O uso de biomarcadores para avaliar o impacto ambiental de contaminantes em rios Mediterrâneos

Doutoramento em BiologiaUm assunto que requer atenção é a avaliação ecológica da qualidade da água de ecossistemas de água doce. Uma abordagem que surge como promissora é a biomonitorização baseada em biomarcadores, porque pode avaliar a saúde dos organismos e obter sinais de alerta precoce acerca dos riscos ambientais. Até agora, porém, o uso de biomarcadores em espécies de invertebrados, para diagnosticar danos ecológicos nos rios, é escasso. Por essa razão, existe uma necessidade urgente de desenvolver biomarcadores nas principais espécies de macroinvertebrados dos ecossistemas fluviais que são alvo de estudo. Esta tese tem como objectivo averiguar se as respostas in situ, aliadas aos biomarcadores, podem ser um método viável para avaliar os danos ecológicos de contaminantes em ecossistemas de água doce. Numa primeira fase, os biomarcadores foram usados para averiguar os mecanismos fisiológicos de adaptação genética de clones de Daphnia magna ao pesticida organofosforado fenitrothion. Numa segunda fase, os biomarcadores foram usados como ferramentas de diagnóstico de poluição em zonas ribeirinhas. Estes estudos foram realizados com três espécies-chave de macroinvertebrados: Daphnia magna, Corbicula fluminea e Hydropsyche exocellata, nos rios Besós e Llobregat e no Delta do rio Ebro (NE Espanha). Além disso, foram realizados com animais capturados nos rios, ou com ensaios de transplantes, e foram complementados com índices biológicos de macroinvertebrados e análises químicas da água e dos animais. Como os contaminantes químicos têm vários modos toxicológicos de acção e, portanto, afectam várias respostas bioquímicas dos organismos, foram analisados nas três espécies um conjunto de biomarcadores pertencentes a diferentes vias metabólicas. A abordagem experimental indica que o uso combinado de biomarcadores e outras medidas, tais como índices biológicos e testes in situ, contribui para diagnosticar os efeitos prejudiciais de contaminantes nas comunidades ribeirinhas.An ecological assessment of water quality in freshwater ecosystems is an issue of major concern. A biomarker based biomonitoring presents a promising approach, because it can be used to assess the health status of organisms and to obtain early-warning signals of environmental risks. Until now, however, the use of biomarkers in invertebrate species to diagnose an ecological impairment in rivers is scarce. Therefore, there is an urgent need to develop biomarkers in key macroinvertebrate species within the river ecosystems that are the object of study. This thesis aims to ascertaining if in situ responses and biomarkers can be a reliable method to diagnose the ecological impairment on freshwater systems due to contaminants. In a first stage, biomarkers were used to find out the physiological mechanisms of genetic adaptation to the organophosphate pesticide fenitrothion in Daphnia magna clones. In a second stage, biomarkers were used as diagnostic tools of pollution in riparian habitats. The latter studies were carried out with three key macroinvertebrate species: Daphnia magna, Corbicula fluminea and Hydropsyche exocellata, in the Besós and Llobregat rivers and in the Ebro Delta (NE Spain). These studies were developed with field collected animals, or with transplants assays, and were complemented with macroinvertebrate biological indices and chemical analyses of river water and animals tissue. As chemical contaminants have multiple toxicological modes of action and hence affect many biochemical responses within the organisms, a battery of biomarkers belonging to different metabolic pathways was analyzed in the three species. The experimental approach indicates that the combined use of biomarkers with different metrics, such as biological indices and in situ tests, improve substantially our ability to diagnose detrimental effects of pollutants in riparian communities.FCT/FSE - SFRH/BD/23269/200

Contribuição para o estudo da anexina V na apoptose celular em concentrados de eritrócitos

