Repositório RCAAP

A 61-year-old man presented with a 3-year history of slowly progressive yellowish plaques involving both eyelids (Fig 1). He also complained of intermittent retro-orbital headache, bilateral lower extremity pain, and increasing fatigue over the preceding year. His medical history was other-wise unremarkable.

Atrial fibrillation (AF), is the most frequent sustained cardiac arrhythmia, described by an irregular and rapid contraction of the two upper chambers of the heart (the atria). AF development is promoted and predisposed by atrial dilation, which is a consequence of atria adaptation to AF. However, it is not clear whether atrial dilation appears similarly over the cardiac cycle and how it affects ventricular volumes. Catheter ablation is arguably the AF gold standard treatment. In their current form, ablations are capable of directly terminating AF in selected patients but are only first-time effective in approximately 50% of the cases. In the first part of this work, volumetric functional markers of the left atrium (LA) and left ventricle (LV) of AF patients were studied. More precisely, a customised convolutional neural network (CNN) was proposed to segment, across the cardiac cycle, the LA from short axis CINE MRI images acquired with full cardiac coverage in AF patients. Using the proposed automatic LA segmentation, volumetric time curves were plotted and ejection fractions (EF) were automatically calculated for both chambers. The second part of the project was dedicated to developing classification models based on cardiac MR images. The EMIDEC STACOM 2020 challenge was used as an initial project and basis to create binary classifiers based on fully automatic classification neural networks (NNs), since it presented a relatively simple binary classification task (presence/absence of disease) and a large dataset. For the challenge, a deep learning NN was proposed to automatically classify myocardial disease from delayed enhancement cardiac MR (DE-CMR) and patient clinical information. The highest classification accuracy (100%) was achieved with Clinic-NET+, a NN that used information from images, segmentations and clinical annotations. For the final goal of this project, the previously referred NNs were re-trained to predict AF recurrence after catheter ablation (CA) in AF patients using pre-ablation LA short axis in CINE MRI images. In this task, the best overall performance was achieved by Clinic-NET+ with a test accuracy of 88%. This work shown the potential of NNs to interpret and extract clinical information from cardiac MRI. If more data is available, in the future, these methods can potentially be used to help and guide clinical AF prognosis and diagnosis.

Presymptomatic gene testing for familial amyloidotic polyneuropathies (FAP) is integrated in genetic counseling protocols common to other "Later onset, hereditary, autosomal dominant, no cure diseases" namely Huntington's Disease (HD) and Machado-Joseph disease (MJD). However, presymptomatic gene testing has specific clinical and therapeutic implications for FAP. Moreover, at least in Portugal, FAP ATTR Val30Met is a serious health problem. The most important implications are: the possibility of family planning including prenatal and preimplantation diagnosis; treatment with liver transplantation (TX); clinical follow-up according to protocols for early diagnosis which will allow patients to access therapy in useful time. This concept of useful time in FAP treatment is discussed. The growing possibilities of different therapeutic approaches are considered. In conclusion, presymptomatic gene testing for FAP may have a positive impact on candidate quality and prolongation of life, and on the future of disease studies.

A previous publication analyzed the clinicopathological characteristics of 105 patients with steatohepatitis: 32 nonalcoholic, 21 ambulatory alcoholics, and 52 hospitalized alcoholics; we now report an up to 12-year follow-up (mean 5.9 +/- 4.7). Between 1988 and 1993, all patients with a histological diagnosis of steatohepatitis were included; necrosis, inflammation, Mallory bodies, and fibrosis were graded. Complete follow-up data were obtained in 78%. Survival curves were similar between nonalcoholic and ambulatory alcoholics; they were, however, better in nonalcoholic than hospitalized alcoholics (P < 0.0001), and in ambulatory relative to hospitalized (P = 0.0001) alcoholics. Nonalcoholics had a better prognosis than the combined alcoholic groups (P = 0.001). Patients with moderate to severe Mallory bodies and severe fibrosis had a significantly worse survival (P < 0.01), whereas severity of hepatocellular damage and neutrophil or mononuclear infiltration had no significant impact. In conclusion, alcoholic patients as a whole had a worse prognosis, yet the ambulatory subgroup had a prognosis similar to nonalcoholic patients.

The objective of this study was to evaluate the long-term predictive value of the haemostatic, inflammatory and haemorheologic disturbances in transmural myocardial infarction (MI). Sixty-four (59 male) consecutive survivors of a MI, with a mean age of 58.3 +/- 12.0 years, were followed over a period of 36 months. Eighteen patients had a cardiovascular event defined as the composite of death, non-fatal MI, unstable angina and stroke. The haemostatic (protein C activity-PtC, antithrombin III, plasminogen activator inhibitor-1), haemorheologic (blood fluidity and components, erythrocyte membrane fluidity) and inflammatory (polymorphonuclear elastase, leukocyte count) profiles were determined at hospital discharge, using standard methodology. Our results can be summarized as follow: (i) at hospital discharge, the subgroup of patients with events had higher leukoactivity, leukocyte count, membrane fluidity, prognosis cyte count (7833.0 +/- 1696.0 vs. 10294.0 +/- 3129.0; p = 0.011), lower PtC (100.65 +/- 19.08 vs.81.25 +/- 19.95; p = 0.002), and lower erythrocyte aggregation (14.26 +/- 5.94 vs. 11.47 +/- 3.45; p = 0.031) in relation to the ones without events; (ii) By Cox regression the protein C activity lower tertile (OR 0.169; 0.045-0.628; p = 0.008); erythrocyte membrane outer layer fluidity upper tertile (OR 0.067; 95% CI 0.011 - 0.393; p = 0.003); and erythrocyte aggregation lower tertile (OR 0.182; 0.038 - 0.876; p = 0.034) were independent predictors of the composite endpoint. We can conclude that some haemostatic, haemorheologic and inflammatory disturbances, at hospital discharge, are long-term independent predictors of recurrent cardiovascular events in transmural myocardial infarction survivors.

Angiogenesis is the de novo formation of blood vessels from pre-existing ones, and is a process regulated by a fine-tuned balance between molecules that either stimulate or inhibit vessel growth. When this balance is disrupted, vessel formation or regression occurs in an exacerbated manner, which can lead to angiogenesisdependent diseases, such as cancer and ischemia. We have previously demonstrated that doses of ionizing radiation lower than 0.8 Gy, defined here as low doses of ionizing radiation (LDIR), induce angiogenesis by activating endothelial cells. In physiological contexts, LDIR promote angiogenesis during zebrafish development and adult fin regeneration. Using experimental models, LDIR stimulated neovascularization after ischemia induction and promoted tumor growth and metastasis formation by enhancing angiogenesis. These LDIR effects are relevant since they are present during radiotherapy in the peritumoral tissues and for that reason, the main aim of this doctoral thesis was to determine if LDIR induce angiogenesis in human tissues. Material from parietal peritoneum, located in peritumoral tissues, was removed from patients diagnosed with locally advanced rectal cancer who underwent neoadjuvant radiotherapy. According to our data, LDIR activate endothelial cells in peritumoral tissues, which is associated with increased microvascular density. This effect should be considered in the treatment plan report for patient follow-up and in future studies to establish a correlation between these doses and tumor dissemination. Additionally, this work focused on the mechanisms through which LDIR induce angiogenesis. Since adipocytes secrete multiple angiogenic factors and adipokines that induce angiogenesis, the effect of LDIR in modulating the pro-angiogenic potential of adipocytes was investigated. An in vitro model of adipocyte differentiation was used, and our data show that LDIR enhance the angiogenic potential of adipocyte-conditioned medium in vitro and in vivo. Lastly, this work reveals that even though regeneration is often regarded as a recapitulation of post-embryonic development, LDIR accelerate post-embryonic development but not regeneration, suggesting that the physiological context could be a major determinant of the specific phenotype promoted by LDIR.

