Repositório RCAAP

As perturbações psicóticas são, pelo seu quadro clínico e impacto na vida dos doentes, as mais graves perturbações em Psiquiatria, com uma prevalência de cerca de 3%. Estudos demonstram que as taxas de mortalidade nestes doentes estão aumentadas 2 a 3 vezes em relação à população geral, vivendo os doentes psicóticos entre 10 a 25 menos anos em comparação com a população geral. O risco de mortalidade nas perturbações psicóticas é particularmente elevado nos primeiros anos de perturbação. As principais causas deste excesso de mortalidade incluem as doenças cardiovasculares, nomeadamente doença isquémica coronária, e o suicídio. Este trabalho investigou a prevalência, evolução e possíveis fatores preditores do comportamento suicida e síndroma metabólica, reconhecidos fatores de risco para suicídio e doença cardiovascular respetivamente, nos 12 meses seguintes a um primeiro episódio psicótico. Trata-se de um estudo longitudinal, que incluiu doentes com primeiro episódio psicótico afetivo e não afetivo de duas equipas de intervenção precoce na psicose na área metropolitana de Lisboa, Portugal. Os doentes foram submetidos a uma avaliação compreensiva que incluiu dados socio-demográficos e clínicos, em dois momentos distintos: uma avaliação realizada na baseline, logo que existiam condições clínicas para esta durante o primeiro episódio psicótico, e uma segunda avaliação 12 meses após a primeira. O comportamento suicida foi investigado por um questionário sobre ideação suicida, plano suicida e tentativa de suicídio. A síndroma metabólica foi avaliada usando os critérios da definição modificada da National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III). Os resultados mostraram uma prevalência de comportamento suicida de 25,4% na avaliação baseline, com uma significativa redução para 13,3% (p=0,035) na avaliação a 12 meses. A análise logística binária multivariada mostrou que a história prévia de comportamento suicida e diagnóstico de depressão no primeiro episódio psicótico foram preditores de comportamento suicida no momento da avaliação baseline, e a história prévia de comportamento suicida e baixos níveis de colesterol total na baseline mostraram ser preditores de comportamento suicida 12 meses após o primeiro episódio psicótico. Em relação à prevalência da síndroma metabólica encontrou-se um aumento de 6,7% na avaliação baseline para 11,7% na avaliação a 12 meses (p=0,250) As prevalências de valores não normais de perímetro abdominal (p=0,039), triglicéridos (p=0.040) e HDL (p=0,019) aumentaram significativamente da avaliação baseline para a avaliação a 12 meses. Todos os parâmetros metabólicos e antropométricos estudados (triglicéridos, HDL, pressão arterial, perímetro abdominal e glucose em jejum) agravaram durante o período de follow-up, mas apenas de forma estatisticamente significativa o perímetro abdominal, triglicéridos e HDL. Apenas o valor médio do perímetro abdominal foi na avaliação a 12 meses não normal (valor médio: 102,50cm±99,41; valores não normais: homem≥102 cm e mulheres≥88cm). Não se identificaram preditores na baseline para o desenvolvimento de síndroma metabólica na avaliação a 12 meses. Este trabalho mostrou uma prevalência elevada de comportamento suicida no ano seguinte ao primeiro episódio psicótico, com uma redução durante o período de follow-up, mas ainda com uma taxa significativa aos 12 meses. Os nossos resultados também mostraram valores elevados das alterações metabólicas com rápido agravamento no primeiro ano após o primeiro episódio psicótico, apesar da não existência de significância estatística para a síndroma metabólica. O perímetro abdominal, indicador de obesidade, foi o parâmetro metabólico estudado que mais significativamente aumentou durante os 12 meses de follow-up, parecendo indicar que este é um fator primário da síndroma metabólica, sendo os restantes elementos secundários aquele. Os nossos resultados mostram que doentes com primeiro episódio psicótico com história prévia de comportamento suicida, depressão e baixos níveis de colesterol total no primeiro episódio psicótico estão em risco particularmente elevado de comportamento suicida, e consequentemente de suicídio, nos 12 meses seguintes ao primeiro episódio psicótico. Este grupo de doentes beneficiará potencialmente de tratamento adequado no sentido da sua proteção e redução do risco de suicídio. Os nossos resultados indicam também que deve existir uma cuidadosa monitorização dos parâmetros metabólicos nos primeiros 12 meses após o primeiro episódio psicótico, aqueles que apresentem critérios de síndroma metabólica, devem ser submetidos a um plano de intervenção sobre os estilos de vida e tratamento. A investigação futura deve procurar replicar os resultados encontrados e ampliar o conhecimento em relação a intervenções que sejam particularmente eficazes neste grupo de doentes com primeiro episódio psicótico e risco elevado de doença cardiovascular e/ou suicídio. Os estudos futuros devem ainda avaliar se estas intervenções têm impacto, como esperado, na redução da mortalidade dos doentes psicóticos, nomeadamente nas fases iniciais destas perturbações.

A saúde ambiental é influenciada por interações complexas indicadores de saúde e indicadores do ambiente natural e construído. Contudo, existe pouca investigação sobre as especificidades dessas relações nas populações que residem em contexto urbano. A crescente urbanização e a industrialização que se tem observado têm criado vários problemas de saúde ambiental e ameaçam não só o desenvolvimento sustentável do ambiente, como afetam a nossa saúde a médio e longo prazo. Melhorar a saúde ambiental e monitorizar as relações entre ambiente natural e construído com a saúde da população tem sido o mote de várias medidas legislativas globais e tornou-se também uma prioridade para as autoridades locais e indústrias. No entanto, é incontestável que existe uma lacuna no conhecimento atual sobre quias os indicadores ambientais adequados para uma monitorização efetiva da saúde ambiental em ambientes urbanos. Esta lacuna dificulta a tomada de decisão por parte dos decisores políticos e indústrias relativamente a intervenções nacionais e locais com vista à melhoria da saúde ambiental. Com o objetivo de contribuir para o aumento do conhecimento sobre a monitorização adequada da saúde ambiental em Lisboa foram realizados três estudos diferentes envolvendo evidência científica e opiniões de especialistas e stakeholders de diferentes áreas de conhecimento. Estudo 1) Este estudo teve como objetivo principal fazer uma revisão sistemática da literatura para identificar os principais determinantes e indicadores ambientais com evidência de estarem associados a impactos na saúde da população, em contextos urbanos. Esta revisão sistemática da literatura permitiu também compreender as potenciais implicações destas interações nas políticas de saúde. Para isso, foi realizada uma pesquisa de literatura publicada nas bases de dados PubMed, Web of Science, Scopus e SciELO Portugal entre 2008 e 2018. Esta pesquisa resultou em 94 estudos em que foram identificadas as associações entre determinantes ambientais e resultados em saúde de populações residentes em ambientes urbanos. Este trabalho permitiu identificar associações positivas entre indicadores socioeconómicos, do ambiente natural, ambiente construídos, serviços de saúde e comportamentais com indicadores de saúde, entre eles mortalidade e morbilidade. Os resultados desta análise permitem inferir que melhorias no rendimento, no nível escolar, na qualidade do ar, no tipo de ocupação laboral, na mobilidade e nos hábitos tabágicos têm um impacto positivo nos indicadores gerais de mortalidade e de morbilidade por doenças crónicas. Estudo 2) Este estudo permitiu selecionar os indicadores adequados para a monitorização e avaliação da saúde ambiental na cidade de Lisboa. Partindo da evidência recolhida no estudo anterior e focando em indicadores de ambiente natural e construído foi realizada uma pesquisa nas bases de dados nacionais e da cidade de Lisboa para complementar a evidência recolhida. Tendo sido identificado um grande número de indicadores, optou-se por fazer uma avaliação prévia da informação através da realização de 12 entrevistas individuais e semiestruturadas com especialistas portugueses. Nestas entrevistas foi pedido a cada um dos especialistas que analisasse a informação recolhida e identificasse quais os indicadores necessários para monitorizar de forma adequada a saúde ambiental em Lisboa. Para validar os resultados das entrevistas foi realizado um processo de Web-Delphi com duas rondas, que envolveu um conjunto alargado de especialistas de diferentes áreas de conhecimento (22 especialistas). Neste processo de Web-Delphi foram validados 17 indicadores, 6 indicadores de ambiente construído e 11 indicadores de ambiente natural. Os resultados deste método participativo apontam para lacunas na recolha dos dados dos indicadores de ruido e mobilidade e levantam questões emergentes relativas a indicadores de habitação que requerem mais investigação. Estudo 3) Este trabalho derivou da necessidade de implementar ferramentas para monitorização da saúde ambiental identificada nos estudos anteriores. Para definir os requisitos para construção de um dashboard para monitorizar a saúde ambiental para a cidade de Lisboa foi implementada uma abordagem centrada no utilizador. Esta abordagem envolveu potenciais utilizadores de instituições locais na identificação e discussão das preferências de visualização dos indicadores de ambiente natural e construído. Foram realizadas três entrevistas de grupo online, com onze potenciais utilizadores. Nessas entrevistas foram utilizados cartões que continham diferentes opções de visualização para cada um dos indicadores validados. O feedback obtido nestas entrevistas foi analisado e utilizado para construir um framework com onze requisitos de design para construir um dashboard de monitorização da saúde ambiental em Lisboa. O framework que resultou deste processo permitirá construir uma ferramenta eficaz e flexível para ser utilizada por decisores políticos, indústrias e organizações locais. Em suma, este trabalho permitiu identificar os indicadores essenciais para monitorizar a saúde ambiental no contexto urbano de Lisboa além de ter permitido identificar uma serie de requisitos para a construção de um dashboard. A combinação de métodos participativos descritos ao longo deste projeto pode ser utilizada para quer para validar informação quer para ajudar a estruturar a construção de ferramentas para apoio à decisão. Podem também servir como referência para outros trabalhos de investigação em outros contextos. Este trabalho demonstra que a melhoria da saúde ambiental pode aliviar a sobrecarga sentida pelos sistemas de saúde e contribuir para melhorias em outros sectores como o desenvolvimento económico, melhoria de processos industriais e mitigação das alterações climáticas. Contudo, deixa também claro que intervenções que visem melhorar a saúde ambiental têm de ter por base a integração de evidência e de ferramentas que incluam os contributos da comunidade científica e institucional.

