DEBA - Teses de Doutoramento

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  • ItemAcesso Restrito
    Threats to the native fish community of the Vilariça River: agricultural activities and the Baixo Sabor Hydroelectric Scheme
    2023-02-17 - Santos , Regina Maria Bessa; Pacheco, Fernando António Leal; Fernandes, Luis Filipe Sanches; Monteiro, Sandra Mariza Veiga
    The freshwater biota in Mediterranean rivers is subject to impacts such as river fragmentation and eutrophication due to hydropower plants and intensive agriculture. The loss of fish biodiversity, whether potamodromous or diadromous, is a mirror of those impacts. Thus, it was selected as a case study of the Sabor River catchment, particularly the Vilariça River, in order to evaluate the effects of human activities, namely river regulation and agricultural activity, on river water quality and the health and life cycles of two target potamodromous species of the Douro River catchment: the Iberian barbel (Luciobarbus bocagei) and the Douro nase (Pseudochondrostoma duriense). The work was divided into three stages. In the first stage, the impact of river damming on water quality and aquatic fauna was assessed in the Sabor River (Chapter 2). Changes in water quality were marked by increases in temperature and electric conductivity downstream of dams and by the input of phosphorus and nitrogen into the two reservoirs, which triggered a growth of algae, an increase in chlorophyll-a, and a drop in water transparency. The consequences of water quality deterioration for aquatic fauna were severe, marked by abrupt declines of native fish species and the invasion of exotic species. The ecological status, determined from ecological quality ratios, changed from good-fair in the unaffected watercourses to fair-poor in the regulated streams. The second stage of the work, developed in the Vilariça River, consisted of relating water quality to agricultural practices and the health status of the fish (Chapters 3, 4, and 5). The water was classified as polluted and extremely polluted in summer and winter, respectively. In summer, the values of physicochemical parameters and metals in the water may be caused by less flow and excessive agricultural fertilization, which reached the river through irrigation and leaching, and in winter by the erosion of soil particles with associated metals. The pronounced and severe histopathological changes observed in both species, such as aneurism, exudate, and hypertrophy in the gills and biliary duct epithelial detachment, hyperplasia of the biliary epithelium, congestion of blood vessels, and degenerative vacuolization in the liver, were statistically related to the poor water quality. The Integrated Biomarker Response, computed from histopathological changes identified in the gonads, gills, and liver, showed higher values in the summer and in the middle and downstream sections of the river compared to the winter season and the upstream section, which is in agreement with the spatiotemporal distribution of concentrations of metals in the river water. In the third stage, also developed in the Vilariça River, the fluvial rehabilitation measures for the reproduction conditions of L. bocagei were evaluated (Chapter 6). The results showed that migration and spawning were improved with incremental flow from a bypass system created for this purpose. Installed submerged weirs also contributed to the success of spawning since they facilitated the upstream migration and the establishment of spawning and resting areas. Additionally, to assess the spawning activity of L. bocagei, a spawn-per-unit-effort (SPUE) methodology was developed. It was proposed to integrate this methodology into the monitoring plan of the compensatory measures of the Vilariça River because it will provide greater knowledge of the spawning activity of L. bocagei, which is essential for improving conservation efforts. In addition, it presents a set of mitigation and management measures directed to the conservation of fish communities in Mediterranean streams in order to reduce the negative impact of agricultural contamination on water quality and fish health, such as improving the irrigation methods, preserving the vegetation cover, promoting reforestation in degraded and burned areas, and improving the riparian vegetation.