A amêijoa-macha (Venerupis pullastra) é um dos bivalves mais consumidos em Portugal. No presente estudo foi avaliada a qualidade microbiológica desta amêijoa capturada na Trafaria através do controle do pH e da contagem de microorganismos indicadores de poluição fecal, tais como Enterobacteriaceae e indicadores de degradação como as bactérias ácido-lácticas, microrganismos viáveis totais e bactérias produtoras de H2S. A amêijoa foi armazenada a diferentes temperaturas (4ºC, 9ºC), e avaliou-se a sua qualidade microbiológica e química (através das técnicas acima referidas) ao longo do tempo nestas temperaturas. Determinou-se que seria benéfico a utilização da temperatura 4 ºC para períodos de armazenamento superiores a 2 dias.

As tartarugas marinhas (superfamília Chelonioidea) são um grupo de 7 espécies de répteis, cujas populações se encontram ameaçadas. A sistemática sobre-exploração levou ao desaparecimento de muitas populações de tartarugas marinhas. Contudo, após intensos esforços de conservação aplicados, algumas populações encontram-se atualmente a recuperar. É fundamental determinar o uso de recursos por parte destas populações em recuperação, uma vez que as tendências populacionais observadas em tartarugas marinhas são reguladas por mecanismos ambientais como a disponibilidade de recursos. A disponibilidade de recursos como fontes de alimento e diversos fatores ambientais, influenciam a abundância de tartarugas marinhas em determinadas regiões e, como tal, a sua distribuição espácio-temporal. Fortemente correlacionados com a disponibilidade de recursos, fatores ambientais como a temperatura da água e o tipo de substrato são responsáveis pela distribuição das áreas de alimentação das tartarugas marinhas e, consequentemente, pela distribuição destes animais em determinadas regiões. No Arquipélago do Havai, a tartaruga-verde (Chelonia mydas) é a espécie de tartaruga marinha mais frequentemente observada. As tartarugas verdes havaianas constituem uma subpopulação geográfica e geneticamente isolada, que foi alvo de intensas medidas de conservação, e encontra-se atualmente com estatuto de conservação Pouco Preocupante. A distribuição da tartaruga-verde no Arquipélago do Havai é determinada pela presença de áreas apropriadas para a sua alimentação, repouso e reprodução. Esta subpopulação habita águas costeiras em redor das principais ilhas havaianas entre migrações efetuadas para as suas áreas de reprodução, situadas em ilhas do noroeste do Arquipélago. Pearl Harbor é um estuário situado em Oahu, uma das principais ilhas havaianas, situadas a sudeste do Arquipélago. Este local, famoso pelas piores razões durante a segunda Guerra Mundial, é um porto estratégico para a Marinha dos Estados Unidos e, como tal, é exigida uma monitorização regular dos recursos naturais presentes em Pearl Harbor pela Sikes Act. Os recursos presentes devem ser elencados num Plano de Gestão Integrada de Recursos Naturais. Este plano deve ser aprovado por agências nacionais responsáveis pela investigação, gestão e recuperação das populações de tartarugas marinhas sob a jurisdição dos Estados Unidos. O nosso estudo tem como objetivos determinar padrões temporais e espaciais no uso de habitat pelas tartarugas-verdes avistadas em Pearl Harbor e determinar a estrutura populacional das mesmas, ao longo de 10 anos de monitorização. Para o primeiro objetivo, foram investigadas tendências anuais nos avistamentos de tartaruga em áreas distintas de Pearl Harbor e dados qualitativos relativos ao comportamento das tartarugas encontradas. Inerente a este objetivo, pretendemos avaliar se existe sazonalidade na presença das tartarugas nesta região, comparando o número de avistamentos entre duas estações gerais no Havai, a fresca (de Novembro a Abril) e a quente (de Maio a Outubro). Relativamente ao segundo objetivo, pretendemos determinar a estrutura populacional das tartarugas observadas, com algumas limitações, através de estimativas visuais do comprimento da carapaça das tartarugas. Para este estudo, Pearl Harbor e o Canal de Entrada de Pearl Harbor foram subdivididos em 21 áreas específicas. De Março de 2000 a Maio de 2011, monitorizações subaquáticas seguindo uma metodologia de transectos lineares ocorreram em Pearl Harbor várias vezes ao longo do ano. Os transectos foram realizados por mergulhadores, com um circuito aberto SCUBA, e conduzidos por cientistas da Marinha. Ao longo dos transectos, foram registados número e espécie das tartarugas marinhas observadas e variáveis oceanográficas como a profundidade e a visibilidade. Durante os avistamentos, os mergulhadores estimaram visualmente o comprimento da carapaça em linha reta (SCL) das tartarugas, possibilitando a inclusão de cada indivíduo numa de três classes de tamanho: indivíduos até 50cm SCL; indivíduos desde 50cm até 1.0m SCL e indivíduos com comprimento superior a 1.0m. A temperatura da superfície do mar foi recolhida remotamente para cada uma das áreas amostradas para o período de tempo examinado, com a mais fina resolução espacial possível, tendo sido associadas temperaturas médias mensais a cada um dos transectos desde 2002 até 2011. Para uma caracterização concisa do tipo de cobertura bentónica das zonas amostradas, recorremos às observações diretas do ambiente marinho durante os transectos e também a monitorizações relativamente mais recentes dos recursos marinhos nesta região. Para a modelação dos avistamentos de tartaruga ao longo de 10 anos de monitorização, usámos um modelo aditivo generalizado hierárquico (HGAM) com uma distribuição Poisson zero-inflacionada. Este foi utilizado para determinar as relações não lineares entre o número de tartarugas observadas por transecto e variáveis temporais, ano e mês, e ambientais, profundidade, visibilidade e temperatura da superfície da água. O comprimento do transecto foi usado como termo offset, para ter em conta o esforço de amostragem não-constante, e as 21 áreas amostradas foram designadas como efeito aleatório no modelo, permitindo assim incorporar variações não explicadas pelas outras variáveis específicas de cada área. Com as predições do modelo escolhido, foi possível construir um perfil de distribuição espacial das tartarugas em Pearl Harbor. Relativamente à análise dos dados qualitativos, recorremos ao teste Qui-quadrado para determinar diferenças nos comportamentos observados entre áreas amostradas, e ao teste Kruskal-Wallis para determinar a existência de diferenças entre as três classes de tamanho relativamente à profundidade a que foram observadas. Entre 2000 e 2011 foi avistado um total de 680 tartarugas marinhas. Do total de avistamentos, 679 eram tartarugas-verdes e apenas uma tartaruga-de-pente (Eretmochelys imbricata) foi positivamente identificada. Os avistamentos de tartaruga-verde ocorreram em 121 transectos (26%), enquanto os restantes 343 transectos tiveram zero avistamentos (n=464). A frequência da presença de tartarugas em transectos variou entre os locais amostrados, tendo esta sido relativamente superior em áreas localizadas no Canal de Entrada. Dentro do porto, verificámos uma presença relativamente menor e, em algumas áreas, não foram avistadas tartarugas durante o período de tempo examinado. Relativamente aos resultados do modelo, considerando um nível de significância de 5%, todas as variáveis à exceção da profundidade influenciaram o número de avistamentos de tartaruga por transecto. Verificámos um aumento generalizado do número de tartarugas-verdes observadas ao longo do tempo, para todas as áreas combinadas. Observámos uma diminuição do número de tartarugas observadas nos primeiros meses do ano. O número de tartarugas observadas aumentou, não surpreendentemente, com o aumento da visibilidade. Relativamente à variável temperatura, observámos dois picos associados com um maior número de tartarugas avistadas, o primeiro entre os 24ºC e os 25ºC e o segundo pico aos 26.5ºC. Relativamente aos dados qualitativos, encontrámos dois comportamentos dominantes exibidos pelas tartarugas avistadas em transectos, nadar e repousar. Através das observações diretas, verificámos que locais para repouso incluíram abrigo como caves submersas na região do canal de Entrada. Nenhuma tartaruga foi observada a alimentar-se. Indivíduos entre os 50cm e 1.0m SCL foram os mais frequentemente observados e, tartarugas superiores a 1.0m SCL foram também relativamente abundantes nos transectos efetuados. Verificámos uma segregação das tartarugas por tamanho quanto à profundidade a que foram observadas em que, a profundidades superiores, foram avistados indivíduos de maiores dimensões. As tartarugas-verdes encontram-se distribuídas de forma não-uniforme em Pearl Harbor. O aumento observado no número de avistamentos de tartaruga por transecto pode ser devido à crescente tendência populacional observada na tartaruga-verde havaiana. A presença de elementos atrativos para repouso, nomeadamente elementos usados como refúgio no Canal de Entrada, pode também ter levado à mais frequente presença de tartarugas nesta zona. Duas áreas forneceram importantes locais de repouso para as tartarugas-verdes, confirmado pelas observações diretas do uso de habitat e um número constante de tartarugas observadas em transectos ao longo dos anos. No entanto, os métodos usados no nosso estudo não permitem avaliar a fidelidade das tartarugas-verdes a estas áreas nem quantificar a disponibilidade de alimento e/ou abrigo em cada uma delas. Consideramos que o baixo número de tartarugas detetadas em transectos dentro do porto pode ter duas origens. A verdadeira ausência das tartarugas nesta zona poderá ser uma das origens. A relativamente menor disponibilidade de alimento e elementos usados para repouso dentro do porto pode levar ao menor uso desta zona pelas tartarugas, que parecem usar maioritariamente algumas áreas do Canal de Entrada, onde a abundância destes recursos pode ser relativamente superior. A segunda explicação poderá estar relacionada com falsos zeros, isto é, as tartarugas estavam presentes durante as monitorizações efetuadas, mas não foram detetadas pelos mergulhadores devido às más condições de visibilidade dentro do porto. Relativamente à sazonalidade encontrada na presença das tartarugas, o menor número de avistamentos nos primeiros meses do ano poderá estar relacionado com a migração reprodutiva da tartaruga-verde havaiana para ilhas a noroeste do Arquipélago. A distribuição das tartarugas por tamanho observada está de acordo com o que já tinha sido encontrado em outras áreas de alimentação da tartaruga-verde havaiana, e também com o tamanho mínimo conhecido a partir do qual se dá o recrutamento de juvenis para áreas de alimentação. O presente estudo permitiu determinar padrões temporais e espaciais de uso de habitat das tartarugas-verdes, ao longo de 10 anos de monitorização. Este tipo de informação é essencial para a gestão dos recursos naturais, em particular das populações de tartarugas marinhas, a uma escala local por parte da Marinha. Em última análise, os nossos resultados contribuíram para o amplo conhecimento existente acerca da ecologia da tartaruga-verde havaiana e para a sua conservação.

