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Obesity and diabetes are widely recognized as pandemic metabolic disorders of the 21st century due to sedentarism and caloric-unbalanced diets. However, there is a silver lining, prediabetes, which precedes diabetes and is characterized by hyperinsulinemia, is a reversible condition. Thus, the prediabetic stage should receive the proper attention. Being prediabetes a multi-systemic disorder, interorgan communication plays a critical role in the disease. Recently, one particular mean of inter-organ communication captured our attention: the extracellular vesicles (EVs), which are released by every cell in the body and transport specific cargo (proteins, DNA, microRNA, and lipids) along the bloodstream, triggering metabolic alteration in distant recipient cells. Interestingly, soluble insulin receptor (InsR) was identified in the systemic circulation. The current thesis aims at revealing a novel aspect of diabetes regulation, supporting a critical role for insulin bound to EVs in disease progression. In this work, it was discovered that in a prediabetic state, there is an increase of insulin receptors in the surface of liver-derived EVs, in relation to the healthy control group. Remarkably, the same does not happen in muscle-derived EVs. Moreover, these InsR harbouring EVs bind insulin in their surface and the liver-derived ones bind ten times more insulin than muscle EVs. It was observed that muscle cells uptake liver-derived EVs, and these EVs modulate glucose uptake. Notably, liver EVs, isolated from prediabetic animals, promote glucose uptake in muscle cells by 2-fold, as opposed to 1,2-fold of healthy EVs. This increase in glucose uptake is only observed when muscle cells are incubated with EVs for a prolonged time, strongly indicating that EVs induce a long-term response. Taken together, this thesis shows that the composition of liver EVs, in insulin and InsR, is modulated by the organismal metabolic state and thus the outcome of the communication between the EVs-producer and recipient cells. A particular focus was given on the communication between liver and muscle in the context of the prediabetic environment, and it was observed that prediabetic liver EVs are performing a compensatory effect by increasing glucose uptake. Our data raise the possibility that EVs harbouring InsR can be used as therapeutic tools in patients.

A vontade evidente de ultrapassar fronteiras rígidas entre áreas do saber levou a que Aires A. Nascimento fosse além da área tradicional dos Estudos Clássicos e se dedicasse sobretudo à filologia latina medieval, à codicologia e à história do livro e das bibliotecas, com dezenas de publicações nomeadamente sobre manuscritos, iluminuras e circulação de códices, sobre as livrarias e os textos de Alcobaça, Santa Cruz de Coimbra, S. Vicente de Fora e Lorvão, sobre hagiografia e relatos de milagres e sobre história medieval e a documentação latina do território português. Do mesmo modo, alargou os seus interesses ao Humanismo Português, onde estudou Pedro Nunes, Damião de Góis, entre tantos outros. Estamos em crer que, sobretudo na filologia latina medieval em Portugal, há claramente um antes e um depois de Aires A. Nascimento. Não tendo sido o único que se dedicou ao estudo de textos latinos medievais, o seu talento tornou-o o principal dinamizador destes estudos em Portugal ao longo de mais de trinta anos. Com uma capacidade de trabalho difícil de ultrapassar, procurou formar discípulos. Este volume surge da ideia de preparar uma homenagem que permita juntar amigos, discípulos e colegas numa obra que celebre não apenas a amizade, mas também a ciência.

Karstic evolution leads to the growth and collapse of cavities by the interaction between geological structures and the hydrogeological framework. Nerja Cave developed within marbles belonging to the Alpujarride complex of the Internal Zones in the Betic Cordillera of Spain. The residual gravity anomaly map of the karstified areas –surrounding the known Nerja Cave– may indicate a likely elongated parallel cave system, N-S oriented, unknown up to present, and formed by both small shallow and large deep caves below a nearby hill located north-westwards to the known cave. At the east hillside, a moderate gravity anomaly minimum and geomorphological evidence (vertical walls and sunken terrains) suggest the presence of an old collapsed cave. At the west hillside, a marked gravity minimum is associated with a strongly folded marble layer without evidence of collapse. An electrical resistivity tomography (ERT) profile across the hill –in the E-W direction– supports an interpretation of several voids, two of the bigger ones located on either side of the hill. The combination of geomorphological, ERT and gravity forward modelling indicate the location of unknown caves, one of them partially collapsed. These caves, located at a higher topographic level than the known Nerja Cave, may represent an early stage of cave development, and suggest the preferred dissolution of some layers in the folded marbles. This field example provides new insights on the interaction of structure in the karstic evolution that determines the cavity stability.