  • ItemAcesso Embargado
    Evaluation of the effects of glyphosate association with copper, zinc, and manganese metals on zebrafish development
    2023-03-03 - Lanzarin, Germano Augusto Bacin; Monteiro, Sandra Mariza Veiga; Venâncio, Carlos Alberto E Silva; Félix, Luís Manuel Lourenço
    The use of herbicides with glyphosate as an active ingredient (a.i.) has increased dramatically in recent years being currently the most used in the world. Due to its inappropriate use, its residues have often been found in soil and water, raising concerns about its potential harmful effects on ecosystems. Recent data indicate that glyphosate can cause adverse effects in vertebrates, although these have been attributed to the presence of adjuvants in commercial formulations and not to the active ingredient. In any case, the underlying ecotoxicity mechanisms are not fully understood. Thus, the evaluation of the toxic effects of glyphosate in the environment and its impact on humans is a priority in ecotoxicology research. The studies developed in this thesis used the zebrafish (Danio rerio) as an animal model. This species has been frequently used in the evaluation of the toxicity profile of compounds in vivo. Early stages of zebrafish development offer a model that fits within the scope of the 3Rs policy, being an alternative to animal experimentation in biomedical and ecotoxicological research. Thus, in the introduction to this thesis (Chapter I), a literature review of the evidence regarding the toxic effects of pure glyphosate or glyphosate-based herbicide (GBH) on embryonic zebrafish stages was elaborated. There are differences in the literature regarding the toxic effects associated with exposure to glyphosate versus GBHs. Additionally, the impact of exposure to GBHs in the early stages of zebrafish development remains unclear. In order to investigate the lethal and sublethal effects of a GBH exposure on developing zebrafish embryos, a 72 h exposure to Roundup® Ultramax (RU) at concentrations of 2, 5, 8.5 μg a.i. mL−1 was performed. The results of this work are presented in chapter II, in which a 72 h LC50 of 8.53 μg a.i. mL−1 and an increased mortality of embryos exposed to concentrations greater than 8.5 μg a.i. mL−1 were reported, as well as an increase in malformations, changes in heart rate and hatching. At concentrations of 2 and 5 μg a.i. mL−1 , no changes were observed in the histological evaluation, namely teratogenic effects, nor in the biochemical parameters analysed, such as generation of reactive oxygen species (ROS), changes in oxidative stress enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), reduced glutathione (GSH), glutathione oxidized (GSSG)), induction of lipid peroxidation (LPO), alteration of acetylcholinesterase (AChE) or lactate dehydrogenase (LDH) activity. The results obtained are suggestive that the toxicological effects on zebrafish are concentration dependent. In chapter III the impact of 72 h exposure to RU at environmentally relevant concentrations (1, 2 and 5 μg a.i. mL−1 ) on different behavioural patterns of zebrafish embryos is reported. Zebrafish embryos were exposed for 72 h and maintained until 144 hpf, when larval behaviour was assessed through general exploratory motility, escape responses, anxiety-related behaviours, social interactions and through the measurement of cortisol levels. Cortisol levels remained similar between treatments, but larvae exposed to 5 μg a.i. mL−1 did not react to the presence of an aversive stimulus, suggesting that RU can induce changes in sensorimotor coordination during zebrafish development, possibly indicative of its neurotoxic effect. Chapter IV presents a study that aimed to evaluate the effects of acute exposure (4 h 30 min) to a commercial formulation of glyphosate, Roundup ® Flex (RF), at concentrations of 1, 5 and 10 μg a.i. mL−1 in Tg(mpxGFP)i114 transgenic and wild-type (WT) zebrafish larvae, with 72 hpf, by evaluating the inflammatory response, oxidative stress, and cell death. None of the concentrations tested induced changes in the number of neutrophils and nitric acid levels. No changes were observed in the activity of antioxidant enzymes SOD, CAT and glutathione peroxidase (GPx), GST, nor in the levels of GSH and GSSG. The results regarding LPO, LDH and AChE were also similar between the groups. However, at a concentration of 10 μg a.i. mL−1 , an increase in ROS levels and cell death was observed. Evidence suggests that toxicological effects are concentration dependent, albeit at concentrations that are not routinely found in the environment. Glyphosate has chelating properties, forming complexes with metal ions, including Cu, Mn and Zn. The synergistic effect between GBH and metals is believed to have a negative impact on ecosystems, however, these mechanisms are currently unclear. To clarify the mechanisms of toxicity involved, in Chapter V the impact of exposure to a GBH (Roundup® Flex) complexed with metals (Copper, Manganese and Zinc) were assessed, during the initial period of zebrafish embryo development (96 hpf). The endpoints of lethal and sublethal development were evaluated. The results showed that metals induced higher levels of toxicity in embryos/larvae, compared to GBH. Cu was able to induce teratogenic effects in the larvae as well as a decrease in CAT activity, an increase in GPx and GST, a reduction in GSH levels and AChE activity, induction of apoptosis and changes in larval behaviour. Zinc increased mortality, ROS levels and SOD activity, and caused a decrease in AChE activity. Manganese increased the activity of SODs enzymes. The combination of GBH/metal also showed teratogenic effects during their development but in smaller proportions than the metal alone. Although more studies are needed, the results suggest that GBH may interfere with mechanisms of metal toxicity in zebrafish. Finally, in Chapter VI, the general conclusions of the studies performed in this thesis on the toxic effects associated with exposure to GBHs in zebrafish embryos and their potential impacts on ecosystems are explored. Possible directions for future research in this area are also proposed.