The medical field related to bacterial infections and cancer are currently facing currently one of the biggest challenges, mostly due to conventional treatments inefficiency after years of overuse and misuse in clinics. Cases of multi-resistant bacterial infections are increasing every year, according to World Health Organization (WHO), explained by resistant microorganisms’ predominance after antibiotic usage and limited pharmaceutical development of new drugs. As for cancer therapeutics, unspecific treatments that promote severe side effects had another reported consequence, increased cancer resistance, prolonging patients’treatment. As a result, new alternatives are necessary to fight these challenges, such as antimicrobial peptides (AMPs) and anticancer peptides (ACPs). These peptides physical-chemical properties, such as small amino acid sequence, amphipathicity and positive net charge, allow them to act selectively at specific cell membranes, mostly due to electrostatic interactions (cationic vs. anionic membranes). Besides, they can be used against different targets, with reported activity against bacteria, viruses, fungi and cancer cells. In the last case, they are dependent of cancer cell membrane phosphatidylserine (PS) translocation from internal to external membrane leaflet, which increases the negative cell surface charge. Throughout the work here presented, we focused on new AMPs designed according to two different strategies: (i) Pa-MAP 2 and Pa-MAP 1.9, synthetic AMPs redesign from a natural protein from the polar fish Pleuronectes americanus (winter flounder), and (ii) EcAMP1R4, PaDBS1R1 and PaDBS1R6, synthetic peptides designed through a bioinformatics algorithm that considers chemical properties and activity efficiency. In both cases, a multidisciplinary approach was performed, using biophysics and cell biology techniques to study their activity in vitro, using membrane models, bacterial and cancer cell lines, and in vivo infection models. Considering the Pa-MAP peptide family (Pa-MAP 2 and Pa-MAP 1.9), with a minimal inhibitory concentration (MIC) of 3.2 and 6.0 μM against Escherichia coli, respectively, they were shown to be efficient against a multi-resistant strain from a clinical isolates, inclusively with promising results demonstrated with an in vivo infection mice model. Nevertheless, only Pa-MAP 1.9 showed to have dual activity (AMP and ACP), being tested in two different cell lines, HeLa and HCT-166. Despite its efficiency in promoting cancer cell death, Pa-MAP 1.9 showed a different mechanistic behaviour for the cell lines tested, promoting total cell death after 6 h of incubation (IC50 of 51.8 ± 1.23 μM) and membrane homeostasis destabilization. As for the synthetic peptides (EcAMP1R4, PaDBS1R1 and PaDBS1R6), their antimicrobial activity was confirmed in vitro, according to bioinformatics studies, with MIC values against E. coli of 11.7, 1.5 and 8.0 μM, respectively. In vivo studies were also performed for the last two peptides, confirming their potential as future antimicrobial drug molecules. Their dynamics after membrane interaction was likewise studied, either using bacteria cells or lipid vesicles, showing that different biomembrane properties are destabilized, which could determinate AMP efficiency. Structure conversion to a-helix after membrane interaction showed to be the first step for peptide activity. Concluding, the work discussed in this thesis resulted in new peptide molecules that are effective AMPs and, one of them (Pa-MAP 1.9), also as ACP. Their activity was characterized in vitro and in vivo through new approaches, with the objective of identifying new insights that may help in future peptide design. Even with the promising results achieved so far, their potential use in therapeutics need to be further tested, considering their efficiency, but also their applicability, focusing on patients and in the pharmaceutical industry needs.