De entre as plantas aromáticas da Flora de um montado inclui-se a família Lamiaceæ, onde encontramos o género Lavandula. A taxonomia do género Lavandula tem sofrido diversas alterações, devido à sua variabilidade morfológica e capacidade de hidridização. Com este trabalho pretendeu-se aprofundar o estudo de duas espécies, L. luisieri e L. pedunculata, realizando uma abordagem conjunta através do estudo químico dos seus óleos essenciais e de análise micromorfológica das suas flores. Os óleos essenciais de várias amostras de ambas as espécies de Lavandula colhidas em anos consecutivos foram isolados por hidrodestilação, examinados por CGL, identificados por CGL/EM e sujeitos a análise aglomerativa usando o programa NTSYS. A morfologia e distribuição do indumento de estruturas florais de ambas as espécies foram analisados usando as técnicas de MEV e MOV. Para o estudo histoquímico das duas espécies, utilizaram-se testes destinados à identificação de determinados grupos químicos, observando-se os resultados em MOV. Observou-se ainda a autofluorescência e fluorescência induzida com reagente de NEU em MOF. Os óleos essenciais das amostras de L. luisieri e L. pedunculata foram obtidos num intervalo de rendimentos de v-1% e 2% (v/ p.f.). Os monoterpenos oxigenados foram detectados em maior percentagem tanto para L. luisieri (33-57%), como para L. pedunculata (90-98%). O conjunto de todos os óleos essenciais foram agrupados em três clusters (cluster I – L. pedunculata, clusters II e III – L. luisieri). O componente com maior percentagem relativa no cluster I foi a fenchona (62-70%), no cluster II o acetato de trans-α- necrodilo (4-20%) e no cluster III o 1,8-cineol (11-38%). Na caracterização micromorfológica das duas espécies verificou-se que apresentam vários tipos de tricomas não glandulares e glandulares, estes últimos distribuídos maioritariamente nas superfícies abaxiais de sépalas e brácteas férteis. Em L. pedunculata identificaram-se tricomas que não foram encontrados em L. luisieri e correspondem a tricomas capitados tipo III, mistos tipo I e tipo II, e ainda um tipo de tricoma peltado com pedúnculo grande que ainda não havia sido referenciado em termos bibliográficos. Do estudo histoquímico concluiu-se que os grupos de compostos existentes em L. luisieri e L. pedunculata foram: polissacáridos totais, pectinas, mucilagens, lípidos (totais, ácidos e neutros e ácidos gordos), terpenóides (óleos essenciais e ácidos resínicos e terpenóides com grupo carbonilo), fenóis e alcalóides. Em MOF foi ainda detectada a presença de flavonóides. A maioria destes compostos foi detectada nas cabeças glandulares dos vários tipos de tricomas.

A memória episódica passa por importantes aperfeiçoamentos até a idade adulta, com estudos anteriores a apresentarem diferentes trajetórias de desenvolvimento para a recuperação automática (familiaridade) e controlada (recoleção) de informação mnésica. A recordação controlada ou estratégica é cognitivamente mais exigente, e o seu desenvolvimento mais prolongado parece alinhar-se bem com a maturação tardia das regiões do cérebro responsáveis pelo controlo executivo (nomeadamente, o córtex pré-frontal). No entanto, a investigação nesta área, tanto ao nível cognitivo como neuronal, tem-se concentrado principalmente na infância, sendo difícil encontrar na literatura diferenças na recuperação mnésica entre adolescentes e adultos/as. Para responder a esta lacuna, investigámos o desenvolvimento da memória episódica, nomeadamente as suas componentes automática e controlada, com recurso a tarefas de memória de item, associativa e da fonte, em adolescentes (13-17 anos de idade) e jovens adultos/as (20-30 anos de idade). Examinámos também como é que o funcionamento da memória episódica interage com a memória semântica e o controlo cognitivo. A nível neuronal, construímos uma rede intrínseca de memória episódica (iEMN, na sigla inglesa) recorrendo à ressonância magnética funcional (fMRI, na sigla inglesa) durante o estado de repouso, e analisámos a maturação dessa rede entre a adolescência e a idade adulta. Os estudos comportamentais demonstraram de forma consistente que, enquanto a recuperação mnésica automática parece já estar consolidada durante a adolescência, a recuperação controlada continua a desenvolver-se ao longo deste período. A elaboração semântica e o controlo inibitório surgiram como mecanismos fundamentais subjacentes ao desenvolvimento tardio de processos controlados de recoleção. Em complemento com estes dados, o estudo de fMRI em estado de repouso revelou a maturação tardia de ligações da iEMN entre o polo temporal direito e o córtex pré-frontal direito. Em conjunto, estes resultados evidenciam trajetórias de desenvolvimento distintas para processos de recuperação mnésica ao longo da adolescência, e que dependem do nível de envolvimento dos sistemas de controlo executivo e de memória semântica. Além disso, os dados de fMRI sugerem que existe uma conectividade funcional ainda em desenvolvimento durante a adolescência, ligando áreas associadas ao processamento semântico (como o polo temporal) e áreas associadas a mecanismos de recuperação controlados (como o córtex pré-frontal). A adolescência evidencia-se assim como um período chave no desenvolvimento neurocognitivo da memória. Os resultados são considerados à luz da literatura existente, discutindo-se o seu contributo para o avanço dos modelos de desenvolvimento da memória.

Ensaiando uma aproximação entre Émile Cioran (1911-1995) e Fernando Pessoa (1888-1935) (e, numa perspectiva mais abrangente, uma fértil articulação entre literatura e filosofia), o presente volume procura equacionar de que modo ambos estes autores convergem numa desconstrução radical do paradigma racional e civilizacional dominante, pensando criticamente nas margens da filosofia e abrindo passagens para os seus outros, desde a poesia e a literatura à experiência extática e mística, sem enquadramento religioso ou teológico. Figuras resolutamente excêntricas (no sentido etimológico do termo: "ekkentro"s, aquele que se situa fora do centro), ambos testaram as aporias dos seus respectivos ofícios, Pessoa forjando uma galáxia literária em permanente mutação, ao passo que Cioran jamais cessaria de interpelar os limites do discurso filosófico.

Aproximação à presença tutelar de Ovídio na poesia de António Franco Alexandre (1944-), com particular ênfase no volume poético "Aracne" (2004), Lisboa, Assírio & Alvim.