Water demand in islands, focused in agriculture, domestic use and tourism, is usually supplied by groundwater. Thus the information about groundwater distribution is an important issue in islands water resources management. Time Domain Electromagnetic (TDEM) provides underground resistivity distribution at greater depths and is of easier application than other methods. In this study TDEM technique was used for groundwater prospection in two volcanic islands with water supply problems, the islands of Fogo and Santo Antão in the Republic of Cape Verde. The 10 islands of Cape Verde Archipelago, located off the coast of Senegal (W Africa), present a semi-arid climate and thus suffer from irregular and scarce precipitation. In the Island of Fogo 26 TDEM soundings, presenting an area distribution, were performed on the SW flank of the volcanic edifice. These allowed obtaining a 3D model composed of 5 layers parallel to the topographic surface separated by 50 m depth down to − 250 m. The results indicate the presence of the water-table at a depth of 150 m in the lower ranges of the W flank of the island, and at > 200 m depth in the area above 250 m above sea level (a.s.l.). In the Island of Santo Antão 32 TDEM soundings, distributed along 5 linear profiles, were obtained on the north-eastern half of the island. The profiles are located in two regions exposed to different humidity conditions to the N and S of the main water divide. The northern flank receives the dominant trade winds first and most of the precipitation and, therefore, the water-table is shallower (~ 50 m depth) than in the S (~ 100 m depth). Our study demonstrates the applicability and usefulness of the TDEM method for groundwater prospection in high resistivity contexts such as in volcanic islands.

One of the most remarkable natural events on Earth are the large lateral flank collapses of oceanic volcanoes, involving volumes of rock exceeding tens of km3. These collapses are relatively frequent in recent geological times as supported by evidence found in the geomorphology of volcanic island edifices and associated debris flows deposited on the proximal ocean floor. The Island of Fogo in the Cape Verde archipelago is one of the most active and prominent oceanic volcanoes on Earth. The island has an average diameter of 25 km and reaches a maximum elevation of 2829 m above sea level (m a.s.l.) at Pico do Fogo, a young stratovolcano located within a summit depression open eastward due to a large lateral flank collapse. The sudden collapse of the eastern flank of Fogo Island produced a megatsunami ~73 ky ago. The limits of the flank collapse were deduced as well from geomorphologic markers within the island. The headwall of the collapse scar is interpreted as either being located beneath the post-collapse volcanic infill of the summit depression or located further west, corresponding to the Bordeira wall that partially surrounds it. The magnetotelluric (MT) method provides a depth distribution of the ground resistivity obtained by the simultaneous measurement of the natural variations of the electric and magnetic field of the Earth. Two N-S magnetotelluric profiles were acquired across the collapsed area to determine its geometry and boundaries. The acquired MT data allowed the determination of the limits of the collapsed area more accurately as well as its morphology at depth and thickness of the post-collapse infill. According to the newly obtained MT data and the bathymetry of the eastern submarine flank of Fogo, the volume involved in the flank collapse is estimated in ~110 km3. This volume –the first calculated onshore– stands between the previously published more conservative and excessive calculations –offshore– that were exclusively based in geomorphic evidence. The model for the summit depression proposing two caldera collapses preceding the collapse of the eastern flank of Fogo is supported by the MT data.

Today, scientific literacy has become increasingly important as a hallmark of citizenship and as a way to facilitate many of life’s daily decisions. Scientific literacy can be achieved through learning, a complex lifelong process that occurs both in school and out-of-school settings. As most adults only engage in formal education about science during compulsory schooling, all the learning that people go through in out-of-school settings and beyond compulsory schooling becomes extremely important toward the scientific literacy of the individual. The learning process in out-of-school settings requires interest from each person and can be facilitated by various agents, for example, museums. This is particularly true for Earth sciences as Geology topics are rarely taught within the science curricula during school. The Museu Nacional de História Natural e da Ciência provides realistic experiences that engage different publics into science and thus enhances the public science literacy. It is expected that by enhancing people’s Earth sciences literacy, it will also enhance their awareness of geological heritage.