  • ItemAcesso Restrito
    Interaction between microplastics and copper: toxicity assessment in zebrafish (Danio rerio) and blackspot seabream (Pagellus bogaraveo)
    2022-09-30 - Santos, Dércia Cabral dos; Monteiro, Sandra Mariza Veiga; Bellas Bereijo, Juan
    Nas últimas décadas, os resíduos de plástico têm sido globalmente reconhecidos como um poluente emergente, com potenciais efeitos negativos sobre os organismos aquáticos e na saúde humana. Embora o tamanho dos plásticos no ambiente aquático varie de pequenas partículas a fragmentos de maiores dimensões ou macroplásticos, atualmente, os microplásticos (MPs) que têm despertado mais atenção. Os MPs, ou seja, partículas sintéticas com formato regular ou irregular e tamanho compreendido entre 1 μm e 5 mm, têm sido reconhecidos como ubíquos nos ecossistemas, persistentes e potencialmente tóxicos para os organismos aquáticos. Devido ao seu pequeno tamanho, elevada área de superfície e hidrofobicidade, os MPs podem adsorver e acumular outros poluentes, como o cobre (Cu), um metal pesado que representa um risco significativo para o ambiente aquático. Por sua vez, quando os MPs interagem com os organismos aquáticos, a biodisponibilidade e toxicidade dos metais pesados adsorvidos pode ser modulada. No entanto, a investigação sobre os efeitos combinados de MPs e metais pesados, nos organismos aquáticos ainda é limitada. Neste contexto, o trabalho desenvolvido nesta TESE tem como principal objetivo avaliar os efeitos toxicológicos de MPs e Cu, isolados ou combinados, em duas espécies de peixes, nomeadamente o peixe-zebra (Danio rerio) e o goraz (Pagellus bogaraveo), contribuindo com novas evidências sobre os impactos ambientais dos MPs nos organismos aquáticos de água doce e marinha. Considerando as limitações atuais de conhecimento sobre os efeitos dos MPs e do Cu em diferentes estágios de vida dos peixes, ou após exposições prolongadas, várias abordagens experimentais foram selecionadas. Peixes-zebra foram expostos a MPs (2 mg/L) e Cu (15 – 125 μg/L, diferentes concentrações de acordo com o bioensaio), isolados ou combinados, desde as 2 horas pós-fertilização (hpf) até às 96 hpf (exposição aguda) ou aos 14 dias pós-fertilização (dpf) (exposição subcrónica), para avaliar, respectivamente, os efeitos independentes ou dependentes da ingestão destes poluentes no desenvolvimento embrionário dos peixes. Similarmente, uma exposição crónica de 30 dias, a MPs (2 mg/L) e Cu (25 μg/L), isoladamente ou combinados, foi realizada em peixes-zebra adultos para avaliar os efeitos de uma exposição prolongada em idade adulta. Adicionalmente, foi realizada uma exposição aguda e subcrónica (3 e 9 dias), a MPs (0,3 mg/L) e Cu (10 – 810 μg/L), isoladamente ou combinados, de larvas recém-eclodidas de goraz, uma espécie marinha, para avaliar o seu efeito nos estágios finais do desenvolvimento larvar. A exposição aguda induziu, em embriões de peixe-zebra, alguns efeitos tóxicos, nomeadamente a diminuição da sobrevivência e da eclosão, stress oxidativo, modulação do sistema antioxidante, inibição da acetilcolinesterase (AChE) e disrupção de comportamentos de aversão e sociais. No entanto, estes efeitos foram observados particularmente nos grupos expostos ao Cu e às misturas (Cu+MPs), não tendo a exposição isolada a MPs produzido efeitos significativos no peixe-zebra. Estes resultados mostram que a ação tóxica da exposição aguda