Malaria and sleeping sickness, infectious diseases caused by Plasmodium parasites and Trypanosoma brucei, respectively, share geographical space in sub-Saharan Africa. Malaria is not only a major threat to public health globally but also the most common infectious disease to occur in patients with sleeping sickness. The host labs have previously shown that a T. brucei infection impairs a secondary P. berghei liver infection and decreases malaria severity in mice. However, whether this effect requires an active trypanosome infection remained unknown. Here we sought to assess whether an active T. brucei infection was required for this impairment, and to unravel the mechanism behind this phenomenon. We found that Plasmodium liver infection can also be inhibited by the serum of a mouse previously infected by T. brucei and by total protein lysates of two life cycle stages of this kinetoplastid. Additionally, mice that received T. brucei total lysates were partly protected from developing severe malaria pathology. Interestingly, the presence of total lysates of T. brucei results in a decrease in the number of infected hepatocytes rather than from an impairment of intrahepatic replication of Plasmodium parasites. Surprisingly, the phenotype observed in this co-infection appears to be independent of liver damage and the of the host’simmune response. Biochemical characterization showed that the anti Plasmodium activity of the total T. brucei lysates depends on its protein fraction, but is independent of the abundant variant surface glycoprotein and other GPI-anchored proteins. Finally, we found that the protein(s) responsible for the inhibition of Plasmodium infection is/are present within a fraction of ~350 proteins that are excreted to the bloodstream of the host. We conclude that the defence mechanism developed by trypanosomes against Plasmodium relies on protein excretion. Our study paves the way to the development of novel antiplasmodial intervention strategies, based on the mechanism involved during the co-infection between T. brucei and Plasmodium.

Type 2 innate lymphoid cells (ILC2s) are essential for the immune response to helminthic parasites, in allergy settings and for the control of adipose tissue homeostasis. However, how ILC2s perceive environmental cues and integrate signals to maintain tissue homeostasis remains poorly understood. Here we show that adipose tissue ILC2s are controlled by the sympathetic tone through the production of neurotrophic factors by mesenchymal stromal cells (MSCs). The gonadal adipose tissue is densely innervated by sympathetic neurons that have discrete central nervous system origins. Ablation of one of such brain centers coordinating the sympathetic output to the adipose tissue, the paraventricular nucleus of the hypothalamus, results in decreased ILC2 function. Thus, ILC2 function in the gonadal adipose tissue is controlled by brain-derived nervous system signals. We dissected the mechanisms behind this regulation, finding that activation of sympathetic neurons leads to an increase in cytokine-producing ILC2s independently of direct β2 adrenergic receptor signaling. Instead, β2 adrenergic receptor activation in a population of MSCs leads to the production of neurotrophic factors by these cells. Conversely, sympathetic ablation or inactivation reduces cytokine-producing ILC2s and production of neurotrophic factors by MSCs. These neurotrophic factors, namely glial derived neurotrophic factor (GDNF), has been described as limiting obesity in previous research. GDNF and the glial family ligands produced by MSCs signal through the tyrosine kinase receptor RET. GDNF activates RET on ILC2s, stimulating cytokine production. Deleting RET signaling on these cells results in reduced frequencies of cytokine-producing ILC2s and RET activation as the opposite effect. Ultimately, RET-dependent cell-autonomous signals on ILC2s control adipose tissue physiology. This is demonstrated by the absence of RET in ILC2s which leads to increased susceptibility to high fat diet-induced obesity with systemic consequences. This work sheds light into a novel neuro-mesenchymal-immune regulatory axis that integrates environmental sensing by the nervous system impacting on adipose tissue homeostasis through the control of ILC2 function by MSCs.

Psychosis is a severe mental condition characterized by a complex set of disturbances of thinking, perception, affect and social behaviour. It is usually preceded by a prodromal phase lasting months to years and in which patients are clinically identified has being ‘At Risk Mental State’ (ARMS). Retrospective studies have showed that ARMS individuals have a 30% risk of transition to psychosis within the first 2 years after presentation to clinical services. Moreover, several neuroimaging, genetic and environmental biomarkers have been independently associated with the onset of psychosis in the ARMS. However, at present there is yet no established method for predicting which individuals will develop the illness and which will not – which would allow cost-efficient targeting of early intervention therapies. Furthermore, a few studies have demonstrated the feasibility to predict psychosis transition from an ARMS using structural magnetic resonance imaging (sMRI) data and machine learning (ML) methods. However, the reliability of these findings is unclear due to possible sampling bias. Moreover, the value of genetic and environmental data in predicting transition to psychosis from an ARMS is yet to be explored. In this study I aimed at predicting transition to psychosis from an ARMS using ML and quantitative data – neuroimaging, genetics, and environment – as predictors. I used several samples (one for each modality – neuroimaging, genetics or environment) drawn from a pool of 246 subjects identified as being at an ARMS when they first sought clinical help (i.e. at baseline). Subjects were clinically identified as transitioned to psychosis (ARMS-T, 60 subjects) if they later presented a first episode of psychosis (FEP) or as nottransitioned to psychosis (ARMS-NT, 186 subjects) if they did not present a FEP within at least a period of 2 years. Structural magnetic resonance imaging, genome-wide genotypes and environmental risk assessment data was collected from the ARMS subjects at baseline. Then, the modality-specific value in predicting transition to psychosis was evaluated using a) several feature types [regional and voxel-based grey matter and white matter volumes, and regional cortical thickness, and brain gyrification, sulci depth and complexity indexes (neuroimaging); a polygenic risk score (PRS) for schizophrenia, a list of psychosisassociated single nucleotide polymorphisms (SNP), and a list of psychosis-associated genes for which several brain tissue-specific expression quantitative trait loci (eQTL) scores were extracted (genetics); and an environmental risk score (ERS) for schizophrenia, and a list of environmental risks factors (environment)], b) several feature manipulation strategies [feature dimensionality reduction through principal component analysis, no feature selection, and forward feature selection (neuroimaging), and embedded feature selection (genetics and environment)], c) several ML algorithms [linear support vector machines (neuroimaging), elastic-net and simple logistic regression (genetics and environment)], d) several cross-validation (CV) strategies [5-fold CV and leave-one scanning acquisition protocol-out (neuroimaging) and leave-one per group, i.e. 1 ARMS-T and 1 ARMS-NT,-out (neuroimaging, genetics and environment)], e) sample balancing, i.e. same number of ARMS-T and ARMS-NT subjects, and f) bootstrapping, i.e. 5 (neuroimaging) or 100 (genetics and environment) semi-random subsamples drawn from the original pool. Then, only the modalities whose classification models showed a balanced accuracy across bootstrapped samples statistically better than chance level were included in a multimodal classification model. Overall, this study’s results showed that only genetics, and when using a set of psychosisassociated SNPs, could predict the transition to psychosis from an ARMS marginally better than chance, albeit with no clinical significance, (balanced accuracy = 53%, diagnostic odds ratio = 3.3 – averaged across bootstrapped samples). Furthermore, the environmental and neuroimaging alone could not predict psychosis from an ARMS, statistically better than chance. Therefore, no multimodal classification model was trained/tested. Moreover, and unexpectedly, I could not replicate previous findings showing the usefulness of structural MRI in predicting transition to psychosis from an ARMS using ML. Therefore, my results suggest that: a) genetic data may be promising for predicting transition to psychosis from an ARMS; and b) the value of structural MRI data in predicting psychosis from an ARMS, as suggested by previous evidence, should be reconsidered. Finally, this study serves as a proofof-concept on how multimodal quantitative data can be used to predict psychosis development already from a prodromal stage and should be replicated in larger ARMs samples.