A doença de Kikuchi-Fujimoto ou linfadenite histiocítica necrosante é uma patologia de etiologia desconhecida, de muito baixa prevalência, afetando principalmente mulheres jovens asiáticas. É geralmente autolimitada e apresenta-se como linfadenite cervical e febre. Pode também causar exantema e artrite. Sendo uma doença rara e afetando com maior incidência uma população específica, muitas vezes esta patologia não é equacionada no diagnóstico diferencial de linfadenite ou febre sem foco. O objetivo deste trabalho é descrever um caso clínico desta patologia e fazer uma breve revisão sobre o tema. Apresenta-se o caso de uma criança, do sexo masculino, com 7 anos, previamente saudável, internado por febre prolongada com 13 dias de evolução, astenia, anorexia, emagrecimento, exantema, odinofagia e artralgias. No exame objetivo detetou-se adenomegália cervical, hepatoesplenomegália, exantema salmão evanescente no tronco e membros e poliartrite. O ecocardiograma era normal. Das análises realizadas destaca-se leucocitose e elevação dos parâmetros inflamatórios (39 980 leucócitos/uL; Neutrófilos 80%; Linfócitos 13%: Proteína C reativa 18,3 mg/dL: VS 92mm/h; ferritina 11 122ng/mL). Excluiu-se infeção por Streptococcus pyogenes, Bartonella henselae, Mycobacterium tuberculosis, toxoplasmose, vírus da imunodeficiência humana, Epstein-Barr, citomegalovírus e herpes. Os anticorpos antinucleares eram negativos e o estudo do complemento normal. O esfregaço de sangue periférico era normal. Realizou biópsia osteomedular, excluindo doença linfoproliferativa. Realizou biópsia do gânglio cervical, tendo-se detetado linfadenite histiocítica necrosante e histiocítica, compatível com doença de Kikuchi-Fujimoto. Iniciou tratamento com prednisolona (1mg/Kg/dia) com rápida resposta, realizando diminuição progressiva da dose. Após 4 meses de seguimento em consulta de Reumatologia Pediátrica encontra-se assintomático e sem terapêutica.

O Síndrome do ovário poliquístico (SOP) é um complexo transtorno endócrino e metabólico frequentemente associado a infertilidade, obesidade e a insulinorresistência. A etiologia da doença permanece mal compreendida, apesar de causas poligenicas e multifatoriais parecerem estar envolvidas, incluindo, em particular, elementos ambientais, onde se destaca a dieta e outros fatores do estilo de vida. Neste contexto, a terapêutica da SOP deve ser ajustada de acordo com a gravidade das manifestações e necessidades da doença e, partindo da modificação do estilo de vida, conjuntamente com potenciais abordagens farmacológicas e/ou cirúrgicas. A presente revisão tem por objetivo fornecer uma revisão compreensiva do estado atual da investigação no que respeita ao tratamento e gestão de mulheres com SOP, focando-se, em particular, nas intervenções nutricionais, pois são estas consideradas opções de tratamento de primeira linha. A dieta e proporção relativa de macronutrientes ideal, em situações de SOP, ainda não foi passível de ser identificada e globalmente reconhecida, já que a heterogeneidade dos resultados encontrados é significativa. Não obstante, uma distribuição de macronutrientes similar à descrita na dieta Mediterrânica mostrou impactos positivos e revelou-se adequada para manutenção a longo prazo. A suplementação de certos elementos em défice pode também desempenhar um papel de interesse na abordagem destes doentes sendo que a vitamina D e os ácidos gordos polinsaturados n3 mostraram os resultados mais consistentes. Mais ainda, a disbiose da microbiota intestinal parece não só influenciar a patogénese da doença como também a sua correção, através de agentes prébióticos e probióticos, parece constituir uma intervenção terapêutica promissora. Apesar da constante publicação de novos dados, esta área de investigação carece ainda de mais estudos de qualidade, visto que devem ser contempladas intervenções com maior duração e com grupos em estudo de maior dimensão e mais diversificados, especialmente no que se refere à etnicidade dos mesmos.