A hemoterapia moderna baseia-se na utilização correcta dos diversos componentes sanguíneos, associados a um maior controle de qualidade do sangue, o que a torna mais segura e, actualmente, muitos doentes sao beneficiados pois, a transfusão de componentes sanguineos, em situaçoes várias, está na linha da frente na manutenção da vida e em casos extremos, o último recurso que salva vidas. A qualidade e a segurança nas transfusões de sangue são grandes preocupações da área médica, autoridades de saúde e doente1. O sangue obtido pelos Centros de Sangue provem de dadores voluntários, dotados de uma enorme sensibilidade social, que periodicamente assumem uma postura benevola e altruista e consequentemente mantêm os bancos de sangue providos de um produto imprescindivel no tratamento de diversas patologias. O produto final disponível – concentrado de eritrócitos (CE´s), plasma e concentrado plaquetário – tem de assumir um carácter seguro e viável de modo a que os riscos para o doente sejam diminutos2. O controlo de qualidade aplicado a todo o sangue doado realiza provas de conformidade nas unidades com especificações previamente definidas, sendo a hémolise um dos parâmetros importantes na avaliação da qualidade dos concentrados de eritrócitos, pois, pode ocasionar implicações clinicas para o receptor. Para além disso a avaliação da concentração de hemoglobina (Hg) no sangue doado mostra-se um controlo imprescindivel que salvaguarda a qualidade e segurança do componente a transfundir3;4.Até se obter um CE há todo um processo moroso e de responsabilidade vital. Todo o sangue obtido passa por várias etapas fundamentais até à obtenção do componente pretendido (analise, produção e armazenamento). Os CE’s obtidos quando armazenados, num ambiente de refrigeração, têm uma vida útil de 42 dias. Após este período, o sangue deve ser inutilizado por se verificar alterações bioquímicas, biomecânicas, e imunológicas nos CE’s e por consequência a sua instabilidade vital no que ao tratamento de patologias, para as quais este componente está indicado, diz respeito5. Foi realizado um estudo experimental com o objetivo de avaliar a contribuição da Anexina V na apoptose celular nos concentrados de eritrócitos, constatando a degradação dos mesmos ao longo de todo o período de armazenamento e validar o paradigma que a ciência preconiza: “Os CE’s após os 42 dias armazenados, em condições específicas (2 a 6º centígrados), são inviaveis para transfundir”6;7. A avaliação dos níveis de apoptose por citometria de fluxo é geralmente realizada por métodos que utilizam Anexina V como marcador vital, que se associa aos resíduos de fosfatidilserina, externalizados no início do processo apoptótico. A Anexina V é uma proteína humana endógena dependente do ião Ca+2, amplamente distribuída intracelularmente em altas concentrações na placenta e em concentrações mais baixas nos eritrócitos, plaquetas e monócitos. Apresenta como principal característica a capacidade de se ligar à fosfatidilserina, um fosfolipído presente na camada interna da bicamada lipídica, que durante a apoptose celular é translocada para a camada externa da membrana celular. A determinação da Anexina V é normalmente utilizada para verificar se as células são viáveis, apoptóticas ou necróticas por meio de diferenças na integridade da membrana plasmática. Assim, ao conjugar a Anexina V ao FITC (Isotiocianato de fluoresceína) é possível identificar e quantificar as células apoptóticas por citometria de fluxo7. Numa amostra de 15 CE’s, a qual foi induzida a hemólise, verificou-se, por citometria de fluxo, que a viabilidade deste componente se desvanesce ao longo do tempo, confirmando assim que o tratamento, manuseamento e armazenamento do sangue compromete a vitalidade terapeutica deste insubstituivel produto vital.Modern hemotherapy is based on the correct use of various blood components associated with a better quality control of blood, which makes it safer and currently, many patients benefit from transfusion of blood components, in various situations, is at the forefront in sustaining life and in extreme cases, is the ultimate resource that saves lives. The quality and safety of blood transfusions are the major concerns in the medical field, health authorities and patients. Blood collected by blood centers comes from volunteer donors, endowed with enormous social sensitivity, which periodically take a benevolent and altruistic posture and consequently maintain blood banks provided with an essential product for the treatment of various diseases. The final product available - red cell concentrate (RCC), plasma and platelet concentrate must take a safe and viable character so that the risks to the patient are minimal. Blood Centers quality control performs tests in accordance with previously defined specifications, and hemolysis is one of the important parameters in evaluating the quality of erythrocytel concentrates, because of the clinical implications for the recipient. In addition the evaluation of hemoglobin (Hg) in blood donated its essential to safeguard the quality and safety of the component to be transfused. Until an Red Cell Concentrade (RCC) is obtained, there is a whole lengthy process and vital responsibility. Whole blood obtained passes through several key steps to achieving the desired component (analysis, production and storage). RCC when stored in a cooling room, have a shelf life of 42 days. After this period, the blood must be discarded as they face biochemical, biomechanical and immunological changes and vital instability in the treatment of pathologies for which this component indicated. An experimental study was conducted in order to verify to contribution of Annexin V in cell apoptosis following their degradation throughout the storage period and to validate transfusion medicine science paradigm that advocates: "RCC after 42 days stored in specific conditions (2-6 degrees Celsius), are not feasible to transfuse”. The evaluation of apoptosis levels of by flow cytometry is usually performed by methods that use Annexin V as a vital marker that is associated with the waste phosphatidylserine, externalized in the early apoptotic process.12 Annexin V is an endogenous Ca2 + ion dependent human protein with placental intracellular high concentrations and in lower concentrations in erythrocytes, platelets and monocytes. As main characteristic it presents the ability to bind to phosphatidylserine, a phospholipid present in the inner layer of the lipid bilayer that is translocated to the outer layer of cellular membrane during apoptosis. Determination of Annexin V is usually used to determine if the cells are viable, apoptotic, or necrotic by differences in membrane integrity. Thus, by combining the Annexin V with FITC (fluorescein isothiocyanate) it is possible to identify and quantify apoptotic cells by flow cytometry. In a sample of 15 RCC, in which hemolysis was induced it was found, by flow cytometry, that the viability of this component vanishes with time confirming that the treatment, handling and storage of blood compromise the vitality of this irreplaceable vital therapeutic product