The genomic revolution marked the beginning of the 21st century in biology and medicine. Completion of the Human Genome Project in 2003 has brought a flood of technological innovations that enabled decoding the entire genetic information in health and disease, leading to the foundation of the precision medicine movement. Genomics has revolutionized cancer research and transformed our understanding of how cancer arises. In recent years cancers have been re-classified based on their mutations, and a multitude of new drugs were developed that target specific molecular features of the tumor. Accurate and sensitive assays for cancer genotyping are crucial to enable precision oncology. Moreover, in addition to genotyping tissue samples from the primary tumor and metastasis, methodologies for cancer genotyping in circulating blood and other biological fluids are attracting much attention. This project was initiated with the goal of implementing and validating cutting-edge methodological approaches for profiling tumor DNA in the clinic. First, we used Sanger sequencing to determine the frequency of rare mutations in the gene that encodes epidermal growth factor receptor (EGFR) that are not detected by the widely used PCR-based Idylla™ EGFR Mutation Assay. Mutations in the EGFR gene are biomarkers that predict how non-small cell lung cancer (NSCLC) patients respond to EGFR-targeted therapies collectively known as tyrosine kinase inhibitors (TKIs). Thus, EGFR genotyping provides crucial information for treatment decision. Both Sanger sequencing and real-time PCR methodologies are used for EGFR genotyping. However, methods based on real-time PCR have limitations, as they may not detect rare or novel mutations. We sought to determine the prevalence of rare mutations in the tyrosine kinase domain (exons 18 to 21) of the EGFR gene not targeted by the most frequently used real-time PCR approaches, i.e., the cobas® EGFR Mutation Test, and the Idylla™ EGFR Mutation Assay. A total of 1228 NSCLC patients were screened for mutations in exons 18 to 21 of the EGFR gene using Sanger sequencing. We observed that 252 patients (~20%) had at least one mutation in the EGFR gene, and 38 (~3%) carried uncommon genetic alterations that could not be identified by the cobas® or the Idylla™ tests. We further found six new single mutations and seven previously unreported compound mutations. Clinical information and patient outcome are presented for these cases. In conclusion, this study highlights the value of sequencing based approaches to identify rare mutations. Our results add to the inventory of known EGFR mutations, thus contributing to improved lung cancer precision treatment. After the detection of an EGFR-TKI-sensitive mutation, at initial diagnosis, and after treatment with 1st or 2 nd generation inhibitors, about half of tumors develop a resistance mutation. To identify this mutation, so that the treatment can be readjusted, it is mandatory to carefully choose the sample and the method to be used. Therefore, we implemented and assessed the performance of a droplet digital PCR (ddPCR) assay for detecting the EGFR T790M mutation in liquid biopsies. Liquid biopsy allows the identification of targetable cancer mutations in a minimally invasive manner. In patients with NSCLC, ddPCR is increasingly used to genotype the EGFR gene in circulating cell-free DNA (cfDNA). However, the sensitivity of this method is still under debate. We optimized a ddPCR assay and used it to detect the EGFR T790M mutation in plasma samples from 77 patients with NSCLC in progression. Our ddPCR assay enabled the detection and quantification of the EGFR T790M mutation at cfDNA allele frequency as low as 0.5%. The mutation was detected in 40 plasma samples, corresponding to a positivity rate of 52%. The number of mutant molecules per mL of plasma ranged from 1 to 6,000. A re-biopsy was analyzed for 12 patients that had a negative plasma test and the mutation was detected in 2 cases. A second liquid biopsy was performed for 6 patients and the mutation was detected in 3 cases. In conclusion, this study highlights the value of ddPCR to detect and quantify the EGFR T790M mutation in liquid biopsies in a real-world clinical setting. Our results suggest that repeated ddPCR tests in cfDNA may obviate tissue re-biopsy in patients unable to provide a tumor tissue sample suitable for molecular analysis. Despite the advantages above described, the major disadvantage of ddPCR is the difficulty in testing multiple targets simultaneously. As such, we explored massively parallel sequencing methodologies for mutation discovery in cfDNA extracted from circulating blood. We analysed a patient with metastatic breast cancer who was selected for enrolment in an open-label clinical phase IIIb trial with ribociclib combined with letrozole. We genotyped cfDNA isolated from blood samples collected before (Pre_cfDNA) and after (Post_cfDNA) treatment with ribociclib and letrozole. Analysis of Pre_cfDNA using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) revealed two alterations: a missense hotspot mutation in the PIK3CA gene (3:178952085, A>G, H1047R) and a copy number amplification of the CCND1 gene. The presence of both molecular alterations was confirmed in the primary tumor. After treatment, the patient presented a significant clinical improvement, and the two alterations were no longer detected in cfDNA (Post_cfDNA). This work underlines that the minimally invasive nature of liquid biopsy allows monitoring of disease progression, demonstrating a practical alternative to tissue biopsy. The use of massively parallel sequencing enables to interrogate of several genes simultaneously, the search for different types of mutation, and identify variants with low allele frequency. Based on the previously mentioned results, we decided to implement a targeted ddPCR assay for detecting PIK3CA mutations in cfDNA. We used this assay to screen urine samples from the patient with a PIK3CA mutation identified in the plasma, but we failed to detect any positive result in the urine. In conclusion, we implemented at GenoMed the main methods that are currently recommended for molecular diagnosis in Precision Oncology, from massively parallel sequencing to ddPCR. We characterized the main advantages and limitations of each technology applied to a variety of biological samples including tumor tissue, blood, and other fluids. Taken together, the results of our work represent an important contribution towards a more achievable and cost-effective Precision Medicine in the context of real world clinical practice.

Integrated care of chronic lung disease management comprises pulmonary rehabilitation (PR), which is an individually tailored intervention including exercise training and self-management education, purposeful on long-term adherence to health behaviour change. There is high scientific evidence that PR improves dyspnea, health status, exercise tolerance and anxiety and depression levels in people with chronic obstructive pulmonary disease (COPD), one of the most prevalent respiratory conditions worldwide. Self-efficacy (SE), defined as one’s belief of being capable of doing something to accomplish a desired goal, is considered a core outcome of self-management interventions, and a clinical and functional determinant of physical activity (PA) in people with COPD. Telemonitoring (TM) and telerehabilitation are telemedicine subsets of digital health supportive environments, that can be designed to meet high ecological validity of assessments and interventions, with potential impact on healthcare effectiveness. The aim of this thesis was to study the impact of PR on SE of people with COPD, including digitally health supportive environments such as TM and pulmonary telerehabilitation (PTR). Study I accomplished the Portuguese validation of the Pulmonary Rehabilitation Adapted-Index of Self-Efficacy (PRAISE) tool, translating, and culturally adapting this SE instrument specific to PR into European Portuguese, by means of forward-backward translation, pilot testing and content validity through a multidisciplinary panel of expert judges. Reliability and construct validity was investigated on a cross-sectional study with 150 Portuguese PR outpatients by means of statistical exploratory factorial analysis. Results were a 4-factor solution suggesting discriminative SE qualities that cumulatively explained 52.3% of SE total variance and showed a PRAISE reliability of Cronbach alpha of 0.78. To refine the accuracy of daily PA assessment methodology, Study II was a cross-sectional study with 100 PR outpatients comparing subjective and objective measures of PA as a vital sign before an exercise-based intervention. Regardless of self-reported PA and International Physical Activity Questionnaire (IPAQ) results, a 4-day continuous combined oximetry-accelerometery TM showed that patients spend at least a mean of 70% of daytime with activities below 2 metabolic equivalent of task (METs) and a mean sedentary time of 5.8 ± 2.7h/day. Moreover, as it was found that 93% of patients had daily episodes of oxygen desaturation, Study III further extended the same research by comparing free-living TM assessments with hospital six-minute walk test (6MWT) results. TM identified 24% more cases of oxygen desaturation compared with the 6MWT, detecting lower 7.2 ± 8.4% peripheral oxygen saturation, higher 9.3 ± 15.5% peak heart rate for more intense 0.3 ± 0.8 METs engaged PA. Also, there were 27% of patients with TM daily oxygen desaturation episodes undetected by the 6MWT, a finding that requires further attention on oximetry-guided interventions, such as telemedicine algorithms, oxygen therapy titration and regular PA assessment in PR. Study IV was a participatory research on PTR including patients within stakeholders, aiming to develop strategies for setting up a person-centred digitally enabled model of care purposeful to enhance SE. This was a proof-of-concept and proof-of-implementation research, providing tacit knowledge about five types of requirements for PTR implementation and related operational strategies: PR core principles, quality and security standards, technological functionality, home environment appropriateness and telesetting skills. Patient’s mean level of satisfaction about PR goals achievement was of 88.1 ± 8.6% and of 95.4 ± 6.3% regarding experienced PTR. Upon the arrival of COVID-19 pandemic, Study V included 100 PR hospital outpatients with suspended face-to-face PR in a prospective quasi-experimental design, including patients discharged and those shifted from face-to-face to PTR. PRAISE was applied as a follow-up screening criteria, together with surveys on the beginning of the outbreak and at 4-month follow-up. Results were that in response to the first ever COVID-19 lockdown, highest follow-up priority should be given to sedentary patients with lower SE, focusing on self-management to increase PA. At 4 months follow-up, 66% of patients suspended breathing exercise routines and 47% discontinued PA, with SE preserved on at least 70% of patients. Patient reported experience about shifting to PTR included «no need for time or means of transportation» and «sense of not being forgotten or left behind» as enablers, whereas «self-execution of the exercises» and «lack of equipment for exercise» as barriers. PTR would be recommended to others by 97.9% of these patients. Overall, 40% of patients who experienced PTR would prefer a hybrid model on a pandemic-free future, whereas 49.2% of those who had been discharged have future interest in PTR. Overall, it can be concluded that PR integrating digital health supportive environments such as TM and telerehabilitation, has potential impact on SE enhancement of people with chronic respiratory disease, including people with COPD.