A expansão urbana não pode ser desligada dos mecanismos de funcionamento do mercado imobiliário. Este alimenta uma indústria de urbanização que transpõem para o território voracidade equivalente aos lucros avultados que mobiliza. A fragmentação do tecido urbano observada desenha nos territórios em torno das áreas consolidadas das cidades médias a fusão grosseira da cidade com campo criando espaços de leitura difícil. Não planeados. Germinam aqui, as cidades novas justapostas nas explorações agrícolas sobreviventes, nos grandes espaços comerciais, nos fragmentos de floresta, nos inúmeros espaços vazios (vazios urbanos/espaços de tudo e de nada), nas vagens de moradias em banda, ou, nas ilhas de loteamentos desgarradas. Tudo isto é amarrado a uma estrutura de circulação deltritica que cola (a post-it) estes espaços hiperfuncionais à cidade e à região metapolitana (?) circundante. Demonstramos que a função reguladora da Administração (pelo lado das regras de uso e transformação do solo e pelo lado da tributação), ou tem sido inoperante, ou tem servido (tem-se servido) a avidez com que o mercado procede à urbanização destes territórios. Reflectimos acerca do tema, discutindo a sua abrangência teórica. Tratamos informação passível de quantificar estes processos à escala de Portugal Continental e, numa segunda parte da dissertação, aprofundamos o caso de estudo da cidade das Caldas da Rainha. Neste laboratório de análise, quantificamos, de vários modos, a dimensão da expansão urbana desencadeada nas últimas décadas. Avaliamos a evolução do mercado imobiliário, nomeadamente, através da criação duma base de dados onde se registaram 2446 imóveis publicitados (entre 1926 e 2008), num jornal local. Procedemos ainda, recorrendo à aplicação de questionários, à avaliação dos moldes definidores da procura residencial dos últimos 15 anos, nas coroas periurbanas e rurbanas da cidade estudada na qual, se identificou espaço urbano, e urbanidades novas, decorrentes do facto de serem valorizadas, e valorizáveis, no mercado.

BACKGROUND: In previous studies, suicidal thoughts in stroke patients were related to depression and recurrent stroke. The aim of this study was to describe the presence and correlates of suicidal thoughts in acute stroke patients. METHODS: We assessed the presence of suicidal thoughts in a sample of 177 consecutive acute stroke patients (≤4 days), using the item "suicidal thoughts" of the Montgomery and Asberg Depression Rating Scale. RESULTS: Fifteen percent of acute stroke patients had suicidal thoughts, 22% of them with explicit plans to complete suicide. Suicidal thoughts were more frequent in patients with a lower educational level (Chi-square 4.69; P = .05), previous mood disorder (Chi-square 12.80; P = .001), diabetes (Chi-square 5.48; P = .04), and acute depression (Chi-square 16.59; P = .001). In logistic regression, a lower educational level (odds ratio [OR] 5.13; 95% confidence interval [CI] 0.80-33.29), diabetes (OR 3.15; 95% CI 0.81-12.28), a previous mood disorder (OR 4.22; 95% CI 1.12-15.86), and depression (OR 7.82; 95% CI 1.58-38.71) were independent factors predicting suicidal thoughts (R(2) = 0.39%). The 23 depressed acute stroke patients with suicidal thoughts were similar in demographic, clinical, and lesion variables, and were similar in profile of depression to the 63 patients with depression but without suicidal thoughts, except for a higher frequency of a previous mood disorder in the former group (Chi-square 7.87; P = .01). CONCLUSIONS: Suicidal thoughts may develop shortly after the onset of acute stroke, especially in patients with a lower educational level, a previous mood disorder, and those who developed depressive symptoms in the acute phase of stroke. The high frequency of suicidal thoughts makes the assessment of suicidal behavior in patients with acute stroke an important clinical issue.