Sepsis and sepsis-associated multiorgan failure represent a systemic inflammatory state mediated by the innate immune system resulting in an excessive cellular response to severe infection, with high levels of morbidity and mortality. Furthermore, patients who survive sepsis, have long-term cognitive and functional impairment. Animal models are essential to clarify the pathophysiological mechanisms of sepsis. There is mounting evidence that inhibition of nuclear factor NFκB activation can reduce sepsis-associated organ dysfunction and injury. Herein, we deeply characterize an animal model of lipopolysaccharide (LPS)-induced acute inflammation, through an integral functional, behavioural, morphological and molecular evaluation as well as tested different possible therapeutic approaches to determine their efficacy on the modulation of systemic inflammation through the NF-kB pathway. Male and female Wistar rats (12-20 weeks) were injected with LPS (E. coli serotype O127:B8; tail IV) and divided into three groups: LPS 6 (6mg/kg), LPS 12 (12mg/kg) and SHAM (NaCl 0.9%). At 6 h and 24 h after LPS administration, an autonomic evaluation was performed in both conscious animals, with continuous radio-telemetry recording of blood pressure (BP) and heart rate (HR), and anesthetized animals with BP, ECG, HR, tracheal pressure, respiratory frequency (RF) and body temperature continuously monitored. Baro- and chemoreflexes were evaluated with phenylephrine and lobeline, respectively. Behavioural changes were also evaluated through the elevated-plus maze (anxiety), open-field (locomotor/exploratory activity) and Y-maze (short-term spatial working memory) tests. Immunohistochemistry and RT-PCR were executed to determine heart and brain inflammatory state. Serum biomarkers levels for organ disfunction were also measured. For the test of potential therapeutic drugs, animals were exposed with the two LPS doses and treated 15 minutes later with four different drugs: erythropoietin, dexamethasone, IKK 16 and adenosine. The functional, behavioural, and molecular analysis were performed. Overall, the characterization model results showed a rise in BP and HR and elevated RF, indicative of tachycardia and tachypnea. The higher RF was concomitant with a higher chemoreceptor reflex sensitivity. The autonomic data reveals an overexcitation of sympathetic tone with a sympatho-vagal imbalance, concomitant with baroreflex impairment and/or increases in BP, all these were strongly marked in the highest LPS dose. At both time-points, both LPS groups show an anxiety-like behaviour, associated with lower locomotor and exploratory capacity and short-term memory impairment. The molecular studies show reactive astrogliosis and microgliosis, due to inflammatory processes in the hippocampus, as well as, an upregulation of pro-inflammatory factors in the heart and brain. Serum analysis yielded higher levels of biomarkers for renal and liver dysfunction, and pancreatic and neuromuscular injury, mainly in the LPS 12 group. The functional, behavioral, and molecular response between female and male rats was not so different. Both sexes present markedly changes when exposed to higher doses of LPS. Indeed, male animals are more sensitive to endotoxin that females, that can fight inflammation more easily. Of the four therapeutic proposals studied, erythropoietin and dexamethasone were the best to modulate the NF-ΚB pathway in order to reduce the dysfunction of multiple organs as well as attenuate the behavioural changes caused by the exposure to endotoxin. Both IKK 16 and adenosine treatments attenuate the organ dysfunction by reducing the serum renal, hepatic, pancreatic and neuromuscular levels, however these two drugs did not improve the cardiac and behavioral modifications. Further studies are needed in order to better understand the role of these drugs in the early stages of sepsis. Summarizing, higher doses of LPS induces strong alterations in both cardiac and neurological systems, as well as, multiple organ dysfunction, that can be attenuated by the administration of specific NF-κB modulators, such as, erythropoietin and dexamethasone. These findings served to better understand the pathophysiological processes involved in these animal model of acute inflammation as well as showed therapeutic evidence that through NF-κB signalling pathway modulation it is possible to attenuate or even reverse the acute stages before this condition evolves to more aggressive stages, namely septic shock and ultimately dead.

O tratamento hormonal adjuvante de doentes com carcinoma da mama melhora a sobrevivência global. O uso em contexto de mundo real, efetividade, tolerabilidade e adesão a inovações recentes da terapia hormonal adjuvante de cancro da mama não está bem caracterizado. Para abordar estes pontos e assim apoiar a tomada de decisão de doentes-médicos e a investigação clínica futura desenvolvemos uma série de projetos com o propósito de: 1) descrever a implementação na prática clínica de mundo real de inovações recentes no campo da terapia hormonal adjuvante de cancro da mama e sumarizar a sua efetividade, 2) quantificar o impacto da terapia hormonal adjuvante na qualidade de vida das doentes e 3) quantificar a adesão e persistência das doentes à terapia hormonal adjuvante. Para concluir estas tarefas utilizámos diferentes estudos de coorte e aplicámos métodos padrão e inovadores de análise de dados. Fazendo recurso de duas coortes retrospetivas derivadas do Registo Oncológico Regional do Sul, a primeira com ~1300 mulheres pós-menopáusicas e a segunda com ~1700 mulheres pré-menopáusicas, identificámos que quer os inibidores da aromatase (IA) quer a supressão da função ovárica (SFO) foram introduzidos com sucesso na prática clínica após publicações científicas de referência em 2005 e 2014, respetivamente. Na coorte pós-menopáusica, 41% das doentes receberam um IA (16% em monoterapia e 25% em sequência) e 59% tamoxifeno com diferenças por centro. Após um acompanhamento mediano de 6.3 anos, os IA associaram-se a melhor sobrevivência global (SG) quando comparados com tamoxifeno (HR-ajustado 0.5, IC 95% 0.37-0.81). Fazendo recurso complementar de uma coorte retrospetiva estado-unidense de ~800 mulheres pós-menopáusicas com tumores lobulares, registámos resultados semelhantes e não foi identificada heterogeneidade de eficácia por tipo histológico, em específico quando comparando carcinomas lobulares puros a carcinomas ductais e lobulares mistos. Na coorte pré-menopáusica, 17% das doentes foram tratadas com SFO com um crescimento substancial do uso de 2014 em diante (16% vs. 25% após 2014), particularmente para a combinação com IA (0.4% vs. 8% após 2014). Após um acompanhamento mediano de ~3 anos, doentes tratadas com SFO tiveram melhor SG que doentes não tratadas com SFO (HR-ajustado 0.44, IC 95% 0.19-0.96). Fazendo recurso de uma sub-coorte de ~4300 doentes com cancro da mama com resultados reportados por doente (patient reported outcomes) disponíveis do estudo CANTO, uma coorte prospetiva francesa, descrevemos um impacto substancial do tratamento na qualidade de vida (QdV) 2 anos após o diagnóstico. Usando o C30 summary score da EORTC, um resultado compósito de várias funções e sintomas, a terapia hormonal, mas não a quimioterapia, teve um impacto persistente na QdV 2 anos após o diagnóstico com diferenças nos domínios impactados. Adicionalmente, expusemos um efeito diferencial do tratamento por estado menopausico: em doentes pré-menopáusicas a quimioterapia parece ser o promotor principal da deterioração de domínios de QdV, enquanto que em doentes pós-menopáusicas foi a terapia hormonal o promotor principal da deterioração da QdV. Finalmente, fazendo recurso de uma segunda sub-coorte de ~1200 derivada do estudo CANTO e de doentes que estavam a tomar tamoxifeno e com avaliação sérica do tamoxifeno, identificámos que 1 em cada 6 mulheres (16%) eram não-aderentes ao tratamento, i.e. tinham os valores séricos de tamoxifeno abaixo da linha de corte de adesão. Esta proporção foi maior que aquela auto-reportada (12.3%). Após um acompanhamento mediano de 2 anos após a avaliação do tamoxifeno sérico, doentes não aderentes quando avaliadas por via bioquímica tiveram uma sobrevivência livre de recidiva à distância mais curta (HR-ajustado de 2.31, IC 95% 1.05-5.06). Este trabalho detalhou a cinética de introdução na prática clínica de inovações recentes na terapia hormonal adjuvante. Adicionalmente, revelou evidência de mundo real da efetividade de IA e SFO adjuvantes. Estes dados sugeriram porém que para uma proporção substancial de doentes a terapia hormonal leva a um impacto persistente e negativo na QdV, especialmente em mulheres pós-menopáusicas, tal como a uma proporção alarmante de não-adesão ao tratamento, até certo ponto relacionada com questões de tolerabilidade.