A sensibilidade à recompensa alimentar tem recebido interesse crescente como deter minante da obesidade. Estudos pré-clínicos sugerem que o valor de reforço de um alimento é determinado não só pelo sabor, mas também pelo seu conteúdo calórico, um processo mediado pela atividade dopaminérgica. Mas, em humanos, os estudos sobre sensibilidade à recompensa, incluindo a percepção do sabor, caracterizaram-se por grande heterogeneidade e inconsistência nos resultados. O reforço pós-ingestivo estava inexplorado nesta condição. Assim, tive como objectivo estudar I) marcadores gustativos e psicométricos de excesso de peso e preditores de perda peso após a cirúrgica bariátrica; II) reforço pós-ingestivo na obesidade e na cirurgia bariátrica. Os estudos que compõem esta tese incluíram vários grupos de participantes, desde uma amostra populacional, voluntários saudáveis e uma cohort de candidatos a modificação de estilo de vida. Incluiu também cohorts bariátricas, uma multicêntrica prospectiva e outra avaliada transversalmente. Ambas incluiram doentes de bypass e sleeve gástrico. Os méto dos consistiram na avaliação multimodal da sensibilidade à recompensa, incluindo medidas psicométricas, testes psicofísicos de sabor, e, em algumas das cohorts um protocolo para estudo do reforço pós-ingestivo bem como a avaliação da disponibilidade de receptores D2 de dopamina (DD2R-like) cerebrais com recurso a medicina nuclear. Os níveis de fome hedónica associaram-se com a obesidade na amostra populacional, algo que confirmamos numa cohort distinta. A percepção da intensidade do sabor doce também se associou positivamente à obesidade. Adicionalmente, os ratings de intensidade do sabor doce antes da cirurgia associaram-se com a perda de peso aos 18 meses, assim como os scores de fome hedónica. Por último, o reforço pós-ingestivo ocorreu em indivíduos saudáveis, na obesidade e após a cirurgia bariátrica. No entanto, a magnitude do condicionamento associou-se com a disponibilidade de DD2R-like cerebral e a percepção da intensidade do sabor doce, apenas no grupo de bypass gástrico. A sensibilidade à recompensa alimentar é da maior relevância para a compreensão da obesidade. Serão necessários mais estudos para explorar novos alvos terapêuticos nesta área para o tratamento e prevenção da obesidade.

Orientation in space is an important determinant of human behaviour. Spatial delusions are a disrupting form of spatial disorientation characterized by a firm conviction of place mislocation. Patients believe they are in a reduplicated, transformed or dislocated place and are rather insensitive to surrounding incongruences. Spatial delusions are frequent after right hemispheric lesions and in neurodegenerative diseases, but empirical evidence about their presentation and pathophysiology is lacking. Here, we aimed to comprehensively study stroke-associated spatial delusions’ clinical features and neural basis. The first study systematically reviewed the reported evidence about lesion-associated spatial delusions. Then, we performed a prospective screening for spatial delusions in right hemisphere acute stroke patients and were able to identify the largest sample of patients with spatial delusion, to our knowledge. The second study described spatial delusions’ clinical and phenomenological features and analysed their neural correlations (n=60). Most places of mislocation were closer to the patient’s home than to the hospital. The duration of the syndrome was short (median: 3 days, interquartile range: 1-7 days) and moderately correlated with structural disruption of left inferior temporal fibres (r=0.39). Each clinical subtype (i.e., place reduplication, transformation or dislocation) had characteristic response patterns, which were reported, and representative examples were provided. In the third study, we performed a case-control analysis of the structural and functional predictors of the syndrome (stroke patients with spatial delusions, n=64; stroke controls, n=233). Spatial delusions were most strongly predicted by the structural disconnection of two distinct streams, connecting right fronto-thalamic and right occipitotemporal structures. The multivariate model also included age, anosognosia and lesion sparing of right dorsal fronto-parietal regions as independent predictors. It was nested cross-validated with a support-vector machine analysis and a good discrimination accuracy was demonstrated (median area under the curve: 0.80, interquartile range: 0.75–0.85). Cognitively, spatial delusions do not seem to be explained by deficits in single cognitive domains but by a dysfunctional combination of multidomain information. The brain’s functional organization comprises multimodal integrative networks, namely the default mode network, and ventral and dorsal attention networks. The neuroimaging study of their anatomy has taken a predominantly corticocentric approach, but clinical, electrophysiological and phylogenetic-derived data suggest that they have essential subcortical components. In the fourth study, we applied neuroimaging methods of functional alignment to healthy subjects’ resting-state functional MRI and demonstrated that the default mode network comprises a subcortical network that matches the anatomical model of the limbic system. Using a similar methodology, the fifth study revealed that the ventral and dorsal attention networks include the pulvinar, the superior colliculi and group brainstem nuclei whose projections are spatially correlated with the acetylcholine nicotinic receptor and dopamine transporter systems. The sixth study explored the relationship between spatial delusions and acute stroke treatment modalities. We performed a subanalysis of the ischemic stroke sample collected for the second and third studies and found an association between endovascular thrombectomy and occurrence of the syndrome (multivariate logistic regression model including age, clinical severity, vascular territory, inter-hospital transfer and endovenous thrombolysis as covariates; odds ratio: 2.46, 95% confidence interval: 1.18 to 5.16). We demonstrated that shared clusters of lesion and structural disconnection, overlapping right thalamo-orbitofrontal fibres and right anterior temporal areas, mediate the association. These regions are irrigated by proximal middle cerebral artery branches, which seem to be particularly prone to ischemia during clot manipulation and extraction. In conclusion, our results provide a broader characterization of stroke-associated spatial delusions’ clinical and phenomenological features, which may support their appropriate diagnosis. They also shed light on the functional and structural predictors of the syndrome and on the subcortical organization of multimodal integrative functional networks. They may contribute to a better understanding of delusional misidentifications and extend our knowledge about the neurobiology of spatial orientation disorders.

Viruses that infect bacteria (phages) can directly drive host death required for their multiplication or alternatively remain in a dormant state (prophage) inside the host, being vertically transmitted to host offspring. Due to host exploitation, phages are usually seen as parasites. However, as a symbiont, the virus can confer some advantages to the host, such as immunity to further infections caused by related virus. Some authors proposed that lysogens (hosts harbouring prophages) can use their pathogens to harm susceptible conspecifics. This behaviour, in which the actor (lysogenic individual) experiences a cost (death) to cause damage to conspecifics is called spite. In a further extent this behaviour can be seen as indirect altruism, because the actor by displacing competitors allows its relatives to have access to more resources, thus benefiting them. Here we show that when competing with susceptible conspecifics in unstructured habitats, there is an optimum initial frequency of lysogens leading to benefit. Above this optimum, the cost of phage production is higher than the benefit associated to the displacement of susceptibles, while under the optimum frequency the viral production is not sufficient to efficiently outcompete susceptibles. However, when competing in structured habitats, spiteful advantage is always seen at short term. At long term, conversion of susceptibles into lysogens predominates. After conversion, the frequency of lysogens decreases if initially rare, or maintains at a certain level if initially common. We also show that after evolution in conditions, phages become more virulent while bacteria become resistant to the phage. Thus, the evolved strain becomes more competitive. In conclusion, we that indeed there is spiteful behaviour at short term. However, the effect of spite in often cancelled because susceptible individuals became phage-immune by lysogenization.