O Acidente Vascular Cerebral (AVC) no adulto jovem, que acomete indivíduos com idade entre os 18 e os 50 anos, representa cerca de 10% a 15% dos casos de AVC. A prevalência de fatores de risco tradicionais nos adultos jovens vítimas de AVC é inferior à verificada em idades mais avançadas, mas, ainda assim, significativa. A variedade etiológica é particularmente ampla, entre doenças infecciosas sistémicas e do SNC, trombofilias hereditárias e adquiridas, entre outros, sendo o diagnóstico etiológico particularmente desafiante. Além dos eventos de etiologia indeterminada, reconhecem-se cada vez mais quadros de etiologia multifatorial que dificultam as decisões terapêuticas e as estratégias de prevenção secundária. A diversidade de etiologias reconhecidas para os eventos vasculares cerebrais em jovens impõe uma investigação exaustiva e um follow-up cuidadoso, permitindo reduzir a incidência de recorrência, as taxas de mortalidade e morbilidade associadas e o consequente impacto socioeconómico desta patologia.

Objetivo: Elaboração de um conjunto de recomendações no que diz respeito ao diagnóstico e abordagem terapêutica da espondilólise e espondilolistese de baixo grau em atletas. Metodologia: Revisão da literatura entre 2000 e 2020, nomeadamente de estudos retrospetivos, séries de casos e revisões sistemáticas com a pesquisa das seguintes palavras-chave: espondilólise, espondilolistese, diagnóstico, tratamento conservador, tratamento cirúrgico, resultados e atletas. Resultados: Foram selecionados 16 artigos(a), 14 com foco no tratamento (5 apenas conservador, 3 apenas cirúrgico, 6 em ambos) e 2 cujo conteúdo principal era a metodologia diagnóstica. Apesar da TC ser o método de eleição para documentar a fratura e a sua consolidação, outros métodos como a SPECT ou a RM têm maior sensibilidade para detetar alterações mais precoces na pars articularis. O tratamento conservador passa pela cessação da atividade física seguida de um programa de fisioterapia, tendo a maioria dos estudos apresentado ótimos outcomes para 85,7-92,2% dos atletas. O regresso à prática desportiva foi possível entre os 3,7 e os 5,9 meses. A maioria dos estudos incluem ortóteses no tratamento conservador, mas evidência recente mostra que esta não altera o outcome. Também o tratamento cirúrgico demonstrou ter excelentes resultados com maior foco nas técnicas de reparação da pars articularis. 80% dos atletas intervencionados, regressam à prática desportiva entre 6-7,9 meses após a cirurgia. Muitos estudos avaliam ainda a taxa de consolidação da lise, com resultados variáveis, no entanto não é um fator essencial para o atleta poder regressar à prática desportiva. Conclusões: A heterogeneidade dos estudos e o baixo grau de evidência dos mesmos não permite chegar a conclusões com elevado grau de evidência. O diagnóstico e tratamento precoces têm importância no regresso mais rápido dos atletas à prática desportiva. Para tal, é importante recorrer a métodos de imagem que documentem alterações ainda antes da existência de uma fratura, tal como a RM ou a SPECT. O tratamento conservador deve ser a abordagem inicial e tem excelentes outcomes, sobretudo com a cessação da prática desportiva. Após seis meses de falência terapêutica, a cirurgia é uma opção viável como segunda linha e apresenta também excelentes outcomes, oferecendo aos atletas uma nova oportunidade de regresso à prática desportiva.