Psoriatic arthritis (PsA) is a chronic inflammatory disease, integrated in the spondylarthritis (SpA) nosologic entity, that exhibits marked phenotypic heterogeneity and complex underlying inflammatory pathways. Tumor necrosis factor (TNF) is the most central inflammatory cytokine implicated in PsA articular and extra-articular manifestations, and TNF inhibitors (TNFi) the most successful therapeutic intervention breakthrough in the last decades for the treatment of this debilitating disease. Despite the remarkable benefits of TNFi for the management of PsA patients, optimal longterm control of disease activity is difficult to achieve at the population level. Furthermore, due to PsA heterogenous phenotypes, some manifestations have been scarcely studied, leaving uncovered evidence gaps for dactylitis, enthesitis and axial PsA symptoms. In this dissertation, we sought to provide new perspectives on TNFi for the treatment algorithm of PsA. Our specific aims were: 1) to describe TNFi positioning in PsA treatment, through a literature review and national rheumatologists consensus; 2) to identify predictors of TNFi’ effectiveness (persistence and response) in PsA patients, using realworld data from the Rheumatic Diseases Portuguese Register (Reuma.pt); 3) to provide high level evidence on TNFi’ efficacy for the treatment of PsA dactylitis (and enthesitis) through an investigator-initiated multicentric randomized placebo-controlled trial; 4) to analyze the effect of TNFi on bone remodeling at the synovio-entheseal complexes using transmembrane TNF transgenic mice (TgA86). Encompassed by the first aim of this doctoral thesis, we performed a comprehensive literature review that included TNFi evidence of efficacy for PsA musculoskeletal manifestations: peripheral arthritis, axial disease, enthesitis and dactylitis; that was integrated in the 2015 update of the recommendations for the use of biologics diseasemodifying antirheumatic drugs (bDMARDs) in the treatment of PsA patients, endorsed by the Portuguese Society of Rheumatology. This review, followed by the assessment of consensus within Portuguese rheumatologists for the established recommendations, reinforced the need to identify clinical predictors of effectiveness that could help clinicians to best select candidates for TNFi in the context of recently approved bDMARDs (non-TNFi) for PsA, aiming at optimal disease control. Furthermore, the lack of consensus on dactylitis and enthesitis treatment indications and the absence of evidence of TNFi efficacy in these manifestations, assessed as primary endpoints in randomized controlled clinical trials, was identified as one of the most relevant knowledge gaps in PsA medical field. The Exchange PsA study addressed the second aim of this thesis and showed that PsA patients registered at Reuma.pt, starting a first TNFi between 2001 and 2017, experienced reduced effectiveness (drug survival and response) when switched within TNFi therapeutic class, for both peripheral and axial phenotypes. We also identified the female gender as a common unfavorable predictor of persistence and response to a first TNFi in this population. We further explored possible interactions and acknowledged that pain, but not disease activity at baseline, contributed to modulate the effect of gender on drug retention of the first-line TNFi, nevertheless gender remaining an independent predictor of discontinuation. The results from our work suggest that the poorer responses to a first-TNFi are not exclusively dependent on patient-reported outcomes. In fact, significantly higher baseline objective measures of peripheral disease activity were observed in females, and these responses were not modified by obesity or disease phenotype, supporting gender-dependent patterns of response. Obesity, a commonly associated PsA comorbidity, was further identified as a predictor of worse response to TNFi, suggesting that concomitant weight reduction plans for obese patients can be determinant to potentiate TNFi effectiveness. The results from the Exchange PsA study created awareness for the early identification of female PsA patients who suffer from delayed therapeutic (TNFi) initiation and are at risk of poor treatment survival and response to TNFi. In line with this, patients’ education combined with nutrition clinics access should be broadly implemented in daily practice and weight reduction programs included in PsA recommendations. The GO-DACT trial was developed under the scope of the third aim of the thesis and assessed dactylitis for the first time as the primary endpoint of a double-blind, randomized, placebo-controlled trial. The results from this multicentre investigator-initiated trial provided first time evidence that the combination of a TNFi (golimumab) and methotrexate (MTX) is superior to MTX monotherapy in improving PsA dactylitis, assessed both clinically and by high-resolution magnetic resonance imaging. Considering the high heterogeneity of outcome measures used to report dactylitis activity in PsA trials, the results from GO-DACT showed that the application of the innovative Dactylitis Severity Score (DSS) and Leeds Dactylitis Index (LDI) response indices (DSS20, 50, 70 and LDI20, 50,70), developed in the framework of this trial, consistently allowed discrimination between treatment arms. The good performance of these newly developed response indices supports their potential usefulness for future PsA trials and for standardization of PsA dactylitis outcomes. In light of the high disease burden of PsA dactylitis, including the risk of structural damage, the lower chance of achieving minimal disease activity and the early benefits from TNFi intervention, it seems reasonable to argue that PsA patients with active dactylitis could benefit from first line TNFi in combination with MTX. The fourth objective of this dissertation was centered on the effect of TNFi at the synovioentheseal complexes, in particular those related to local dichotomic bone remodeling disturbances observed in PsA pathogenesis (erosions and new bone formation). It required the identification of a mouse model developing SpA-like manifestations and, specifically, axial new bone formation. This was possible through a literature review on mouse models mimicking SpA characteristics, which lead to the selection of transmembrane TNF transgenic mice (TgA86) as the best option to address this research question. In an in vivo experimental setting, we were able to demonstrate that tmTNF-dependent pathways induce a SpA-like phenotype with peripheral and axial inflammation and deformity, and axial new bone formation in mice. Furthermore, we showed that the inhibition of tmTNF signaling, using a TNFi like mouse equivalent antibody, reduced not only inflammation but also new chondrogenesis/bone formation in the axial spine of tmTNF transgenic mice (TgA86), and this effect was more notorious in an early therapeutic intervention. Despite the limitations in showing a clear effect of TNFi in hampering syndesmophytes progression in human PsA/SpA, blocking new chondrogenesis/new bone formation in mice seems an attainable objective, especially in early disease interventions. With this doctoral thesis, we provide new evidence for the use of TNFi in clinical practice, which is likely to have implications for future guidelines and recommendations in the treatment of PsA patients.

Malaria, which is caused by Plasmodium parasites, is one of the most prevalent infectious diseases worldwide, with an annual death rate around 435,000, accounting for more than half of all deaths by a vector-borne disease, despite innumerous efforts to eliminate it. The infection initiates when the liver form of the parasite, called sporozoites, are injected into the host skin through a bite by the female Anopheles mosquito. Hepatocyte infection by Plasmodium sporozoites is the first natural step towards the establishment of malaria disease. Inside hepatocytes, the parasite lives in a specialized compartment enveloped by a membrane called parasitophorous vacuole membrane (PVM). The PVM represents the interface between the parasite and the host cell, and most likely plays different roles that may range from protection and signalling to waste elimination. Nevertheless, little is known about the molecular players and interactions occurring at this interface. A subset of proteins encoded by the “Upregulated in Infective Sporozoites” (UIS) genes have been identified, and it has been shown that some of them are exported to the PVM. One such protein is UIS4, which has been shown to be essential for parasite survival. For instances, sporozoites of the rodent malaria parasites species, Plasmodium berghei (P. berghei) and Plasmodium yoelii (P. yoelii) lacking UIS4 can invade liver cells and begin to grow. However, they do not efficiently develop and replicate. Despite its important role for malaria parasites during the liver phase of the disease, the biological function of UIS4 remains to be elucidated. The aims of this work were twofold: Aim 1. To study the biological function of the UIS4 in the liver stage on the PVM, by revealing its interaction partners. Aim 2. To search for new host or parasite proteins localized on the PVM during the liver stage of infection. Our working hypothesis to fulfil the first aim is that UIS4 protects the parasite and that this role is achieved through a network of interacting proteins. Although UIS4-KO parasites have been previously generated in the NK65 parasite line, they did not infect the mosquito efficiently. Two new UIS4-KO parasites in WT and GFP-expressing P. berghei ANKA lines were created. To generate these knock-out parasite lines, the P. berghei ANKA UIS4 open reading frame (ORF) was replaced by the hDHFR gene (human Dihydrofolate Reductase) using a double cross-over recombination strategy. The modified parasite line has resistance to pyrimethamine. The UIS4-KO parasite lines were genotyped to ensure that the transfection constructs integrated into the correct locus. The absence of UIS4 expression in mutant parasites was confirmed by RT-PCR (for RNA expression), immunofluorescence and Western Blot (WB), for protein expression. Assessment of parasite load agreed with previously published studies, both in vivo and in vitro. To identify the UIS4 interacting partners, immunoprecipitation using an antibody against UIS4 followed by mass spectrometry and WB of infected cells with UIS4-KO and wild-type parasites was performed. Independent experiments identified actin as a major UIS4 interacting partner. Moreover, a similar immunoprecipitation experiment was carried out on mammalian cells transfected with a plasmid expressing the soluble domain of UIS4. Again, actin was immunoprecipitated with UIS4. Notably, in vitro assays of actin polymerization revealed that UIS4 promotes actin polymerization. In order to find new PVM proteins, an approach based on proximity-dependent biotin labelling, named BioID, was designed. To that end, a parasite line expressing a UIS4-BirA* fusion protein, PbUIS4-BirA*-HA, was generated by transfecting P. berghei ANKA parasites, via a single cross-over recombination, with a plasmid containing approximately 800bp of the UIS4 gene sequence in frame with the BirA*-coding sequence, followed by pyrimethamine treatment and clonal selection. The transgenic parasites were characterized to ensure the correct size of the fusion protein by WB and localization to the PVM by microscopy. Most importantly, the data show that PbUIS4-BirA*-HA parasites behave as wild-type parasites throughout the entire parasite life cycle, and that the biotin supplementation leads to specific biotinylation of proteins on the PVM. Such immunoprecipitation approach followed by mass spectrometry analysis revealed several novel parasite and host proteins as potential PVM-resident candidates. Altogether, this study revealed: a) a previously unknown interaction between a critical parasite protein essential for the success of the liver stage of infection and host actin, and b) it also identified new parasite and host proteins that may play a critical role in the interface between Plasmodium parasites and its first obligatory host cell on the PVM.