aos MPs, durante o período embrionário, pode ser limitada, possivelmente devido à presença do córion que atua como uma barreira protetora contra os poluentes. Quando expostos subcronicamente, durante 14 dias, observou-se que os MPs e as misturas Cu+MPs tiveram maior efeito tóxico nas larvas de peixe-zebra, em comparação com a exposição aguda, sugerindo que a toxicidade das partículas de plástico pode surgir após exposições mais longas e estar ainda relacionada com a sua ingestão. Em resumo, os resultados da exposição subcrónica mostraram que os MPs e Cu, isolados ou combinados, causaram uma elevada mortalidade, stress oxidativo e neurotoxicidade nas larvas de peixe-zebra. Em relação aos efeitos associados à neurotoxicidade, foi observada uma inibição da atividade da AChE e uma diminuição da expressão de genes relacionados com a proliferação neuronal (sox2, pcna), neurogénese (neuroD, olig2), desenvolvimento dos neurónios motores (islet) e DNA metiltransferases. Por sua vez, estes efeitos tóxicos podem ter contribuído para as alterações comportamentais observadas ao nível da competência locomotora e escape a predadores, nas larvas de peixe-zebra expostas a MPs, Cu e Cu+MPs. Adicionalmente, foram avaliados os efeitos crónicos dos MPs e Cu em peixes-zebra adultos, particularmente em dois principais órgãos-alvo – brânquias e cérebro. Nas brânquias, foi observado um aumento dos níveis de espécies reativas de oxigénio (ROS), com a consequente a indução de stress oxidativo, bem como um aumento da expressão de genes relacionados com a síntese de serotonina e apoptose. Em relação aos efeitos no cérebro do peixe-zebra, não foram observados sinais de indução de stress oxidativo. Porém, notou-se um aumento da atividade da AChE em todos os grupos expostos, alterações na expressão génica relacionada com a neurogénese adulta, sistema dopaminérgico e apoptose, bem como alterações no comportamento locomotor e social dos peixes. Estes resultados, por sua vez, destacam as diferentes interações dos MPs e Cu em diferentes órgãos. Por fim, a exposição aguda e subcrónica do goraz a MPs e Cu, provocou stress oxidativo, dano celular e modulação do sistema antioxidante. À semelhança do observado em peixe-zebra, uma espécie de água doce, os resultados demonstraram também no goraz, uma espécie marinha, que a toxicidade dos MPs em fases embrionárias e larvares surge principalmente após exposições mais longas. No entanto, ao contrário do peixe-zebra onde os MPs e Cu, isolados ou combinados, desregularam a atividade da AChE e a proliferação neuronal, no goraz não foram observados tais efeitos neurotóxicos. Isto, por sua vez, indica que a resposta dos peixes aos MPs e Cu é também dependente da espécie. De uma forma geral, os resultados evidenciam que a exposição a MPs e Cu, isoladamente ou combinados, pode promover toxicidade no desenvolvimento, stress oxidativo e dano celular, alterações em termos de atividade metabólica, neurotoxicidade e comportamental. Os dados aqui apresentados proporcionam um conhecimento mais aprofundado sobre as interações toxicológicas dos MPs e metais pesados em peixes, principalmente em fases iniciais de vida. Esta TESE enfatiza que novas pesquisas sobre os efeitos dos MPs, isolados ou combinados com outros poluentes, devem incluir exposições mais prolongadas para avaliar adequadamente o potencial risco das partículas de plástico na saúde dos organismos.