Introduction: The role of intrapartum ultrasound as an ancillary method to instrumental vaginal delivery is yet to be determined. This study aimed to compare the use of transabdominal and transperineal ultrasound with routine clinical care before performing an instrumental vaginal delivery, regarding the incidence of adverse maternal and neonatal outcomes. Material and methods: A randomized controlled trial was conducted between October 2016 and March 2019 in two tertiary care maternity hospitals in Lisbon, Portugal. Women at term, with full cervical dilatation, singleton fetuses in cephalic presentation, and with an established indication for instrumental vaginal delivery, were approached for enrollment. After informed consent was obtained, randomization into one of two groups was carried out. In the experimental arm, women underwent transabdominal ultrasound for determination of the fetal head position and transperineal ultrasound for evaluation of the angle of progression, before instrumental vaginal delivery. In the control arm, no ultrasound was carried out before instrumental vaginal delivery. Primary outcomes were composite measures of maternal and neonatal morbidity. Composite maternal morbidity consisted of severe postpartum hemorrhage, perineal trauma, and prolonged hospital stay. Composite neonatal morbidity consisted of low 5-minute Apgar score, umbilical artery metabolic acidosis, birth trauma, and neonatal intensive care unit admission. Results: A total of 222 women were enrolled (113 in the experimental arm and 109 in the control arm). No significant differences between the two arms were found in composite measures of maternal (23.9% in the experimental group vs 22.9% in the control group, odds ratio 1.055, 95% CI 0.567-1.964) or neonatal morbidity (9.7% in the experimental group vs 6.4% in the control group, odds ratio 1.571, 95% CI 0.586-4.215), nor in any of the individual outcomes. Conclusions: In this small randomized controlled trial that was stopped for futility before reaching the required sample size, transabdominal and transperineal ultrasound performed just before instrumental vaginal delivery did not reduce the incidence of adverse maternal and neonatal outcomes, when compared with routine clinical care.

Water depths and flow velocities decisively influence the damage caused by flash floods. Geographic Information System (GIS) is a powerful and useful tool, allowing the spa-tial analysis of results obtained by hydraulic modelling, namely from the HEC-RAS/HEC- GeoRAS software. The GIS spatial analysis performed in this study seeks to explain and quantify the spatial relationships between the stream channel features and flow components during flash flood events. Despite these relationships are generically known, there are few studies exploring this subject in different geographic contexts. A 1D hydraulic model was ap-plied in a small watershed in Portugal, providing good results in the definition of floodable areas, water depths and longitudinal velocities. No direct relationship was found between water depths and velocities in the floodable areas; however, negative strong correlations were found between the two flow components along the stream centerlines. Bed slope, channel and flood width, and roughness prove to be highly relevant on the longitudinal varia-tions of water depths and velocities and on the location of maximum values. Increasing peak discharges and return periods (RT) can change the relationships between water depths and velocities at the same location. Results can be improved with more accurate elevation data for stream channels and floodplains.

In the course of a study of lymphocyte responses to microbial antigens in HIV-infected patients, we detected a previously unrecognized phenomenon of inhibition of lymphocyte baseline proliferation, induced by the presence of tetanus toxoid and Escherichia coli in the cultures. The effects of tetanus toxoid and Escherichia coli on lymphocyte proliferation in vitro were assessed by comparing the 3H-thymidine uptake by lymphocytes cultured without stimulant with the uptake of lymphocytes cultured in the presence of the antigens. Twenty-six patients with HIV infection (20 asymptomatic/persistent generalized lymphadenopathy, 2 AIDS-related complex, 4 AIDS) were investigated and the controls were 33 healthy individuals without evidence of HIV infection. Eight out of 22 asymptomatic/PGL and ARC patients progressed to full-blown AIDS in the mean follow-up of 26 months. The inhibition of proliferation was considered to be significant when the uptake of 3H-thymidine was reduced by 20% in the presence of the antigens. Using these criteria, 50% of the patients studied with tetanus toxoid and 36% of those studied with E. coli had evidence of the inhibitory phenomenon. Seven of the eight patients who developed AIDS during the observation period had the inhibitory phenomenon. In the group of patients without the inhibitory signs only one evolved to AIDS during the follow-up. The possibility of this phenomenon being related to an induction of suppressor cell activity by the antigens is discussed.