O voluntariado na adolescência : um estudo exploratório sobre o impacto na auto-eficácia e na concepção positiva de si

Tese de mestrado, Psicologia (Secção de Psicologia Clínica e da Saúde - Núcleo de Psicoterapia Cognitivo-Comportamental e Integrativa), Universidade de Lisboa, Faculdade de Psicologia, 2011A ideia de que um indivíduo invista o seu tempo e esforço em favor de outra pessoa, particularmente quando essa pessoa é um estranho, tem suscitado o interesse daqueles que se dedicam ao estudo do comportamento humano e motivado os estudos em torno da influência do voluntariado nos que a ele se dedicam. Este é mais um estudo que pretende contribuir para o conhecimento nesta área, investigando a relação da prática de voluntariado com a auto-eficácia geral percebida e a concepção positiva de si, em adolescentes. A hipótese que se coloca é que o voluntariado reforce as expectativas de auto-eficácia e que estas, por sua vez, funcionem como facilitadoras da capacidade de alcançar a concepção positiva de si. A investigação partiu da análise qualitativa de entrevistas realizadas a oito participantes com idades compreendidas entre os 16 e os 18 anos, numa perspectiva exploratória. Os resultados apontam para o potencial benéfico do voluntariado, nomeadamente em termos de satisfação, crescimento pessoal e aumento da compreensão do mundo e de realidades diferentes daquelas em que os participantes vivem. No entanto, o estudo é inconclusivo no que diz respeito à relação da prática de voluntariado com a auto-eficácia geral percebida e com a concepção positiva de si.The idea that an individual invests his time and effort on behalf of another person, especially when that person is a stranger, has aroused the interest of those who dedicate themselves to the study of human behavior and motivated their studies about the influence of volunteering in those who are engaged. This is another study that aims to contribute to knowledge in this area, investigating the relationship of the practice of volunteering with the perceived general self-efficacy and best possible self in adolescents. The hypothesis aroused is that volunteerism strengthens expectations of self-efficacy and that these, in turn, work as facilitators of ability to achieve best possible self. The investigation was based on the qualitative analysis of interviews with eight participants aged 16 to 18 years, in an exploratory perspective. The results indicate the potential benefit of volunteering, particularly in terms of satisfaction, personal growth and increased understanding of the world and different realities than those in which participants live. However, the study is inconclusive regarding the relationship of the practice of volunteering with the perceived general self-efficacy and best possible self