  • ItemAcesso Restrito
    Fungal bioconversion of Agro-Industrial by-products and modeling of Laccase Kinetics
    2020-06-18 - Pinto, Paula Alexandra da Costa Sousa Botelho; Dias, José Albino Alves; Bezerra, Rui Manuel Furtado
    The study of enzymatic processes involved in fungal bioconversion of lignocellulosic byproducts is the main objective of this thesis. Since cellulose and lignin are the main components of most of these byproducts, the mathematical modeling of bioconversion processes can be used to predict/evaluate the behavior of the interveners in these processes allowing their optimization. In a first phase (Chapter 2), the bioconversion of two byproducts, wheat straw and chestnut shell, was evaluated in its enzymatic component and effect on the hydrolysis of the pretreated substrate, using a commercial mix of holocellulases. Lignocellulolytic enzymes, as fundamental catalysts in bioconversion processes, should be among the study priorities, not only to clarify their mechanisms, but also to search for multiple inhibition relationships. Following this perspective, the lignocellulolytic activity of the fungi and the enzymatic saccharification were evaluated. To clarify the relationships within the general mechanisms of bioconversion/saccharification, the effect of some variables (hydrolysis time, enzymatic activity and holocellulase concentration) was estimated whiles a general predictive model of saccharification. The findings showed that Trametes versicolor was able to significantly increase saccharification in both pretreated substrates. The covariance analysis showed a significant effect between lignin peroxidase and increased straw saccharification. A high consistency was also found, relating the effects of xylanase and laccase activities on the final release of reducing sugars from the chestnut shell. These results can be used for optimization planning in saccharification of this substrate. In a second phase (Chapter 3), the development of the work focused on one of the major enzymes of bioconversion, laccase. Several Michaelis-Menten models (linear and nonlinear) were applied to clarify the inhibition mechanisms that may influence the optimization of the processes. For the mathematical modeling of the inhibition mechanism, the oxidation of a lignin-derived natural substrate, (ferulic acid) in the presence of chlorine, was used. The models were submitted to a ranking methodology, using as estimator, of the relative quality of the different kinetic models, the Akaike Information Criterion (AIC) with respective Akaike Weights. At the end of this chapter it was concluded that, analysis of AIC with Akaike weights allowed the discrimination and quantification of probabilities associated with each model, with only one inhibitor concentration, confirming a competitive inhibition of laccase in the presence of chlorine. Continuing the study of laccase and possible inhibitions (Chapter 4), Bi-Bi substrate models were tested in two features of substrate inhibition: no inhibition and inhibition by reducing and/or oxidizing substrate. During delignification, many intermediates (e.g. 2,6-dimethoxyphenol, DMP) may serve as substrates and inhibitors at the same time. Generally, in the laccase oxidation, the dioxygen is assumed under saturating conditions, simplifying the mechanism and its study. It is also commonly assumed, that the reducing substrate and dioxygen are related via ping-pong mechanism. In order to clarify the laccase mechanism under non-saturating oxygen conditions, two substrates: ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and DMP, were used. The discrimination of the models was done based on the ranking methodology previously described (AIC). It was concluded that the most likely models for ABTS and DMP oxidation are Ping-Pong and Theorell-Chance, respectively. The catalytic efficiency of oxygen conversion to water appears to be compatible with a process relatively independent of reducing substrates and the bisubstrate mechanism type. In addition, in the DMP oxidation mechanism laccase is not oxygen-saturated which, in turn, may lead