A number of studies have shown the benefit of hypertensive treatment even though the most common forms of the disease are mild-to-moderate in severity. Considering the overall aging of the world's population, it is of particular interest to study hypertension and its treatment in geriatric patients. A short-term study of isradipine was conducted to assess its effectiveness and tolerability in patients with mild-to-moderate hypertension. The study was carried out by general practitioners and involved 3343 patients, aged > or = 18 years, with diastolic blood pressures (DBPs) ranging from 95 to 114 mm Hg. A 4-week wash-out and placebo run-in phase was followed by a 12-week active treatment period with isradipine at 1.25 or 2.5 mg/day, depending on the blood pressure response. Posttreatment results in a subgroup of 1092 patients (444 men and 648 women), aged > or = 60 years, showed decreases in systolic blood pressure (SBP) from 173.1 to 149.2 mm Hg (mean decrease, 20.9 mm Hg) and, in DBP, from 102.0 to 85.0 mm Hg (mean decrease, 16.9 mm Hg). The majority (84.6%) of these patients showed DBP reductions of > 10 mm Hg, and 82.3% achieved normalization (DBP < 90 mm Hg) at the end of treatment. The mean dosage was 1.74 +/- 0.69 mg twice daily, and 37% of patients doubled their initial 1.25 mg twice daily dosages. There were no significant changes in either heart rate or major metabolic parameters. Adverse events were reported by 3.1% of the patients, and 90% of both patients and physicians expressed satisfaction with the therapy. There were no differences between men and women with regard to adverse events or efficacy, nor were the results in patients > or = 60 years different from those in younger patients. Thus, isradipine was effective and well tolerated in these geriatric patients.

The diagnosis of drug-induced hepatitis is usually based on clinical criteria, with emphasis on both the temporal relationship between drug intake and liver injury and the exclusion of alternative causes. In vitro tests of lymphocyte sensitization to drugs are considered to have a low sensitivity. We investigated the possibility of detecting lymphocyte reactivity to drugs in drug-induced hepatitis by analyzing the lymphocyte proliferative responses to ex-vivo drug or metabolite antigens to improve the sensitivity of the in vitro test. Lymphocyte proliferative responses to five different concentrations of the drug and to ex-vivo drug antigens (serum collected from normal subjects after the ingestion of the drugs) were analyzed in 25 patients with a clinical diagnosis of drug-induced hepatitis, 27 healthy subjects and 10 individuals with a recent exposure to the same drugs without development of adverse drug reactions. In seven of the 25 patients, lymphocyte reactivity to drugs was detected (28%). The use of sera collected from healthy volunteers after drug intake (ex-vivo drug antigens) and the addition of a prostaglandin inhibitor to the cultures allowed the detection of lymphocyte sensitization in seven additional cases, increasing the detection ability from 28% to 56%. We suggest that the use of ex-vivo drug antigens may represent a significant contribution to the identification of the drug involved in cases of drug-induced hepatitis.

