Repositório RCAAP

Parkinson’s disease (PD) is the most frequent neurodegenerative movement disorder, affecting 1-2% of the world population above 65 years of age. Although identified in a minority of patients, mutations in the LRRK2 gene, in particular the G2019S mutation, are the most frequent known cause of PD identified so far. Research on LRRK2 related PD epidemiology and associated phenotypes is of the utmost importance for clinical practice and counseling and for uncovering new molecular pathways and pharmacological targets. The present investigation aims to identify the frequency of LRRK2 G2019S mutation in different populations, to characterize LRRK2 mutations in Portuguese PD patients, to study associated clinical phenotypes, and to contribute to the evaluation of a possible imaging biomarker. Methods To study the frequency of the LRRK2 G2019S mutation in different PD populations we conducted a systematic review of all published papers regarding G2019S frequency in which a PD population with a common ethnic background could be independently analyzed. The quality of published methodology and related susceptibility to bias were assessed through a checklist approach. To clarify the frequency of LRRK2 mutations in Portuguese PD patients, a comprehensive screening of LRRK2 mutations in a consecutively recruited cohort of familial PD probands was performed. Findings were evaluated in a larger PD cohort and in controls, ascertained at the Movements Disorders Outpatient Clinic of the Hospital de Santa Maria in Lisbon. A case-control cross-sectional study was designed to compare the motor and non-motor clinical phenotype between PD patients carrying a LRRK2 gene mutation and PD patients with no mutations identified, matched for age of onset, disease duration and gender. The primary outcome was the Movement Disorders Society- Unified Parkinson’s disease Rating Scale (MDS-UPDRS) part III (motor part) and secondary outcomes concerned other questionnaires and scales for the study of motor and nonmotor symptoms in PD. Retrospective data on motor and nonmotor symptoms was additionally collected and analyzed. A logistic regression model was used to identify PD patients carrying LRRK2 mutations. For the evaluation of the use of neuromelanin-Magnetic Resonance imaging (Neuromelanin-MRI) as biomarker in LRRK2-related PD, we performed a comparative imaging study between PD patients carrying an LRRK2 mutation vs. controls with no known neurodegenerative disorder and PD patients with no mutations identified. Results Sixty-eight studies from 32 countries were included in the systematic review on reported G2019S frequency in different PD populations. Heterogeneity in frequency in different PD populations was observed, ranging from no cases to 35.7% in sporadic and 42% in familial North-African Arab patients. Only one paper from one sub-Saharan country was found. Methodological pitfalls were identified. Sixty-one familial PD probands were included in the analysis of the entire LRRK2 coding region and respective exon-intron boundaries. The G2019S mutation was found in 16.4% of the probands, the R1441H mutation in 3.3% and five novel coding LRRK2 variants were identified, each in one patient. Identified mutations were tested in additional familial and sporadic patients, and together with previous results, we found a frequency of G2019S carriers of 14% for familial cases (12/86) and 3.5% for sporadic cases (8/226). The five novel coding variants were absent from 191 sporadic PD patients. No mutations or novel variants were identified in 138 control individuals. For studying the LRRK2 associated clinical phenotype, 342 PD patients were additionally screened for LRRK2 mutations. 24 PD patients carrying a LRRK2 mutation (20 G2019S, 4 R1441H) and 48 PD controls with no mutations identified were included in the case-control cross-sectional study. With a mean disease duration of 16.0±8.7 years, PD patients with a LRRK2 mutation presented no significant difference in MDS-UPDRS III and significantly less frequent nocturia when compared with PD patients with no mutations identified. REM sleep behavior disorder (RBD) related symptoms were significantly less reported in PD patients carrying a LRRK2 mutation. The Odds Ratio of a PD patient not carrying a LRRK2 mutation was 7.96 if RBD related symptoms were ever reported. Neuromelanin-MRI was performed in 13 PD patients with a LRRK2 mutation (10 G2019S, 3 R1441H), in 13 PD patients with no LRRK2 mutations identified and in 10 controls with no known neurodegenerative disorder. The SN neuromelanin signal area was significantly decreased in both LRRK2-related PD and PD patients with no LRRK2 mutations when compared to controls. There was no significant difference in neuromelanin signal areas between PD groups with and without LRRK2 mutations. The neuromelanin-MRI area measurement had a 92.3% sensitivity and 100% specificity in discriminating LRRK2-related PD patients from controls. Conclusions Worldwide frequencies of G2019S LRRK2 PD mutation carriers are heterogeneous and highly variable, reflecting differences in populations and possible methodological discrepancies. In order to improve accuracy in reporting, recommendations on the methods of selection of participants and on the definition of familial PD are made. Concerning Portugal, our comprehensive genetic screening study confirms G2019S as the most important genetic cause of PD known so far in Portuguese PD patients and supports R1441H as pathogenic. Together with our first study, our results show that Portugal has one of the highest frequencies of G2019S PD carriers in Europe. Concerning LRRK2-related PD clinical expression, our results confirm that also for patients with long disease duration, the LRRK2-associated motor phenotype largely overlaps with idiopathic Parkinson’s disease. Nocturia and RBD were significantly less frequent in LRRK2-PD patients and emerged as main differences to be further investigated. Neuromelanin-MR images of the SN can accurately differentiate LRRK2-related PD from control individuals. Our results may have important implications for clinical practice, counseling, molecular and clinical research, in preclinical, prodromal and post diagnostic phases of the disease.