Rare occurrence of severe blindness and deafness in Friedreich ataxia: a case report

Background: Friedreich ataxia is the most frequent hereditary ataxia worldwide. Subclinical visual and auditory involvement has been recognized in these patients, with co-occurrence of severe blindness and deafness being rare. Case report: We describe a patient, homozygous for a 873 GAA expansion in the FXN gene, whose first symptoms appeared by the age of 8. At 22 years-old he developed sensorineural deafness, and at 26 visual impairment. Deafness had a progressive course over 11 years, until a stage of extreme severity which hindered communication. Visual acuity had a catastrophic deterioration, with blindness 3 years after visual impairment was first noticed. Audiograms documented progressive sensorineural deafness, most striking for low frequencies. Visual evoked potentials disclosed bilaterally increased P100 latency. He passed away at the age of 41 years old, at a stage of extreme disability, blind and deaf, in addition to the complete phenotype of a patient with Friedreich ataxia of more than 30 years duration. Discussion: Severe vision loss and extreme deafness has been described in very few patients with Friedreich ataxia. Long duration, severe disease and large expanded alleles may account for such an extreme phenotype; nonetheless, the role of factors as modifying genes warrants further investigation in this subset of patients.info:eu-repo/semantics/publishedVersio

Novel MAG Variant Causes Cerebellar Ataxia with Oculomotor Apraxia: Molecular Basis and Expanded Clinical Phenotype

Homozygous variants in MAG, encoding myelin-associated glycoprotein (MAG), have been associated with complicated forms of hereditary spastic paraplegia (HSP). MAG is a glycoprotein member of the immunoglobulin superfamily, expressed by myelination cells. In this study, we identified a novel homozygous missense variant in MAG (c.124T>C; p.Cys42Arg) in a Portuguese family with early-onset autosomal recessive cerebellar ataxia with neuropathy and oculomotor apraxia. We used homozygosity mapping and exome sequencing to identify the MAG variant, and cellular studies to confirm its detrimental effect. Our results showed that this variant reduces protein stability and impairs the post-translational processing (N-linked glycosylation) and subcellular localization of MAG, thereby associating a loss of protein function with the phenotype. Therefore, MAG variants should be considered in the diagnosis of hereditary cerebellar ataxia with oculomotor apraxia, in addition to spastic paraplegia.This work was funded by National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P.,under the project UIDB/04293/2020. It was also funded by FEDER funds through the Programa OperacionalFactores de Competitividade—COMPETE 2020 and by Nacional funds through the FCT [COMPETE:POCI-01-0145-FEDER-007440]. This work was also funded in part by the FCT grant FCT-ANR/BEX-GMG/0008/2013and the Porto Neurosciences and Neurologic Disease Research Initiative at the i3S (Norte-01-0145-FEDER-000008),supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020Partnership Agreement, also through FEDER. The authors also acknowledge the support of the i3S ScientificPlatform Advanced Light Microscopy, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122) and GenomePT(POCI-01-0145-FEDER-022184). MS was the recipient of a fellowship (SFRH/BPD/116046/2016) from the FCTsupported by POPH/MCTES funding.info:eu-repo/semantics/publishedVersio

a case report

personBACKGROUND: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive solid tumors. ATC is frequently diagnosed at advanced stages with unresectable disease and palliative care is often indicated. Recently, several patient-tailored therapies for ATC are emerging due to advances in molecular profiling of these tumors. Entrectinib is a potent oral selective inhibitor of neutrotrophic tropomyosin receptor kinase (NTRK), ROS1, and anaplastic lymphoma kinase fusions. The experience regarding ATC and other thyroid carcinomas, particularly in the neoadjuvant setting, is minimal. CASE REPORT: We present a case of a 51-year-old female patient presenting with a bulky mass of the left thyroid lobe measuring 100 × 108 × 80 mm that was considered surgically unresectable. While waiting for next-generation sequence (NGS) profiling, lenvatinib was initiated. There was an initial clinical and imagiologic response; however, progression occurred after 12 weeks, and at this time NGS identified an ETV6-NTRK3 fusion and entrectinib was started. After 12 weeks, tumor diameters reduced to a minimum of 68×60×49 mm, and the patient underwent total thyroidectomy plus central lymphadenectomy. Histological diagnosis confirmed an ATC (pT4a R2 N1a). Adjuvant radiotherapy (RT) (60 Grays) with weekly paclitaxel (45 mg/m2) was then administered followed by maintenance entrectinib 600 mg daily. Fluorodeoxyglucose positron emission tomography performed 3 months after completion of RT showed only non-specific uptake in the posterior wall of the hypopharynx and larynx, suggestive of inflammation. CONCLUSION: We report the first case of an ATC with a dramatic response to neoadjuvant therapy with entrectinib, which enabled surgical resection of an ab initio unresectable tumor.publishersversionpublishe