Thyroid cancer (TC) is the most common endocrine neoplasia. Thyroid carcinomas derived from follicular cells or thyrocytes are designated as nonmedullary thyroid carcinomas (NMTCs). The most undifferentiated subset of NMTC are anaplastic thyroid carcinomas (ATCs) which, although rare, are the most aggressive and lethal type of TC. ATCs are highly invasive, and lost the intrinsic functions of normal thyrocytes, presenting a downregulation of the sodium-iodide symporter (NIS), which makes the usual treatments for differentiated thyroid cancer, such as surgery and radioactive iodine, much less effective. Therefore, it is necessary to search for new therapeutic options, such as targeted therapies. The most promising targeted therapy for ATCs is the combination of dabrafenib (BRAF inhibitor) plus trametinib (MEK inhibitor) which, in a clinical trial, showed a significant response rate in ATC patients with the BRAF p.V600E mutation. This combination (DT) was recently approved only by the Food and Drug Administration (FDA) for this indication. Since some patients may develop resistance to DT, it is important to investigate the mechanisms involved and also new molecular therapeutic targets, in order to improve their overall survival. More than 50% of ATC’s tumour microenvironment (TME) is comprised by tumour associated macrophages (TAMs), which are associated with ATC aggressiveness, and may represent a possible novel target for ATC treatment. In previous proteomic studies developed by the Molecular Endocrinology group, on ATC-TAM crosstalk, Sprouty RTK Signaling Antagonist 4 (SPRY4) was identified as being a potential modulator of these interactions. The main aim of this work was to investigate the molecular mechanisms involved in ATC aggressiveness and response to DT therapy. To achieve this goal, two major approaches were used: (1) characterization of the molecular profiles of nine ATC BRAF p.V600E-positive patients, through the analysis of a multigene panel by Next Generation Sequencing (NGS) and Sanger sequencing, in order to investigate the alterations that could be associated with DT response and resistance, and to define treatment options in these cases; (2) in vitro experimental procedures, using 2D transwell cultures of ATC cell lines C3948 and T235, and a monocyte cell line (THP-1), in monocultures (without THP-1) and in co-cultures (with THP-1), to further study the role of SPRY4 in the ATC-TAM crosstalk. In the first task, we identified telomerase promotor (TERTp) mutations in all patients, which in concomitance with BRAF p.V600E, were likely contributing to the excellent initial DT therapy response. In one patient, a NRAS activating mutation was identified, representing an acquired off-target resistance mechanism, as previously described in ATCs. PIK3CA mutations were also detected in three patients and could be related with the selection of resistant clones to DT, as described in melanoma. A novel likely pathogenic mutation in MAP2K1, which encodes for MEK1, (p.E120Q), was identified in one patient, so far responding to DT. A MTOR mutation was identified in one case and may represent a novel therapeutic target in ATCs. TP53 mutations were detected, in five patients and, although known to be related with ATC development, did not appear to be directly associated with DT efficacy. Although ATC is a rare disease, the characterization of additional cases and molecular targets, may further support and extend the present findings. In the second task, western blot analysis showed that SPRY4 was downregulated in T235 co-cultures and upregulated in C3948 co-cultures, confirming previous proteomic data. Using SPRY4 siRNA-mediated silencing (> 50% of silencing), it was possible to observe that, in both cells lines, the invasion capabilities significantly increased in co-culture, similar to that observed in T235 using the non-target control (siNT) but contrasting with the decrease observed for C3948 cells with siNT. Also, the analysis of ATC cells’ cytoskeletal alterations and morphology, through immunofluorescence with phalloidin to detect actin filaments, confirmed the spreading behavior under siSPRY4 conditions in both cell lines, consistent with invasion assays results. Taken together, these results unveiled a tumour suppressor gene role for SPRY4 in ATC, in the presence of TAMs, with an influence in ATC-TAMs interactions and ATC aggressiveness. Additional studies are warranted to further elucidate SPRY4-related signaling mechanisms in this context. In conclusion, this study contributed to improve the present knowledge on the cellular and molecular mechanisms involved ATC response to therapy, and TME/TAM interactions, that are associated with ATC aggressiveness, which may eventually lead to new therapeutic approaches capable of improving the prognosis and overall survival of ATC patients.

There are still more than 1,400 Mendelian conditions whose molecular cause is un known. In addition, almost all medical conditions are somehow influenced by human genetic variation. This challenge also presents itself as an opportunity to understand the mechanisms of diseases, thus allowing the development of better mitigation strategies, finding diagnostic markers and therapeutic targets. Deciphering the link between genes and diseases is one of the most demanding tasks in biomedical research. Computational approaches for gene-disease associations prediction can greatly accelerate this process, and recent developments that explore the scientific knowledge described in ontologies have achieved good results. State-of-the-art approaches that take advantage of ontologies or knowledge graphs for these predictions are typically based on semantic similarity measures that only take into consideration hierarchical relations. New developments in the area of knowledge graphs embeddings support more powerful representations but are usually limited to a single ontology, which may be insufficient in multi-domain applications such as the prediction of gene-disease associations. This dissertation proposes a novel approach of gene-disease associations prediction by exploring both the Human Phenotype Ontology and the Gene Ontology, using knowledge graph embeddings to represent gene and disease features in a shared semantic space that covers both gene function and phenotypes. Our approach integrates different methods for building the shared semantic space, as well as multiple knowledge graph embeddings algorithms and machine learning methods. The prediction performance was evaluated on curated gene-disease associations from DisGeNET and compared to classical semantic similarity measures. Our experiments demonstrate the value of employing knowledge graph embeddings based on random walks and highlight the need for closer integration of different ontologies.