Kyotorphin (KTP, L-tyrosyl-L-arginine) is an endogenous dipeptide, described for the first time in 1979, as a potent analgesic molecule. Its naloxone-reversible opioid-like analgesic effect is indirectly mediated by the inducing the release of Met-enkephalin (Met-enk). It is currently accepted that KTP acts through a specific Gi-coupled receptor (KTPr), inducing Ca2+ influx in a phospholipase C-mediated process. Moreover, this KTPr-mediated action can be antagonized by the dipeptide L-Leucine-L-Arginine (KTPant). Over the last decades, several studies have been revealing KTP role in the modulation of several mechanisms in the central and peripheric nervous systems. KTP has been described as having an antiepileptic, thermoregulatory, anti-hibernation, behavioral and stress modulatory actions, being these non-opioid-mediated effects. However, the vast majority of KTP research has been explored its potential application in pain treatment. More recently, the link between pain and Alzheimer’s Disease (AD), two conditions with high epidemiologic relevance, supported the use of KTP-related drugs as new therapeutic strategies for AD. Evidence shows that chronic pain aggravates AD, and the limited capacity of AD patients to verbally express and perceive pain can worsen disease progression. AD has proven to be a highly complex neurodegenerative disease, of which brain amyloid plaques, mainly constituted by amyloid beta (Aβ) peptide, and neurofibrillary tangles formed by hyperphosphorylated tau (p-Tau) protein, are the two histopathological hallmarks of this disease. Additionally, excitotoxicity, as well as dysregulation of brain-derived neurotrophic factor (BDNF) signaling, are known to be involved in neurodegenerative disorders such as AD. Similarly to what was observed in patients with persistent pain, AD patients have decreased cerebrospinal fluid (CSF) KTP levels, which are inversely correlated to the increase of p-Tau levels in CSF of those patients. Recently, KTP was suggested to be an endogenous neuroprotective agent. In particular, when intracerebroventricular (i.c.v.) injected, KTP ameliorated memory impairments in a rat model of sporadic AD. However, KTP has a limited capacity to cross the blood-brain barrier (BBB). The potential therapeutic value of KTP as a central nervous system (CNS) drug led to the development of synthetic KTP derivatives, which might cross the BBB. Accordingly, the KTP amidated-derivative, the Amidated-Kyotorphin (KTP-NH2), was designed and produced to overcome the BBB. In this work, the main goal was to explore the therapeutic use of KTP-NH2 as a new drug for AD treatment. It started with the characterization and comparison of the impact of KTP and KTP-NH2 in synaptic function under physiological mimetic conditions. Then, given the particular interest of this work in AD treatment, the neuroprotective potential of both peptides was evaluated upon Aβ-induced AD (Chapter 3). Finally, this work correlated the synaptic mechanisms protected by KTP-NH2 action against Aβ-induced toxicity, and the ameliorated memory impairments observed after the systemic administration of KTP-NH2 in a model of sporadic AD in rat (Chapter 4). Electrophysiological recordings obtained in the CA1 area of hippocampal slices prepared from adult male C57BL/6J mice, pre-exposed or superfused with KTP and KTP-NH2, allowed the characterization of the effects of both peptides on synaptic function under non pathological conditions. Results revealed that for concentrations ranging from 5 nM to 50 µM, the peptides affected the basal synaptic transmission in a concentration-dependent manner. While KTP slightly increased synaptic transmission, with a maximal effect measured at 50 nM, KTP-NH2 had a gradual inhibitory effect. At concentrations of 5 mM, largely in excess of what is likely to occur endogenously, peptides’ action rapidly inhibited synaptic transmission, being these effects reversible. In fact, this inhibitory effect was totally or partially eliminated with the respective removal of KTP or KTP-NH2. Thus, under nonpathological conditions, these findings suggested a different effect on synaptic mechanisms. However, neither KTP (50 nM), nor KTP-NH2 (50 nM), significantly affected: 1) synaptic transmission efficiency, evaluated by input/output curves; 2) short-term plasticity, evaluated by post-tetanic potentiation and paired-pulse facilitation; or 3) glutamate release. Together, these findings suggested that the effects of both peptides on synaptic transmission were likely not directly mediated by pre- or post-synaptic mechanisms. Synaptic plasticity is a key mechanism in memory and learning processes. Long-term potentiation (LTP) was evaluated in hippocampal slices, which were either pre-exposed for 3h or acutely superfused with KTP (50 nM) or KTP-NH2 (50 nM). For testing mimetic AD pathophysiological conditions, hippocampal slices were pre-treated with oligomeric Aβ peptide species (200 nM), one of the most soluble toxic Aβ species. Additionally, dendritic spines were evaluated in cultured cortical neurons, after the treatment with KTP (50 nM) or KTP-NH2 (50 nM), and in the presence or absence of Aβ peptide (25 µM) for 24h. Results demonstrated that KTP-NH2, but not KTP, had a neuroprotective effect against Aβ-induced impairments on LTP magnitude. However, these differences contrasted with the similar molecular neuroprotective effect. The action of both peptides restored the density of dendritic spines affected by the action of the Aβ peptide, without inducing toxic effects on neurons. To evaluate whether the neuroprotective effect of KTP-NH2 (50 nM) over LTP could be antagonized by KTPant, slices were pre-treated for 30 min with KTPant (250 nM). The results revealed that KTPant antagonized the neuroprotective effect of KTP-NH2 over Aβ peptide-induced impairments in LTP. However, it is still unclear if KTPant, alone, had a preventive neuroprotective action against Aβ peptide, or which affected mechanism might prompt toxicity when KTPant was added prior to KTP-NH2. Elevation of intracellular Ca2+ levels are a hallmark of KTP-mediated processes. Thus, calcium imaging technique was used to understand whether the increase in Ca2+ levels could be directly caused by KTP activity and/or whether such increases would be necessary for the emergence of KTP-mediated actions. The results revealed that neither the presence of KTP (50 nM) nor KTP-NH2 (50 nM) affected Ca2+ intracellular levels in neurons, so there were no significant changes in neuronal calcium homeostasis. Calpain overactivation happens due to increased intracellular Ca2+ levels, usually as a consequence of Aβ peptide-induced excitotoxicity. As such, the action of both peptides (50 nM) over calpain in vitro activity was assessed using mice cortical tissue homogenates. Results revealed that neither KTP nor KTP-NH2 impacted calpain activation and, consequently, did not prevent calpain-induced cleavage of the BDNF receptor, the tropomyosin receptor kinase B-full length (TrkB-FL), which has a well-known neuroprotective role, and it is a substrate for calpains under Aβ-induced toxicity. Finally, in a rat model of sporadic AD, systemic administration of KTP-NH2 protected against spatial working-memory and episodic memory deficits, without affecting motor activity or inducing an anxiety-like behavior in the animals. Moreover, this KTP-NH2-induced neuroprotective effect was correlated with the prevention of Aβ-induced deficits, in both LTP magnitude of pre-treated hippocampal slices and spine density of cortical neuronal cultures. In conclusion, the present work collected novel evidence of KTP and KTP-NH2 neuromodulatory effects over synaptic function, highlighting the neuroprotective actions of KTP-NH2. Under physiological mimetic conditions, the absence of effects over synaptic function bolsters the therapeutic potential of both peptides, suggesting the absence of sideeffects upon synaptic transmission. At molecular and functional levels, these findings supported the KTP-NH2 neuroprotective effect (Chapter 3), confirming the results obtained through its systemic administration in a rat model of sporadic AD (Chapter 4), which provides important evidence for the use of KTP-NH2 as a drug for treatment of AD and, eventually, other diseases. In light of the complex pathophysiology of AD, in the future, it will be important to determine whether KTP-NH2 neuroprotective effect is potentiated as a standalone treatment, or if its actions should be included as a broader multidrug treatment regimen. Among other aspects, in the treatment of AD, identifying the time-window for therapeutic intervention with KTP-NH2 will be essential to ascertain whether its action relies on the prevention or if this drug is able to recover the molecular and cognitive deficits present in AD pathophysiology.