Sleep disturbances in Parkinson's disease are associated with central parkinsonian pain

Introduction: Sleep disturbances and pain are common non-motor symptoms in Parkinson's disease (PD). This study aimed to explore the association between these two symptoms in a cohort of patients with PD. Materials and methods: The Parkinson's Disease Sleep Scale (PDSS-2) was used to identify sleep disturbances in a series of 229 PD patients. The identification and characterization of pain was performed by a semi-structured interview and by the application of the Ford classification and the Brief Pain Inventory (BPI). The Unified Parkinson's Disease Rating Scale-III, Hoehn & Yahr (H&Y), and Schwab and England Independence Scale were used to assess motor symptoms and functional independence in off and on conditions. The Hospital Anxiety and Depression Scale (HADS) and SF-36 were applied to screen for anxiety and depression and to evaluate the quality of life. Non-parametric tests were used for group comparisons and logistic regressions were applied to explore predictors of sleep disturbances. Results: Seventy-five (33%) patients had clinically relevant sleep disturbances (PDSS-2≥18) and 162 patients (71%) reported pain. Of those with pain, 38 (24%) had central parkinsonian pain. PD patients with sleep disturbances experienced more pain and had more severe motor symptoms, lower functional independence, more anxiety and depression symptoms, and worst quality of life. Among patients with pain, central parkinsonian pain was the subtype of pain with the highest odds of sleep disturbances, even when taking into account motor symptoms (H&Y off), motor fluctuations, intensity of pain (BPI), and symptoms of anxiety and depression (HADS). Conclusions: The association between pain and sleep disturbances in PD appears to be dependent on subtype of pain. The close relationship between central parkinsonian pain and sleep disturbances in PD raises the possibility of common pathophysiological mechanisms. A better understanding of the relationship between sleep disturbances and central parkinsonian pain may contribute to the development of new care strategies in PD patients.info:eu-repo/semantics/publishedVersio

Molecular characterization of Portuguese patients with hereditary cerebellar ataxia

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Hereditary cerebellar ataxia (HCA) comprises a clinical and genetic heterogeneous group of neurodegenerative disorders characterized by incoordination of movement, speech, and unsteady gait. In this study, we performed whole-exome sequencing (WES) in 19 families with HCA and presumed autosomal recessive (AR) inheritance, to identify the causal genes. A phenotypic classification was performed, considering the main clinical syndromes: spastic ataxia, ataxia and neuropathy, ataxia and oculomotor apraxia (AOA), ataxia and dystonia, and ataxia with cognitive impairment. The most frequent causal genes were associated with spastic ataxia (SACS and KIF1C) and with ataxia and neuropathy or AOA (PNKP). We also identified three families with autosomal dominant (AD) forms arising from de novo variants in KIF1A, CACNA1A, or ATP1A3, reinforcing the importance of differential diagnosis (AR vs. AD forms) in families with only one affected member. Moreover, 10 novel causal-variants were identified, and the detrimental effect of two splice-site variants confirmed through functional assays. Finally, by reviewing the molecular mechanisms, we speculated that regulation of cytoskeleton function might be impaired in spastic ataxia, whereas DNA repair is clearly associated with AOA. In conclusion, our study provided a genetic diagnosis for HCA families and proposed common molecular pathways underlying cerebellar neurodegeneration.This work was funded by National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04293/2020. It was also supported in part by the FCT grant FCT-ANR/BEX-GMG/0008/2013; the Porto Neurosciences and Neurologic Disease Research Initiative at the i3S (Norte-01-0145-FEDER-000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement, also through FEDER; and by GenomePT (POCI-01-0145-FEDER-022184). M.S. was the recipient of a fellowship (SFRH/BPD/116046/2016) and acknowledges funding from FCT through program DL 57/2016—Norma Transitória.info:eu-repo/semantics/publishedVersio