O diagnóstico da Doença de Ménière é clínico. Consiste numa síndrome caracterizada por episódios recorrentes de vertigem espontânea, hipoacúsia neurosensorial para frequências médias e baixas e sintomas audiovestibulares flutuantes, como acufeno, plenitude auricular e instabilidade postural. A hidrópsia endolinfática é o substrato anatomo-patológico da doença e pode ser demonstrada por ressonância magnética na maioria dos ouvidos sintomáticos com doença de Ménière, mas também em ouvidos assintomáticos ou pré sintomáticos, por vezes precedendo em vários anos a instalação do diagnóstico clínico. Com este trabalho pretendemos explorar o papel da ressonância magnética no estudo do doente com sintomas audiovestibulares, em particular na doença de Ménière, focando-nos na avaliação da hidrópsia endolinfática e em fatores anatómicos do ouvido interno. O trabalho inicia-se com uma revisão da literatura sobre a ressonância magnética na doença de Ménière (capítulo 1), um tema controverso, que seguramente terá desenvolvimentos a curto prazo. Este trabalho foi publicado na revista da sociedade europeia de neurorradiologia – Neuroradiology. Seguem-se o estudo imagiológico com componentes transversal e longitudinal na doença de Ménière (capítulo 2), publicado recentemente na revista científica European Archives of Oto-Rhino-Laryngology. Trata-se de um trabalho com limitações inerentes à época pandémica com o qual coincidiu, sobretudo em relação à dimensão da amostra e à caracterização clínica. Apesar disso, traz algumas informações ainda pouco exploradas, a merecer confirmação em estudos controlados e com maior número de doentes. Por ressonância magnética caracterizámos a hidrópsia endolinfática ao longo do tempo numa população com doença de Ménière definitiva, provável e em doentes monosintomáticos. Avaliámos também doentes com sintomas audiovestibulares sem características de doença de Ménière e indivíduos assintomáticos. A hidrópsia endolinfática foi universal e mais acentuada nos doentes com doença de Ménière definitiva, mantendo-se estável ao longo do tempo de seguimento. Os resultados apontam para um agravamento acelerado da hidrópsia endolinfática em alguns doentes em fases mais precoces da doença, sobretudo em quadros inicialmente monosintomáticos. Porque a hidrópsia endolinfática por si só não explica a grande variabilidade clínica na doença de Ménière, e dada a estreita relação anatómica e funcional do modíolo com o labirinto membranoso, colocámos a hipótese de existir uma remodelação óssea do modíolo em ouvidos com hidrópsia marcada. Assim, avaliámos pela primeira vez o modíolo coclear por ressonância magnética numa população com doença de Ménière definitiva, procurando relacionar as dimensões do modíolo coclear com a hidrópsia endolinfática (capítulo 3). Este trabalho foi publicado na revista Frontiers in Surgery. Encontrámos uma redução dimensional do modíolo coclear em alguns doentes com hidrópsia extrema, aspeto a explorar em estudos controlados e com amostras maiores. Seguidamente apresentamos os 3 artigos referidos e, adicionalmente, um comentário publicado em coautoria, focando a qualidade da imagem de ressonância magnética no estudo da hidrópsia endolinfática. Este conjunto de trabalhos corroboram a ideia de que a hidrópsia endolinfática tem um papel central na doença de Ménière, a qual parece ser multifatorial. Num futuro próximo, é expectável uma atualização dos atuais critérios de diagnóstico, exclusivamente clínicos, de forma a viabilizar um diagnóstico e uma intervenção mais precoces em doentes monosintomáticos. A ressonância magnética assume um papel inequívoco na identificação e caracterização imagiológica da hidrópsia endolinfática (no diagnóstico e ao longo do curso da doença), bem como de outros potenciais biomarcadores da doença, alguns dos quais anatómicos, podendo contribuir para uma melhor estratificação e compreensão da doença.