Non-infectious uveitis (NIU) comprises a heterogeneous group of inflammatory diseases of the uvea and is commonly believed to result from an imune-mediated response to ocular antigens. Complications of uveitis can be sight-threatening, severely impairing quality of life (QoL). Hence, NIU brings a significant burden for both healthcare systems and affected patients, who tend to be young and professionally active. Regarding the treatment of NIU, the ultimate treatment goal is to reduce ocular inflammation, thus preventing damage to ocular structures and consequent vision loss. Although corticosteroids are the mainstay of therapy, such drugs are often incapable of proper inflammation control and have long-term systemic side effects. Therefore, systemic immunomodulatory treatment, including conventional synthetic (csDMARDs) and biological disease modifying anti-rheumatic drugs (bDMARDs), is currently considered in the therapeutic stepladder approach. Despite scarce evidence on the use of systemic immunomodulatory therapy in NIU, bDMARDs are placed at the top of the treatment algorithms, with evidence supporting its efficacy and safety. Encompassed in bDMARDs, tumour necrosis factor-inhibitors (TNFi) are still used off-label for NIU, except adalimumab (ADA), which has recently been approved for adult non-infectious intermediate, posterior uveitis and panuveitis dependent on and/or resistant to corticosteroids and paediatric chronic NIU. However, some patients present incomplete response and/or intolerance to ADA. Also, most of the available evidence does not come from real-world conditions, and almost all published trials and/or case series focusing on the role of bDMARDs in NIU exclude anterior NIU, for which high-quality evidence is still lacking. Thus, a gap in the existing knowledge has been identified regarding the role of bDMARDs in NIU. In this dissertation, which includes four chapters (Introduction, Results, Integrated Discussion and Scientific and Personal Acknowledgements), we sought to provide novel multiapproach insights into the treatment of NIU with bDMARDs. Our specific aims were: i) to provide state-of-the-art evidence for the use of TNF inhibitors (TNFi) in current and future clinical practice (considering novel drug delivery routes), ii) to describe the bDMARDs’ positioning in the NIU treatment algorithm developed by Portuguese ophthalmologists and rheumatologists and generate national guidelines on the use of bDMARDs in this disease, iii) to present real-world evidence on the role of anti-TNF in NIU (first, by studying the currently only approved drug for NIU [ADA], and then, by exploring the role of another TNFi (golimumab, [GLM]) in the treatment and prevention of uveitis flares in an underlying systemic inflammatory disease, and iv) to develop a ground-breaking tool to pave the way for future research. Encompassed by the first aim of this thesis, we described the state-of-the-art role of bDMARD therapy in NIU using systematic approaches. In the first systematic review and meta-analysis, we assessed the efficacy and safety of TNFi drugs for chronic adult NIU. We concluded that there is high-quality evidence demonstrating that ADA decreases the risk of worsening visual acuity (VA) in active and inactive NIU and very low-quality evidence that the risk of etanercept (ETN) worsening VA in inactive NIU is no different from placebo. With regard to the risk of drug withdrawal, moderate quality evidence suggests that TNFi drugs are not different from placebo. The second approach included a systematic review investigating the role of a possible intravitreal administration of TNFi drugs in view of minimising patient morbidity and systemic adverse effects while maintaining therapeutic effectiveness. Since the available evidence is not sufficiently solid to draw a conclusion on the clinical effectivity of intravitreal TNFi in NIU, no recommendations can be made. The intravitreal injection of TNFi remains a possible treatment option to be explored through robust clinical investigation. To unveil the national positioning of bDMARDs in the NIU treatment algorithm and to emanate valuable recommendations, the second aim of this thesis was built through collaborative multidisciplinary work. Applied to the case of juvenile idiopathic arthritis associated-uveitis (JIA-U), and through a systematic literature review, we performed a national ophthalmologists’ and paediatric rheumatologists’ consensus following a modified Delphi approach. This document, which represents a coordinated effort at a national level, delivers a comprehensive and easy-to-read systematic review on the topic and 26 guidelines focusing on the following topics: general management (3), screening and follow-up of uveitis (4), treatment (17) and health education in JIA-U among patients and families (2). These guidelines were designed to support the shared medical management of patients with JIA-U and to emphasise the imperative need for a multidisciplinary approach in Ophthalmology and Paediatric Rheumatology. Two real-world studies addressed the third aim of this thesis. The first project, a retrospective study on the role of TNFi according to the European label in adults with NIU, used real-world data from the Manchester Uveitis Clinic, including efficacy, safety, ability to spare corticosteroids and predictors of response. To permit comparisons with published evidence, while acknowledging heterogeneity in the study cohorts, we used established composite treatment failure endpoints as the primary goal, as used in VISUAL studies. This study included 51 patients (102 eyes). Treatment failure occurred in 9/51 patients (10 eyes) after six months and 13/51 (20 eyes) patients after 12 months. The need for rescue treatment in the 12 months before ADA was significantly associated with treatment failure at 12 months (p<0.05). The mean prednisolone dosage was <10 mg/day at 6 and 12 months. There were no serious adverse events. Patients with NIU who received ADA therapy for 12 months were likely to achieve disease control, stabilise or improve VA, experience a reduction in immunosuppression and reduce corticosteroid dosage. The GO-VISION observational study (VISION-related quality of life in spondyloarthritis [SpA] patients with a history of acute anterior uveitis under treatment with GOlimumab) was also developed under the scope of the third aim of this thesis and assessed health-related QoL (HRQoL) outcomes for the first time as the primary endpoint of a prospective study in NIU. The GO-VISION study was able to prospectively explore the efficacy of an additional TNFi drug (GLM) in acute anterior uveitis (AAU) recurrence in patients with SpA, including the description of the efficacy, safety and change in general HRQoL and vision-related QoL (VRQoL). By the time of this thesis completion, this study was still ongoing, and we herein present an interim analysis. During the first six months of GLM treatment, the AAU incidence rate was reduced from 1.54 to 0.11 per 100 patient-years (incidence rate-ratio 13.46, 95% CI 2.15–558.00; p < 0.01). At baseline and 24 weeks after GLM onset, the overall mean index National Eye Institute Visual Functioning Questionnaire 25 (NEI VFQ-25) total score was 70.2 and 83.2, respectively; improvement in the NEI VFQ-25 total score was +13±18.2. Preliminary data from the GO-VISION study suggest that GLM is safe and effective in patients with SpA and history of AAU, reducing the AAU occurrence rate and potentially increasing QoL. As a closure to our efforts and imbued with the hindrances we experienced in gathering data for the two observational studies, in the fourth aim, we created an innovative platform, called Uveite.pt, which enables follow-up of uveitis patients at a countrywide level. Working as a fully structured electronic medical record, the platform provides robust evidence in this infrequent disease. The usefulness of Uveite.pt was further confirmed in a proof-of concept paper, which included the description of our multidisciplinary clinic and demonstrated the added value of the tool in a dedicated, real-world NIU setting. This platform can be utilised for both clinical practice and future research. In conclusion, not only does this thesis provide new evidence of the current challenges in NIU management, but it also unveils the blossoming future ofbDMARD therapy over the next few years, and hopefully nourishes future research with Uveite.pt.

The adult mammalian brain is endowed with a considerable amount of regenerative potential through the generation of new cells, particularly neurons, throughout adulthood from adult neural stem cells (NSCs). This adult neurogenesis phenomenon mainly operates in two regions – the neurogenic niches – in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). In the latter, in a process of adult hippocampal neurogenesis (AHN), adultborn neurons participate in hippocampal-dependent functions such as cognitive processing and emotional flexibility by promoting circuitry remodelling and endogenous brain plasticity. This process is tightly regulated by several factors present in the surrounding extracellular milieu that shape the dynamics of NSCs and progenitors. Among these, neurotrophic factors are key molecules that support neural growth and survival and contribute to NSC regulation. On the other hand, cannabinoids are known modulators of homeostasis, neuroplasticity and proliferation, differentiation and maturation of NSCs and progenitor cells. Nevertheless, there is still a lack of knowledge about the mechanisms by which these molecules control adult neurogenesis and NSC activity. This work aimed to evaluate and dissect the individual and combined actions of both neurotrophic factors and cannabinoids in the regulation of adult NSC dynamics in physiological and pathological contexts. In the first study of this thesis (chapter 4), we show that cannabinoid type 1 (CB1R) and type 2 (CB2R) receptors and brain-derived neurotrophic factor (BDNF) are key players responsible for fine-tuning early SVZ and DG postnatal neurogenesis. Furthermore, we demonstrate that BDNF is essential for cannabinoid-mediated neurogenesis. Importantly, in DG neurogenesis, we show that BDNF-mediated actions in cell proliferation are dependent on CB2Rs while CB2R-mediated effects in neuronal differentiation require endogenous BDNF, depicting an interdependence between these two systems. These findings led us to investigate the actions of other exercise-associated neurotrophic factors, namely vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) as well as the role of CB2Rs in the regulation of DG postnatal neurogenesis. In the second study of this thesis (chapter 5), we found that a cocktail of these neurotrophic factors, composed of BDNF+VEGF+IGF-1, significantly enhanced DG cell proliferation, an effect that was partially abrogated by CB2R blockage. Moreover, the actions of this exercise-mimicking cocktail in early neuronal commitment, but not late neuronal differentiation, were abrogated by CB2R blockage, suggesting that CB2Rs have a determinant role in the regulation of early stages of DG neurogenesis. Taking this into consideration, and aiming at a translational rationale with a putative stress-countering strategy, in a third study (chapter 7) we evaluated the combined actions of CB2Rs together with physical exercise (PE), which is a known inductor of neurotrophic factor upregulation, in the context of depression. For this, we have submitted adult mice to an unpredictable chronic mild stress (uCMS) protocol to induce core symptoms of depressive-like behaviour. At the end of uCMS, animals were submitted to a PE regimen and treatment with CB2R modulators, independently or in combination with PE, for 2 weeks. We found that CB2R blockage, but not CB2R activation, together with PE treatment significantly ameliorated chronic stress-evoked emotional changes (anxious- and anhedonic-like behaviours) as well as cognitive deficits (long-term memory). Importantly, we show that this combined strategy critically influences stress-induced changes in AHN dynamics, leading to a significant increase in the rates of proliferation and differentiation of newborn neurons. Moreover, CB2R blockage together with PE reduced the overall neuroinflammatory tone, counteracting the deleterious effects exerted by chronic stress. This reveals a synergistic effect of CB2R blockage and PE treatment that is critical to counter the detrimental effects of chronic stress and sheds light on the potential of multitargeted approaches for stressrelated pathologies. Altogether, the data herein revealed a novel layer of interaction between different adult NSC modulators, namely neurotrophic factors and cannabinoids, showing for the first time how CB2Rs fine-tune adult neurogenesis in collaboration with exercise-associated neurotrophic factors. These results also contribute to a better understanding of NSC dynamics and the importance of these neurogenic modulators in physiological and pathological contexts. Finally, this work provides new insights on the potential of CB2Rs and PE as promising targets for the development of brain repair and regeneration strategies for stress-related disorders such as depression.

