodon filii (Hochst. ex Seub.) Paiva & Ormonde seeds after seven years of storage; c) the germination characteristics of in situ harvested seeds of Luzula purpureo-splendens Seub.; and d) the feasibility of harvesting seeds from transplanted flowering Bellis azorica Hochst. plants to initiate in vitro cultures on Murashige and Skoog (MS) medium. For each of these four species, we estimated the percentage of germination, the number of days for radicle emergence, and the mean time to germination in different experimental conditions. The best germination percentages obtained were: a) 81.6 % for Spergularia azorica 18 year-old seeds and 97 % for the next generation of ex situ-produced seeds; b) 91% for Leontodon filii after seven years’ storage; c) 73% for in situ harvested seeds of Luzula purpureo-splendens; and d) 81% for Bellis azorica ex situ-produced seeds. Also, in vitro cultures of Bellis azorica were initiated on MS medium

Falar, Ler e Escrever Notícias com as crianças no Jardim de Infância

O presente relatório surge no âmbito do Mestrado em Educação Pré-Escolar e Ensino do 1.º Ciclo do Ensino Básico e encontra-se dividido em duas partes. A primeira parte refere-se à prática de ensino supervisionada em contexto de educação de infância e a segunda parte à prática de ensino supervisionada em contexto de 1.º ciclo. Na primeira parte abordam-se assuntos relacionados com a educação de infância nomeadamente, o papel das dimensões pedagógicas: o tempo e o espaço e o ciclo de ação pedagógica. A metodologia de trabalho por projeto é vivenciada em contexto de Jardim de Infância e por isso, apresenta-se um breve relato sobre o projeto e uma reflexão acerca das aprendizagens realizadas com as crianças, como também o papel da documentação pedagógica como suporte à aprendizagem. Ainda nesta parte, apresenta-se um estudo de caso realizado em torno da temática da educação para os media, com um grupo de quatro crianças com idades compreendidas entre os 3 e os 6 anos. Este estudo de índole qualitativo visa conhecer as conceções das crianças do pré-escolar acerca do universo dos media, especificamente, as notícias, seus meios de difusão com vista a uma ação pedagógica sustentada na investigação-ação. Na generalidade, os resultados apontam para a importância de o educador de infância potenciar precocemente aprendizagens em torno dos media, desenvolvendo a sensibilidade e conhecimentos que permitam alargar os conhecimentos das crianças, expandindo o seu universo de referências, enquanto consumidores e produtores de texto noticioso. Na segunda parte, respeitante à dimensão reflexiva em contexto de 1.º ciclo, apresenta-se um relato reflexivo em torno de uma experiência interdisciplinar realizada com uma turma de 3.º ano de escolaridade

Relatório de estágio auditoria financeira

Num cenário marcado pela instabilidade e globalização da economia, as entidades encontram-se, cada vez mais, expostas a uma grande diversidade de riscos que colocam em causa os objetivos pré-estabelecidos, existindo por isso uma grande variedade de interessados na produção e no uso de Demonstrações Financeiras auditadas. Neste contexto e atual paradigma, o revisor oficial de contas na sua função de utilidade pública, assume papel de destaque. Dotados de informação priveligiada pelo conhecimento das entidades e dos mercados que operam, os revisores oficiais de contas revestem a informação finaceira com uma credibilidade acrescida, o que pode também sujeitar a sua atividade a diversos riscos. O presente relatório tem por base um estágio curricular no âmbito do Mestrado em Auditoria, lecionado no Instituto Superior de Contabilidade e Administração do Porto, tendo como principal objetivo descrever os objetivos e procedimentos exigidos na concretização da atividade de auditoria financeira, no âmbito do esclarecimento e abordagem de todos os aspetos essenciais para o desenvolvimento do trabalho de um auditor externo. Encontra-se dividido em quatro partes. Iniciando-se com um enquadramento teórico do processo de auditoria financeira, passando pelo seu planeamento, materialidade, risco de auditoria, e a sua finalidade, a emissão da Certificação Legal de Contas. Posteriormente procurou-se demonstrar a importância e impacto do controlo de qualidade, terminando com uma apresentação da Empresa acolhedora e todas as atividade desempenhadas ao longo do estágio. A última parte do presente relatório é referente às conclusões obtidas.In a overview marked by the instability and globalization of the economy, entities are increasingly exposed to a great diversity of risks that jeopardize the pre-established objectives, therefore there is a wide variety of stakeholders in the production and use Audited Financial Statements. In this context and current paradigm, the statutory auditor in his public utility role, assumes a prominent role. Endowed with privileged information due to the knowledge of the entities and the markets they operate, statutory auditors provide financial information with added credibility, which can also subject their activity to various risks. This report is based on a curricular internship in the scope of the Master in Auditing, taught at the Higher Institute of Accounting and Administration of Porto, having as main objective to describe the objectives and procedures required in the accomplishment of the financial audit activity, in the scope of clarification and approach. all aspects essential to the work of an external auditor. It is divided into four parts. Starting with a theoretical framework of the financial audit process, going through its planning, materiality, audit risk, and its purpose, the issuance of the Legal Certification of Accounts. Subsequently, an attempt was made to demonstrate the importance and impact of quality control, ending with a presentation by the welcoming Company and all the activities performed during the internship. The last part of this report refers to the conclusions reached