O excesso de peso (incluindo pré-obesidade e obesidade) na adolescência é uma preocupação de saúde pública, uma vez que está associado a condições de saúde adversas, a curto e longo prazos. Comportamentos de saúde inapropriados, como um aporte energético excessivo e níveis insuficientes de atividade física (AF), podem conduzir ao excesso de peso. Existe uma necessidade urgente em encontrar estratégias que possam influenciar positivamente os comportamentos alimentares e de AF. O projeto PAC-MAnO (The effect of a Physical Activity Consultation in the management of adolescent overweight) foi desenhado com o objetivo de investigar o impacto de uma Consulta de Atividade Física, com recurso a técnicas de entrevista motivacional, no comportamento de AF e z-score do índice de massa corporal (IMC) em adolescentes com excesso de peso seguidos em consulta de Obesidade Pediátrica. De acordo com o nosso conhecimento, este é o primeiro estudo realizado em Portugal a analisar, de forma vasta, o impacto de uma Consulta de Atividade Física na gestão do peso em adolescentes com excesso de peso. Entre setembro de 2016 e dezembro 2018, os adolescentes com excesso de peso (IMC ≥ p85), em consulta de primeira vez (n=165), que consentiram em participar no projeto PAC-MAnO, foram alocados por amostragem consecutiva em três grupos: Grupo de controlo (GC), Grupo experimental I (GEI), e Grupo experimental II (GEII). O GC seguiu o plano assistêncial base (consultas de Pediatria e Nutrição) durante 12 meses. Os GEs foram expostos à Consulta de AF adicionalmente ao plano assistêncial base durante os mesmos 12 meses. Os participantes incluídos no GEII foram adicionalmente convidados/encorajados a participar em duas sessões semanais de exercício físico, durante os primeiros 6 meses do projeto (fase I). No momento inicial, aos 6 (final da fase I) e aos 12 meses (final da fase II), foi realizado um conjunto de avaliações antropométricas, clínicas, nutricionais e de AF. Dos participantes recrutados, 132 completaram a fase I (GC n=41, GI n=45, GEII n=46) e 102, a fase II (GC n=28, GEI n=36, GEII n=38). Apenas 43,5% (n=20) dos participantes do GEII cumpriram pelo menos 80% das sessões de exercício agendadas (fase I). Aos 6 meses, quer o GEI, quer o GEII mostraram melhorias significativas no z-score do IMC (d=0.75, p=.002; d=0.60, p=.045), composição corporal, aptidão cardiorespiratória (ApCR) (d=1.48, p=.012; d=1.32, p=.022), e AF moderada (d=0.81, p=.005; d=1.39, p<.001), vigorosa (d=0.83, p=.001; d=1.04, p<.001) e moderada-vigorosa (d=0.90, p=.001; d=1.43, p<.001). O GEI mostrou ainda melhorias no espessamento do complexo íntima-média da artéria carótida primitiva (d=1.09, p=.044), bem como no tempo em comportamento sedentário (d=0.70, p=.019) aos 6 meses, comparativamente ao GC. Aos 12 meses foram observadas melhorias adicionais no z-score do IMC (d=0.95, p<.001; d=0.86, p=.015), composição corporal e ApCR (ml/kg/min) (d=1.58, p=.033; d=1.17, p=.043) em ambos os GEs, comparativamente ao GC. O custo líquido atribuído ao GEI foi de 12,41 e 7,78 € por participante (representando um acréscimo de 19.5 e 24.1% comparativamente ao GC) para a fase I e II, respetivamente. Estimou-se uma redução de 0,002 e 0,009 no z-score do IMC aos 6 e 12 meses, para cada Euro (1€) gasto no GEI. O custo líquido do GEII foi de 252,37 e 7,78 € por participante, para a fase I e II, respetivamente. De acordo com os resultados observados, a inclusão de uma Consulta de AF num programa clínico multidisciplinar de gestão do peso em adolescentes (com recurso a técnicas de entrevista motivacional) pode melhorar de forma eficaz os níveis de AF, z-score do IMC, composição corporal e ApCR dos adolescentes, comparativamente àqueles expostos ao plano assistêncial base (consultas de Pediatria e Nutrição), com um bom custoeficácia. Estes resultados são observáveis a 6 meses, com melhorias adicionais a 12 meses. A inclusão de sessões de exercício físico poderá representar uma mais-valia, desde que se assegure a presença dos participantes nas referidas sessões.

Cerebral Venous Thrombosis (CVT) is a less common form of stroke that mostly affects young women. Despite the great progress made in the last decades, significant evidence gaps persist in the understanding of the pathophysiology of brain damage, the mechanisms underlying the benefit of the available treatment strategies and the management of secondary prophylaxis. This work was focused on two of these questions, namely: (1) the mechanisms underlying the pathogenesis of brain lesion and their evolution in relation with the therapeutic intervention; and (2) the risk of pregnancy-related venous thrombotic events and unfavourable foetal outcomes in women with history of CVT, and the most appropriate approach to prevent them. We have performed two systematic reviews (and one systematic review update) and three original studies. The available evidence was appraised in the systematic reviews and the main research gaps were identified. To further address the first aim, we have conducted a multicenter prospective cohort study of patients treated with anticoagulation that included serial standard magnetic resonance imaging and blood collection at several time-points, in order to assess early venous recanalization and biomarkers of blood-brain barrier (BBB) disruption and inflammation. We found an association between persistent venous occlusion and early worsening of brain lesions and diffusion restriction was often associated with tissue recovery in patients with early recanalization. Patients with CVT and parenchymal brain lesions had abnormal levels of matrix metalloproteinase 9 (MMP-9) and the levels of this marker of BBB disruption following treatment start were related with the early recanalization status. Patients with CVT had higher levels of Interleukin-6 (IL-6) and this was a predictor of unfavourable functional outcome at 90-days. Also, we have pooled two registries in order to describe that a MRI marker of hypoperfusion and dilated collateral circulation, the brush sign, can be identified in CVT and is associated with brain lesion and others makers of severity. For the second aim, we performed a follow-up study of women at fertile age with history of CVT and assessed recurrent venous thrombotic events and foetal outcomes, according to antithrombotic prophylaxis. Our results add new evidence on the mechanisms of disease, providing insights into the role of early venous recanalization in the progression and recovery of brain lesions associated with CVT in patients treated with anticoagulation, and indicating new promising markers, as the brush sign, MMP-9 and IL-6. We also collected substantial evidence that support the use of antithrombotic prophylaxis to reduce the pregnancy-related venous thrombotic events and miscarriage among women with history of CVT.