A presente tese foca-se no estudo da tomada de decisão baseada no esforço. Para tal, desenvolvemos uma tarefa computacional e usamo-la em dois projetos diferentes. No primeiro projeto, usamo-la para investigar se as pessoas executam mais esforço para evitar perdas do que para ganhar. Este estudo foi motivado pela longa evidência acerca da aversão à perda (isto é, que perdas têm maior impacto do que ganhos). Na nossa tarefa os participantes escolhem e executam esforço em situações em que podem evitar perder pontos e em situações em que podem ganhar pontos. A tarefa foi desenhada de forma a minimizar a influência do desconto temporal e de risco. Analisando os resultados, observámos que as pessoas executaram mais esforço para evitar perder do que para ganhar. Este comportamento manteve-se mesmo após transformando os ganhos e as perdas para a mesma escala de magnitude usando os coeficientes individuais de aversão à perda (estimados através de uma tarefa com um procedimento clássico na área de neuro economia). Uma possível explicação para este resultado é que haja diferentes sistemas cerebrais em relação a gostar/decisão e querer/execução de uma ação. No segundo projecto, usámos novamente a tarefa desenvolvida e testamo-la em crianças e adolescentes com Perturbação de Atenção/Défice de Hiperactividade (PHDA) com e sem medicação (metilfenidato). Neste estudo, crianças e adolescentes saudáveis com sexo e idades semelhantes também realizaram a tarefa duas vezes, permitindo o estudo do efeito da doença e da medicação. A doença levou a uma pior execução quando comparando doentes na sessão sem medicação com controlos; e a medicação reverteu este efeito. Estes resultados são congruentes com a evidência que relaciona níveis de dopamina (DA) mais elevados com maior execução de esforço e, também, com a two-factor theory que se baseia na ideia que o término de um estímulo aversivo é visto como uma recompensa. Acreditamos que estudos como os aqui detalhados são de grande importância para estudar processos de decisão baseados em esforço e, consequentemente, défices motivacionais presentes em várias doenças psiquiátricas.

The brain is one of the organs with the highest energy demands in the body and the majority of these energy requirements are fulfilled by mitochondria. Most of the energy used in the brain is required for synaptic transmission, either in propagation of action potentials or for neurotransmission (Harris, Jolivet and Attwell, 2012). Being synapses one of the highest energy-consuming brain structures, as well as one of the most molecularly dynamic sites in the brain able to change within milliseconds from a resting to a stimulated state, it is expected that mitochondria population at synapses – synaptic mitochondria - are adapted to the synaptic environment and present different properties from other brain mitochondria - non-synaptic mitochondria. Indeed, morphological (Chavan et al., 2015a), proteomic (Kelly L. Stauch, Purnell and Fox, 2014; Völgyi et al., 2015), lipidomic (Kiebish et al., 2008), activity of OxPhos complexes (Almeida et al., 1995; Villa, Gorini and Hoyer, 2006) and pathological (Du et al., 2010) differences have been found between synaptic and non-synaptic mitochondria. Unexpectedly, not much is known on the functional consequences of these differences and more specifically whether synaptic mitochondria have specialized bioenergetic properties adapted to the synaptic environment. Therefore, in the first part of this thesis, we aim at dissecting the functional bioenergetic fingerprint of synaptic mitochondria in comparison with non-synaptic mitochondria, by providing an extensive bioenergetic characterization of synaptic mitochondria. We observed that synaptic mitochondria have a higher energetic flexibility to respond to different respiratory stimuli. They demonstrate higher activity of Complex IV and more drastically of Complex V, whose organization in oligomers is favourable in this mitochondrial population. On the other hand, a lower enzymatic Complex I activity was found in synaptic mitochondria, which is accompanied by a lower FMN content, NAD+ content and lower N-module core subunits expression pointing towards a lower N-module function. Despite overall Complex I activity being lower in synaptic mitochondria, the combined enzymatic activity of Complex I and III was significantly increased in this population and when dissecting Complex I expression and activity, it was demonstrated that, in synaptic mitochondria, Complex I is preferentially integrated into supercomplexes in comparison with non-synaptic mitochondria. We hypothesised that differential arrangement of Complex I is a functional bioenergetic signature of synaptic mitochondria and may constitute a protective mechanism so that synaptic mitochondria in resting conditions are still able to provide ATP for basal synaptic activity but keeping low ROS damage derived from individual Complex I activity. Our results seem to indicate that under non-stimulating conditions mitochondria at synapses have already a prepared “energetic machinery” on-hold to help responding to the higher energetic demands upon synaptic stimulation. Additionally, to study mitochondria in synapses and neurons, in vitro primary neuronal cultures are widely used and, hence constant protocol optimizations and new culture media are formulated, as is the case of BrainPhys medium (Bardy et al., 2015). BrainPhys medium was formulated to mimic physiological brain environment (Bardy et al., 2015). Therefore, in the second part of this thesis we aimed to assess the effects of BrainPhys medium on mouse neuronal maturation and to characterize how this physiologically-formulated medium impacted on neuronal bioenergetics in comparison with widely-used Neurobasal medium. Our findings demonstrate that neurons maintained in BrainPhys medium formed dense neuronal networks, abundantly stained for neuronal and synaptic markers and are functionally active. Additionally, mouse neurons in BrainPhys medium have significantly higher ATP levels, as well as, increased mitochondrial activity and Complex IV subunit expression, especially at latter maturation stages. Overall, our results corroborated previous data (Bardy et al., 2015; Jackson et al., 2018; Satir et al., 2020) showing the adequacy of BP medium to generate robust neuronal networks in mouse primary neurons and elucidated that neurons maintained in BP medium are more reliant on mitochondrial activity, as occurs in physiological conditions.

Multiple Sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease with a multifactorial undetermined etiology that causes demyelination and neuroaxonal loss in the central nervous system (CNS), leading to disability, particularly in women. Mitochondria are pivotal for MS disease activity and progression: (I) in the CNS, mitochondrial dysfunction is the main trigger for neuroaxonal loss and brain atrophy; and (II) in the periphery, immunometabolism is critical for CNS autoimmunity, while patients with MS (PwMS) show inappropriate mitochondrial phenotypes and/or oxidative phosphorylation (OXPHOS) insufficiency, particularly in CD4+ T cells. Nonetheless, no studies have explored if mitochondrial genotype could be causing the mitochondrial dysfunction observed in CD4+ T cells from PwMS. In contrast, most studies have opted for whole blood analysis, despite the significant tissue-specificity of mitochondrial DNA (mtDNA) variants, including T cells. Overall, next-generation sequencing (NGS) approaches to mtDNA have been hindered by fewer resources and less uniformity, in comparison with nuclear DNA (nDNA). A notable example is the Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, Waltham, MA, USA), mostly used in forensic research, which achieves mtDNA whole genome sequencing (WGS) with very low DNA amounts. Despite this, its bioinformatic processing occurs in the enterprise Ion Torrent Suite™ Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), thus impeding further bioinformatic processing with third-party tools, with a relatively high heteroplasmy threshold level of 10%. In order to better understand the role of mitochondrial genotype in MS activity and progression, I aimed to: (I) devise a novel approach for the Precision ID mtDNA Whole Genome Panel, which yielded an output in the correct rCRS and a lower heteroplasmy threshold; (II) explore differences in mtDNA in CD4+ T cells between newly diagnosed PwMS and healthy controls (HC); and (III) analyze baseline and longitudinal mtDNA changes regarding MS clinical phenotypes, enabling a correlation with disease activity and progression. Accordingly, an alternative customizable and open-source pipeline, the PrecisionCallerPipeline (PCP), was developed for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, PCP achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. These findings were further validated with 50 samples from an independent cohort of stroke patients, with PCP finding 98.31% of TSS’s variants (TSS found 57.92% of PCP’s variants), with a significant correlation between the variant levels (VLs) of variants found with both pipelines. Regarding the MS/Clinically Isolated Syndrome (CIS) cohort, it was possible to fully sequence the mtDNA of 61 PwMS at two clinical visits, six months (V1) and 36 months (V2) after disease onset, as well as their age- and sex-matched HC, following the successful magnetic enrichment in CD4+ T cells — mean fold change in paired samples of 2.59, 95% confidence interval (CI) [2.31–2.87]. Ultimately, however, there were no statistically significant differences in mitochondrial genotype between PwMS and HC, as well as between clinical visits V1 and V2 within PwMS. Thus, mitochondrial genotype does not seem to determine a higher risk of developing MS, nor a higher disease disability in PwMS. Furthermore, mitochondrial dysfunction in CD4+ T cells is unlikely to derive from mitochondrial genotype.