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by sustained synovitis. Recently, several studies have proposed neutrophils and Th17 cells as key players in the onset and perpetuation of this disease. The main goal of this work was to determine whether cytokines driving neutrophil and Th17 activation are dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of disease duration and before treatment (VERA). Methods: Cytokines related to neutrophil and Th17 activation were quantified in the serum of VERA and established RA patients and compared with other very early arthritis (VEA) and healthy controls. Synovial fluid (SF) from RA and osteoarthritis (OA) patients was also analyzed. Results: VERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1b and IL-6), as well as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to induce neutrophil-mediated inflammation. In established RA this pattern is more evident within the SF. Early treatment with methotrexate or corticosteroids led to clinical improvement but without an impact on the cytokine pattern. Conclusions: VERA patients already display increased levels of cytokines related with Th17 polarization and neutrophil recruitment and activation, a dysregulation also found in SF of established RA. 0 Thus, our data suggest that a cytokine-milieu favoring Th17 and neutrophil activity is an early event in RA pathogenesis.

The advance of knowledge about the pathogenesis of inflammatory bowel disease (IBD) has allowed for the discovery of potential treatments aimed at biological targets. Thus, in the early 90’s, IBD treatment entered a new era, with the development of biological therapies. However, some patients do not respond to induction treatment (primary loss of response) or lose response during maintenance treatment (secondary loss of response) over time. This loss of response may be related to these treatments being highly immunogenic, thus leading to the development of anti-drug antibodies (ADAs), which can neutralize drug-target binding or increase drug clearance, resulting in sub-optimal drug concentrations and shorter response times. Usually, this loss of response is managed empirically. However, this empirical approach increases the risk of irreversible tissue damage and health care costs, and may delay effective IBD treatment. Therefore, therapeutic drug monitoring (TDM) is an emerging strategy in the treatment of IBD patients. Assessing both drug and ADAs levels can help clinicians adjust therapy on individual basis. However, its proper use depends on knowledge of the pharmacokinetic properties that influence loss of response and on the correct use of methods for detecting drug and ADA levels. The global aim of this work was to understand the impact of the methodological approach of TDM and the consequent biases in its interpretation in IBD patients treated with anti-TNFα drugs. This work demonstrated that the presence of the drug influences the detection of ADAs and this impact is methodology-dependent. Therefore, the inability of some assays to determine ADAs in the presence of the drug may hamper the clinical interpretation of TDM. Moreover, this work also demonstrated that the methodologies used for TDM of Remicade® can also be used to monitor the levels of Flixabi® (Remicade-biosimilar). In addition, we also demonstrated that Remicade®, Remsima® and Flixabi® show a high cross-immunogenicity, which supports their high similarity, but prevents their exchange, as an efficacious therapeutic option, in patients who do not respond to anti-TNFα therapy. This work shows that TDM can be advantageous in: i) identifying medication adherence problems in patients who have lost response, allowing clinicians to discriminate between pharmacokinetic and pharmacodynamic reasons for treatment failure; ii) identification of the most appropriate dosing regimen to achieve the optimal response with minimal toxicity; iii) help clinicians identify patients who will and will not benefit from treatment. However, the use of TDM should always be integrated with the identification of possible methodological biases and the clinical assessment of the patient.