Alzheimer’s Disease (AD) is a chronic and progressive neurodegenerative disease, being the most common form of dementia worldwide (60-70% of all cases). AD is considered a protein misfolding disease and the accumulation of amyloid-beta (Aβ) peptide in the brain is considered one of the main hallmarks of this disease. Accumulatoin of this peptide induces a multiplicity of neurotoxic consequences, which ultimately supports the neuronal death, tightly associated with AD pathophysiology. In line with these alterations promoted by Aβ peptide also alterations in the signaling mediated by the brain-derived neurotrophic factor (BDNF) have been described. BDNF actions on neuronal survival, differentiation and plasticity are mediated by the activation of its full-length receptor (TrkB-FL). The impairment of BDNF signalling in AD might be explained by the decreased protein levels of BDNF and TrkB-FL, together with an increase of truncated TrkB isoforms (TrkB-Tc), which act as negative modulators of BDNF effects. In the last years, we have been establishing a direct relationship between Aβ peptide accumulation and TrkB-FL cleavage. Indeed, we observed, using different experimental approaches and several biological resources, that Aβ peptide, due to a dysfunctional activation of extrasynaptic N-methyl-D-aspartate receptor (eNMDAr), leads to an increase of intracellular calcium. As a consequence, calpains became overactivated and cleave TrkB-FL receptor, forming a new truncated receptor (TrkB-T’) and an intracellular fragment (TrkB-ICD), promoting the loss of BDNF signalling previously reported in the literature. Therefore, the Aβ-triggered cleavage of TrkB-FL receptor might be a strong contributor to neurodegeneration and to an overall loss of neuroprotection in AD patients. This project aimed i) to deeply assess TrkB-ICD fragmentimpact upon normal physiology, ii) to evaluate TrkB-FL cleavage in human post-mortem brain samples from AD patients, and iii) to develop and test a newly developed drug to prevent TrkB-FL cleavage. By the transfection of primary neurons, its was possible to investigate the role of TrkB-ICD. We found that TrkB-ICD i) is a stable fragment, ii) has tyrosine kinase activity and iii) accumulates in the nucleus, indicating a possible propagation of the initial Aβ toxicity. Furthermore, TrkB-ICD overexpression, by viral transduction of primary neuronal cultures with pFCK-CamKII-TrkB-ICD-IRESZsGreen induced an up-regulation of genes involved in neurotransmitter activity and in neural development, promoting in paralel a down-regulation of genes involved in chromosome structure stability and telomere organization, as analysed by whole trancriptome analysis (next generation sequencing). Remarkably, through in vivo intrahippocampal injection of the same viral particles in wild-type (WT) animals (C57BL/6, 12 weeks), we observed a disruption in recognition memory (Novel Object Recognition test) in TrkB-ICD-expressing animals. TrkB-ICD overexpression did not affect locomotion and anxiety-like behaviour. Intriguingly, we observed an increase in long-term potentiation magnitude, the molecular basis for learning and memory, recorded from hippocampal slices of TrkB-ICD-expressing-animals. This evidence is apparently not in line with the cognitive impairments detected in vivo. This discrepancy may indicate that TrkB-ICD promotes a dysfunctional increase in synaptic plasticity and could underlie, at least in part, the memory impairment well-described in AD patients. Regarding TrkB-FL cleavage validation in human brain samples, we used post-mortem inferior temporal cortical samples from human AD patients at different stages of disease severity. Results indicate that, over AD progression, TrkB-FL protein levels decreased, while TrkB-ICD protein levels increased. We must reinforce that this set of data is of an extreme importance, since it validated the enhancement of TrkB-FL cleavage in human patients diagnosed with AD. Finally, following 2D and 3D structural predictions, we designed a new compound to prevent TrkB-FL cleavage. Importantly, we validated the action of the new compound as a strategy that prevents TrkB-FL cleavage, through in vitro and ex vivo studies. Importantly, in addition to molecular studies, functional assays reinforced the potential therapeutic value of the new compound, since the prevention of TrkB-FL cleavage avoided the loss of BDNF function upon LTP and neurotransmitter release. Due to the promising collected data, this compound has a provisional patent application filled in UK. In summary, the data here presented i) indicates that TrkBICD might contribute or aggravate the Aβ toxicity, ii) validates the exacerbation of TrkB-FL cleavage in AD human brain samples, and iii) demonstrates a possible therapeutic value for our new created compound designed to prevent TrkB-FL cleavage. Interestingly, TrkB-FL cleavage was already described in other pathologies, including epilepsy, stroke and ischemia. Therefore, it should be indicated that the evidences described in this work might be transposed to other diseases where excitotoxicy plays a central role.

Satisfazer a crescente procura energética mundial é um dos maiores desafios que a humanidade atravessa atualmente. O aumento da poluição constitui outro grande problema que merece atenção por parte dos governos mundiais e público em geral. O processo de copirólise pode ser muito útil na solução desta situação, uma vez que permite o aproveitamento de resíduos e a sua transformação em biocombustível. Existe bastante informação sobre o processo de pirólise uma vez que já vem sendo estudado há muitos anos, no entanto, dependendo dos produtos a pirolisar, essa informação pode tornar-se escassa. Posto isto, não foi encontrado nenhum estudo de copirólise de resíduos de plástico, borracha de pneu e óleo de lubrificante usado, que constituem o objeto de estudo desta dissertação. O objetivo principal é o de pirolisar misturas destes resíduos com diferentes composições e avaliar a sua influência no rendimento dos produtos sólidos, líquidos e gasosos bem como na composição destes. Para tal, foram realizados ensaios experimentais numa autoclave, sob uma atmosfera de reação de azoto e sendo a pressão inicial a atmosférica. A temperatura de reação foi 420 ºC, a taxa de aquecimento foi 16 ºC/min e o tempo de residência foi 30 minutos. Estes parâmetros foram definidos com base na literatura e também na realização de ensaios preliminares. A quantidade de pneu favorece a produção de sólido, em detrimento da produção gasosa e líquida. O rendimento líquido variou entre 70,5 e os 82,3 %, o sólido entre 15,3 e 27,7 % e o gasoso entre 1,9 e 3,5 %. O uso de borracha de pneu implica a libertação de enxofre durante o processo de pirólise. Por este motivo, o produto gasoso recolhido foi amostrado por um processo de retenção de enxofre. Em média, quanto mais borracha se utiliza, maior a libertação de enxofre. O gás foi analisado através de cromatografia gasosa, tendo-se observado que a sua composição apresentava essencialmente alcanos, em particular, metano mas também etano, propano e propeno. O teor em alcanos diminuiu com o aumento do número de átomos de carbono. A borracha de pneu e o óleo favoreceram a produção destes compostos. O poder calorífico superior do gás variou entre 48,80 e 54,16 MJ/kg e o índice de Wobbe variou entre 55,04 e 60,84 MJ/Nm3. Tanto um como o outro, aparentaram aumentar consoante a quantidade de pneu e de óleo e diminuir com a quantidade de polipropileno. Os líquidos recolhidos foram submetidos a uma destilação de modo a obter duas frações, T [ºC] < 150 e 150 < T [ºC] < 270, respetivamente. Todas as curvas de destilação ficaram situadas entre curvas típicas da gasolina e a do gasóleo. A temperatura a que cada líquido começou a destilar aproximou-se à da gasolina. A análise cromatográfica efetuada aos líquidos, mostrou que ambos os destilados, apresentaram uma maior concentração de ramificados e cíclicos e uma menor concentração de alcenos. Por último, calculou-se o poder calorífico superior das duas frações sendo este mais elevado para o 1º destilado (entre 25,99 e 35,64 MJ/kg) do que para o 2º (9,89 e 14,74 MJ/kg).