TOMATE PARA INDÚSTRIA ESTRATÉGIAS SUSTENTÁVEIS NO COMBATE A TUTA ABSOLUTA

O objetivo deste estudo foi desenvolver um sistema de apoio à decisão com base no acompanhamento técnico dos inimigos da cultura e na construção de mapas de risco para as principais zonas de produção. Outros objetivos foram o estabelecimento de uma metodologia de observação e avaliação, nomeadamente métodos expeditos de estimativa do risco, estabelecimento de regras de tomada de decisão e o estudo da bioecologia da espécie em tomate para indústria no Ribatejo. Os resultados deste projeto são uma contribuição para o uso sustentável dos produtos fitofarmacêuticos e, consequentemente, para uma maior ecoeficiência, conservação da biodiversidade e garantia de qualidade e segurança alimentar do produto final

Mitochondrial diseases in the next generation sequencing era: study of 450 patients

As doenças mitocondriais (DM) são doenças raras, clínica e geneticamente heterogéneas, de difícil diagnóstico, para as quais não existe uma terapia eficaz. O desenvolvimento da tecnologia de sequenciação de nova geração (NGS) revolucionou o diagnóstico molecular deste grupo de doenças, permitindo a identificação de novos genes associados a estas patologias. Nesta nova era genética, através da utilização da tecnologia de NGS, estudamos um grupo de 450 doentes suspeitos de DM, sem etiologia molecular. A nossa estratégia combinada de NGS, englobou a sequenciação de um painel de 213 genes nucleares associados a DM e do DNA mitocondrial completo. Neste estudo, identificamos variantes causais em 134 (30%) doentes analisados, 88 dos quais apresentaram variantes no DNA nuclear e 46 no DNA mitocondrial, tratando-se na maioria de doentes pediátricos (66%). Neste grupo de doentes, identificamos 72 variantes patogénicas descritas na literatura e 20 novas variantes provavelmente patogénicas, assim como 62 variantes de significado indeterminado. Como laboratório nacional de referência para o estudo e investigação das DM, demonstramos o contributo da tecnologia de NGS para esclarecer a etiologia molecular destes doentes, para expandir o espectro mutacional associado a estas patologias e oferecer um diagnóstico pré-natal e aconselhamento genético aos casais em risco.Mitochondrial diseases (MD) are rare disorders, clinically and genetically heterogeneous, making their diagnosis a challenge and with hither to no ef fective therapy options. The development of Next Generation Sequencing (NGS) technologies has revolutionized the understanding of many genes involved in the pathogenesis of MD. In this new genetic era, using a NGS approach, we studied a cohor t of 450 Por tuguese patients suspected of MD, and without a molecular etiology. Our combined NGS approach was first based on analysis of a custom-made targeted mitochondrial panel of 213 nuclear genes followed by the whole mitochondrial genome. In this study, we identif ied disease related variants in 134 (30%) analyzed patients, 88 with nuclear DNA and 46 with mitochondrial DNA variants, being most of them pediatric patients (66%). The molecular analysis of this cohor t revealed 72 already described pathogenic and 20 novel probably pathogenic variants, as well as 62 variants of unknown signif icance. As a national laboratory for the study and research of MD, we demonstrated the power of NGS to achieve a molecular etiology of these patients, to expand the mutational spectrum associated to MD and to propose a prenatal diagnosis as well as an accurate genetic counseling for af fected families.Fundação da Ciência e Tecnologia (PTDC/DTP-PIC/2220/2014, Genetic Defects of Mitochondrial Diseases: a Next Generation Sequencing Approach); Ao Programa Nor te 2020 (NORTE-01-0246- -FEDER-000014, DESVENDAR “DEScobrir, VENcer as Doenças rARas”)info:eu-repo/semantics/publishedVersio