to potential increased reaction rates resulting from the use of oxygen-pressurized bioreactors. The Chapter 5 describes the development of a new methodology and a new integrated equation, which allows the determination of kinetic parameters for two mutually non-exclusive inhibitors, when one of them is produced during the reaction. The determination of kinetic parameters in these circumstances, without the integrated Michaelis-Menten equations, was referred as containing increased errors in parameter estimation. Phosphate and urea, as alkaline phosphatase inhibitors, were used to illustrate the study of this methodology. The results obtained were subjected to a comparative analysis of previously published data to assess: (i) whether the inhibitors were mutually exclusive or non-exclusive inhibitors; ii) if non-exclusive inhibitors, determine the nature of the interaction (facilitation, impediment or independence); iii) determine whether they were non-exclusive unique only to E (enzyme) or ES (enzymesubstrate complex), or both enzyme forms; iv) the type of inhibition and v) the kinetic constants of each inhibitor. The methodology developed, with the new integrated equation, allowed not only to obtain the inhibition constants of each inhibitor, but also to determine the interaction between the two inhibitors. This interaction study is not possible using the Michaelis-Menten initial velocity equations whenever one of the inhibitors is a reaction product.
  • ItemAcesso Aberto
    A perspetiva ciência, tecnologia e sociedade no ensino das ciências naturais: Um estudo com alunos do 7ºano de ciências naturais
    2012 - Carvalho, Andreia Marlise Carneiro de; Favas, Paulo Jorge de Campos
    Vivemos num mundo em que o desenvolvimento científico e tecnológico ocorre a um ritmo cada vez maior, pelo que diversos autores defendem que a perspetiva Ciência, Tecnologia e Sociedade (CTS), no Ensino das Ciências, pode ser importante para incrementar o interesse dos alunos pela aprendizagem da Ciência, levando-os a compreender quer os benefícios, quer as limitações da Ciência e da Tecnologia, assim como as influências recíprocas entre estas componentes e a Sociedade e o Ambiente. Desta forma, a perspetiva CTS poderá contribuir para a formação de cidadãos cientificamente informados e intervenientes na Sociedade democrática, tomando decisões conscientes e fundamentadas. O presente trabalho visa a obtenção do grau de mestre em ensino de Biologia e de Geologia no 3º ciclo do Ensino Básico e no Ensino Secundário e teve por base o estágio pedagógico efetuado na Escola S/3 S. Pedro, em Vila Real. Este relatório reflete a prática pedagógica desenvolvida no 7º ano de Ciências Naturais e no 11º ano de Biologia e Geologia. Pretende-se com este trabalho referir a importância do Ensino das Ciências e da literacia científica na aprendizagem dos alunos e realçar os métodos, estratégias e recursos de ensino-aprendizagem aplicados segundo a orientação CTS. O estudo de caso, de natureza qualitativa, foi efetuado no 3º ciclo do Ensino Básico (7º ano) e pretendemos com ele demonstrar a importância da orientação CTS no Ensino das Ciências e na aprendizagem dos alunos. Apresentámos a revisão da literatura de duas das unidades lecionadas na prática letiva, as planificações elaboradas para as turmas onde incidiu o estudo, a reflexão da prática pedagógica, os resultados obtidos com a aplicação da orientação CTS no Ensino das Ciências e a respetiva análise dos resultados na turma de estudo e na turma de controlo. A realização deste relatório, assim como a prática pedagógica, foi importante para a nossa formação profissional. Além disso, permitiu-nos pôr em prática a perspetiva CTS no Ensino das Ciências e verificar o seu efeito na aprendizagem dos alunos e, também, adquirir experiência no campo da investigação em educação. Este trabalho pode, ainda, ajudar outros professores que pretendam realizar investigações e/ou aplicar, nas suas aulas, metodologias novas e comprovadamente úteis para o desenvolvimento pessoal e profissional dos alunos, assim como para uma melhor aprendizagem dos conteúdos científicos.