A Organização Mundial de Saúde (OMS) é um organismo internacional, do sistema das Nações Unidas, totalmente dedicado às questões da saúde. Foi estabelecida em 1948 e dotada de uma Constituição que consagra a saúde como um estado de completo bem-estar físico, mental e social e não apenas como a ausência de doença ou enfermidade1. A missão da OMS é, pois, justamente, trabalhar para que todas as pessoas possam atingir o nível mais elevado de saúde possível, promovendo para isso actividades próprias e a cooperação entre os Estados. O principal órgão de decisão é a Assembleia Mundial de Saúde (AMS), abrangendo actualmente 193 Estados-membros, que reúne em Maio de cada ano, em Genebra, para discutir temas da maior relevância, segundo uma agenda preparada pelo Conselho Executivo (CE).

Com base nas premissas de que os instrumentos de gestão e de ordenamento territorial de diferentes escalas não abordam adequadamente o risco e de que análises mais aprofundadas de avaliação do risco deveriam ser incorporadas nos planos de manejo e de ordenamento de áreas protegidas para que os objetivos de proteção e de conservação sejam atingidos, este trabalho analisa a abordagem ao risco nos instrumentos de ordenamento e de apoio à gestão de duas áreas protegidas (AP): a Floresta Nacional de Ipanema, no Brasil, e o Parque Natural do Alvão, em Portugal. Apesar de se inserirem em políticas de ordenamento territorial, os Planos de Manejo (PM), no Brasil, e Planos de Ordenamento de Áreas Protegidas (POAP), em Portugal, relacionam-se direta e indiretamente com outras políticas (de desenvolvimento regional, ambientais, agrícolas etc.). Os PM e os POAP atuam em espaços geográficos definidos e gerenciados, permeados por uma rede sociopolítica complexa. Esses instrumentos de ordenamento devem levar em consideração as políticas, programas e planos territoriais e setoriais dos diferentes níveis (nacional, regionais e locais), verificando se esses planos afetam de maneira positiva ou negativa a gestão dessas áreas e buscando formas de compatibilizá-los com seus objetivos (Esteves, 2015). Ao mesmo tempo, os planos de ordenamento territorial devem observar a presença de áreas protegidas e levar em conta os seus planos de gestão (Phillips, 2002). A primeira metade desta tese é dedicada a analisar se os planos e políticas se integram e se esses instrumentos abordam a gestão do risco às áreas protegidas. Em estágio seguinte, após a definição de metodologias de apoio, este trabalho propõe um modelo de análise do risco e de elaboração de cartografia de risco de incêndio florestal para a Floresta Nacional de Ipanema e o Parque Natural do Alvão, tendo como base principal os planos de manejo e de ordenamento dessas áreas. Os incêndios florestais em áreas protegidas interferem na conservação desses territórios e originam outras ameaças, como a fragmentação florestal e a perda de biodiversidade. As relações entre as ameaças às duas áreas protegidas foram identificadas e analisadas integradamente, com base nos planos de manejo e de ordenamento, em publicações científicas, em relatórios de oficinas participativas e em entrevistas com gestores e técnicos, o que possibilitou estabelecer uma rede de ameaças e suas origens. Em seguida, foi mapeada para cada AP a localização das ameaças internas e externas e seus graus de influência, gerando cartas de criticidade que puderam, então, ser cruzadas com as cartas de capacidade de suporte, de perigosidade e de valor, para a criação das cartas de risco de incêndio florestal. A partir delas, foram elaboradas, também, cartas de risco de fragmentação florestal das áreas estudadas, revelando os setores prioritários para intervenções de proteção e de conservação. Com a possibilidade da aplicação da metodologia de avaliação de risco de incêndio florestal proposta por este trabalho em outras AP do Brasil e de Portugal, esta tese se configura como uma ferramenta de apoio na execução de políticas públicas voltadas à gestão de áreas protegidas.

Land Use and Cover Change (LUCC) occurs as a consequence of both natural and human activities, causing impacts on biophysical and agricultural resources. In enlarged urban regions, the major changes are those that occur from agriculture to urban uses. Urban uses compete with rural ones due among others, to population growth and housing demand. This competition and the rapid nature of change can lead to fragmented and scattered land use development generating new challenges, for example, concerning food security, soil and biodiversity preservation, among others. Landowners play a key role in LUCC. In peri-urban contexts, three interrelated key actors are pre-eminent in LUCC complex process: 1) investors or developers, who are waiting to take advantage of urban development to obtain the highest profit margin. They rely on population growth, housing demand and spatial planning strategies; 2) farmers, who are affected by urban development and intend to capitalise on their investment, or farmers who own property for amenity and lifestyle values; 3) and at a broader scale, land use planners/ decision-makers. Farmers’ participation in the real estate market as buyers, sellers or developers and in the land renting market has major implications for LUCC because they have the capacity for financial investment and to control future agricultural land use. Several studies have analysed farmer decision-making processes in peri-urban regions. These studies identified agricultural areas as the most vulnerable to changes, and where farmers are presented with the choice of maintaining their agricultural activities and maximising the production potential of their crops or selling their farmland to land investors. Also, some evaluate the behavioural response of peri-urban farmers to urban development, and income from agricultural production, agritourism, and off-farm employment. Uncertainty about future land profits is a major motivator for decisions to transform farmland into urban development. Thus, LUCC occurs when the value of expected urban development rents exceeds the value of agricultural ones. Some studies have considered two main approaches in analysing farmer decisions: how drivers influence farmer’s decisions; and how their decisions influence LUCC. To analyse farmers’ decisions is to acknowledge the present and future trends and their potential spatial impacts. Simulation models, using cellular automata (CA), artificial neural networks (ANN) or agent-based systems (ABM) are commonly used. This PhD research aims to propose a model to understand the agricultural land-use change in a peri-urban context. We seek to understand how human drivers (e.g., demographic, economic, planning) and biophysical drivers can affect farmer’s intentions regarding the future agricultural land and model those intentions. This study presents an exploratory analysis aimed at understanding the complex dynamics of LUCC based on farmers’ intentions when they are faced with four scenarios with the time horizon of 2025: the A0 scenario – based on current demographic, social and economic trends and investigating what happens if conditions are maintained (BAU); the A1 scenario – based on a regional food security; the A2 scenario – based on climate change; and the B0 scenario – based on farming under urban pressure, and investigating what happens if people start to move to rural areas. These scenarios were selected because of the early urbanisation of the study area, as a consequence of economic, social and demographic development; and because of the interest in preserving and maintaining agriculture as an essential resource. Also, Torres Vedras represents one of the leading suppliers of agricultural goods (mainly fresh fruits, vegetables, and wine) in Portugal. To model LUCC a CA-Markov, an ANN-multilayer perceptron, and an ABM approach were applied. Our results suggest that significant LUCC will occur depending on farmers’ intentions in different scenarios. The highlights are: (1) the highest growth in permanently irrigated land in the A1 scenario; (2) the most significant drop in non-irrigated arable land, and the highest growth in the forest and semi-natural areas in the A2 scenario; and (3) the greatest urban growth was recognised in the B0 scenario. To verify if the fitting simulations performed well, statistical analysis to measure agreement and quantity-allocation disagreements and a participatory workshop with local stakeholders to validate the achieved results were applied. These outcomes could provide decision-makers with the capacity to observe different possible futures in ‘what if’ scenarios, allowing them to anticipate future uncertainties, and consequently allowing them the possibility to choose the more desirable future.