Neste artigo propomo-nos a analisar os fatores políticos que contribuíram de uma forma decisiva para que a Indonésia procedesse ao rompimento formal de relações diplomáticas com Portugal. Analisaremos ainda, os sinais de bastidor, externos e internos, dados por Jacarta. Pretendemos dissecar os rumores infundados acerca da invasão do Timor português pela autodenominada União da República de Timor (Uni Republic Timor, URT). Finalmente iremos ver as repercussões do golpe de Estado em Jacarta, em 1 de outubro de 1965, quer para o Timor português, quer nas relações indonésias-portuguesas. Quando o regime português passou a democrático e procedeu à descolonização do império português, o regime autoritário de Jacarta pôs em marcha a ocupação de Timor-Leste por javaneses, que durou 24 anos.

The most immediate and direct impact of global warming on human health is recognised by the consistent increase in the average global temperature and increase frequency, intensity, and heatwaves duration. This example of extreme heat events is already considered one of the most important climatic hazards and constitute a leading cause of mortality worldwide, particularly in southern Europe with pronounced impacts on cities. The respective health and economic burdens are of growing concern and have attracted various authorities and institutions to find new public health measures to protect citizens. Evidence supports that heat hazard related health outcomes are associated with the populations' exposure, vulnerability, and adaptive capacity. The literature identifies individual risk factors such as age (individuals over 65) and people with pre-existing cardiovascular and/or respiratory diseases. In addition to health and demographic factors, the community's vulnerability is also associated with low socioeconomic conditions, lack of urban vegetation, low education attainment, building characteristics, among others. Less studied are the population adaptation and adaptive capacity. Portugal has a well-documented heat-related morbidity and mortality history. It has a set of well-established models for heat-related mortality either for the city of Lisbon and for Portugal mainland (joining models for four ad-hoc constructed geographical regions) – known as ÍCARUS' models. Based on these models Portugal has a well-established Heat Health Warning System (HHWS) since 1999 – the ICARUS Surveillance System, recognised in scientific literature as the first of such systems in Europe. The studies present in this thesis aimed: 1) to analyse the eventual need to update the existing ICARUS′ models and surveillance system, 2) and to investigate the potential advantage of studying mortality at the neighbourhood scale of Lisbon's municipality, the respective spatial patterns of heat-related mortality and their potential explanatory factors. The performed work building from the evolution in Geographic Information Systems (GIS) and the currently associated sets of analytical and statistical methods went beyond conventional geographical scales analysis to align with the real people's needs and improve health outcomes' spatial patterns knowledge and accuracy. This dissertation's work showed that the national models for heat-related mortality, particularly the Lisbon model, and inherently the existing Surveillance System, do not need updating. The heat-related mortality metric for Lisbon models GATO IV (Generalized Accumulated Thermal Overload) has a good predictive power to prevent health heat-related risks performing better for Lisbon than the international metric EHF (Excess Heat Factor), more broadly known, cited, and referenced in the scientific community. It was also showed how to geocode deaths in Portugal using the addresses present on e-death certificate and, subsequently, its mapping elderly's heat-related cardiorespiratory mortality at a neighbourhood scale for Lisbon. It was possible to understand the potential spatially-varying factors, from medical facilities to socioeconomic, urbanistic and environmental factors also associated with each neighbourhood. To our knowledge, this is the first spatial modelling of heat-related mortality at this scale, and with such a wide range of variables.

Memory complaints are considered the initial and the most prominent symptom of Alzheimer’s disease (AD), leading the patients to seek for clinical assistance. Recent revised criteria that incorporate biomarkers maintain the report of memory decline by patients or informants as part of the core diagnostic features for AD. Subjective memory complaints (SMC) thus represent an important symptom in clinical practice, as individuals with SMC are at greater risk to develop dementia. On the other hand, memory complaints are very common in the general population and in the community besides elderly people many young individuals consider they are forgetful. The clinical significance of SMC may depend upon the settings where the participants are recruited and may also depend upon the way they are assessed. Patients with amnestic Mild Cognitive Impairment (aMCI) have memory complaints and objective impairment in memory and/or other cognitive domains, without major repercussions in daily life, and are not demented, although the aMCI is a condition that often progresses to dementia, mainly AD. The clinical use of biomarkers has allowed the possibility of diagnosing AD in patients that present with aMCI, as they reflect pathological alterations in the brain characteristic of AD, in particular amyloid-β (Aβ) biomarkers that ascertain whether an individual is in the Alzheimer’s continuum. However, it is recognized that is unfeasible that all patients with aMCI could presently undergo testing of the whole set of biomarkers. Therefore, we aimed to investigate the hypothesis that aMCI patients with biomarkers of amyloid pathology have more SMC than those without amyloid pathology. More specifically, we test whether the Aβ+ aMCI patients present a higher SMC scale total score compared to the Aβ- aMCI patients. The participants were selected from a cohort of nondemented patients with cognitive complaints (CCC) and a comprehensive neuropsychological evaluation on the basis of a diagnosis of aMCI, a detailed assessment of memory difficulties with the SMC scale and known amyloid status that was determined through either CSFAβ 1–42 concentration or amyloid PET imaging. We studied 176 patients with aMCI (90 were Aβ+ and 86 were Aβ-). We observed that the total score of the SMC scale was not significantly different in the Aβ+ aMCI patients (9.48±4.18) when compared to the one of the Aβ- aMCI patients (10.52±4.57). Interestingly, a statistically significant positive correlation was observed between the SMC total score and the GDS (Geriatric Depression Scale) total score in both Aβ+ aMCI (r = 0.489; p = 0.001) and Aβ- aMCI patients (r = 0.594; p < 0.001). The SMC have been extensively studied in the context of AD and other neurodegenerative disorders, but less is known about the characteristics of SMC in patients with MDD. The SMC along with cognitive deficits are frequently observed in patients with Major Depressive Disorder (MDD). For sure, SMC are not specific of MDD as are frequently reported by healthy adults as well as by people in the community who seek for primary clinical care. It was suggested that patients with MDD might report complaints qualitatively different from other disorders. Thus, we investigated SMC and the relationship with objective memory performance in patients with MDD in comparison with patients with Mild Cognitive Impairment due to AD (MCI-AD) and healthy controls (HC). Patients with MDD (n=47), MCI-AD (n=43) and HC (n=45) were assessed with a self-report memory complaints scale (SMCS) and underwent a comprehensive clinical and neuropsychological assessment. A discrepancy score between episodic memory and the SMCS total score was calculated as a measure of memory awareness. We observed that patients with MDD (12.5±4.4) and patients with MCI-AD (10.9±4.1) had not significantly different SMCS total scores, whereas HC showed significantly lower scores (4.0±3.0). As much as 74.5% of patients with MDD patients and 65.1% of patients with MCI-AD reported prominent memory complaints, whereas only 4.4% of HC did. Therefore, patients with MDD had relatively preserved memory tests and showed inaccurate memory self-awareness as they under-estimated their memory functioning, a pattern distinct from both patients with MCI-AD and HC. In the present thesis, we investigated and discussed the role of the SMC to identifyamong the patients with aMCI those who have AD. The results pointed out that evaluating the SMC may not be helpful at this stage of the disease. A discussion was made regarding the possible reasons and other perspectives to study the SMC were advanced. On the other hand, although we found differences in memory awareness between patients with MCI-AD and patients with MDD, it remains unclear whether memory awareness might be useful to discriminate betwen MCI-AD and MDD. Some other perspectives to investigate the relationship between SMC with depressive symptoms, MDD, objective memory performance and ansognosia were debated.

Ageing of population is a worldwide reality and Portugal is one of the most ageing -leading countries. The prevalence of dementia increases with age, almost doubling every five years when over 60 years old. With the increase in human longevity, the global number of people with dementia is expected to grow significantly upon incoming years. Following increasing demographic ageing, the prevalence of dementia is particularly worrying in Portugal. Dementia prevalence has undergone a vast increase in recent years, and it has become one of the greatest healthcare challenges of the 21st century due to the high demand for medical, social, and institutional care. Dementia is a clinical syndrome characterised by a cluster of symptoms manifested by impairment in higher brain functions such as memory, language and cognitive functions, changes in behaviours, entailing difficulties in activities of daily living (ADL). As a result, there is an increasing demand for informal care that play an important role for people with dementia (PwD), helping them in their daily activities, whose dependence increases as disease progresses. The task of caregiving is complex and can lead to physical, mental, and financial stress for caregivers. Emotions such as guilt, resentment, sadness, and anticipated loss of the cared one can emerge. Therefore, informal caregivers significantly endure levels of psychological morbidity, depression, stress and burden. It is widely reported that dementia informal care affects the health, well-being and quality of life (QoL) of caregivers. Therefore, the burden experienced by caregivers might result in low capacity or mood to care for their relatives with dementia, affecting patient’s well-being as well. The studies of this thesis aimed to help characterize the daily impact of dementia in a Portuguese sample of PwD and their informal caregivers at the individual, familiar and social level. To measure the QoL and dependency level of the patients, while describing their living context and formal care services utilization. To describe, as disease progresses, how it is like to care for a PwD in a familial context and the costs involving dementia were also objectives of the work, as well as, to measure the QoL and physical, emotional and social burden of the informal caregivers and describe the main factors involved. This thesis is a synergy of qualitative and quantitative methods. The mixed use of these methodologies empowered caregivers, allowing a voice to better describe how is caring for a PwD and its impact on QoL. The quantitative part of this study allows data characterization of patient’s QoL, cognitive and functional ability and, informal caregivers QoL and physical, emotional and social burden, together with study of the influence of sociodemographic variables of the dyads that influenced PwD QoL, informal caregivers QoL and overall burden. The work in this dissertation showed that our sample of Portuguese PwD were referred by their caregivers as having extreme low levels of QoL and high impairment on their functional and cognitive ability. Of note, dementia informal care included a wide range of experiences referred by the caregivers, as well as drastic changes introduced in their personal lives. Altogether, informal care resulted in high burden and low levels of QoL for the informal caregiver. Factors such as the PwD educational level, the changes in everyday life and habits were identified as potential explanatory factors of PwD HRQoL. Different explanatory factors were identified for caregivers QoL and burden, however, in both cases the familiar relationship with the PwD and PwD pain/discomfort were identified. Planning to decrease the burden of dementia informal care and the impact on caregivers and patient’s well-being, will likely mean that the articulation and cooperation of different public services (formal care services, neurology, psychiatric, psychology, social and financial support) should be re-thought. In the future, more studies are needed to further characterize the hurdles and needs of dementia care to further develop multicomplex interventions aimed to reduce the negative effects of dementia on patient’s QoL, while promoting caregiver’s QoL and decrease the overall burden.

Due to the unique mode of action and high selectivity, plant defensins (PDs) constitute a group of therapeutic candidates worthy of note. PDs are involved in the first-line defense system in plants having strong antimicrobial action on a wide variety of pathogens. Numerous studies highlighted the activity of several PDs against human infectious pathogens including resistant species of bacteria and viruses, and even cancer. This remarkable set of biological activities is additionally enriched by the low toxicity of healthy mammalian cells. This set of notable facets makes plant defensins interesting pharmaceutical candidates for further development. However, entry to the clinical pipeline requires a thorough characterization of the mode of action of a drug lead. Despite a reasonable understanding of the mode of action of plant defensins on microorganisms, there are very few reports elaborating on mechanisms in which PDs exert a harmful effect on cancer cells. To this end, the major pitfall concerns the production of PDs in quantities enabling detailed investigation on relevant in vitro and in vivo models. Chemical synthesis is the most frequently used method in the pharmaceutical manufactory. Nevertheless, several concerns discourage using this technique to produce more “complicated” peptides, such as plant defensins. PDs have a long sequence containing multiple cysteine residues. These residues are involved in intramolecular covalent disulfide bonds thus regulate peptide’s 3D structure. Consequently, due to the highly knotted folding, PDs have been mostly obtained through isolation from the plant extract or by heterologous expression systems. Natural peptide PvD1 is a representative of plant defensins family originating from common bean (Phaseolus vulgaris) from Brazil. This peptide shares common structural facets with other members of PDs such as the canonical CSαβ motif. PvD1 shows strong antifungal activity, yet it has been also shown to have antiprotozoal and anticancer properties, concurrently presenting a low toxicity profile on healthy human cells. Such a remarkable multifunctional action of this peptide calls for deepening the understanding of PvD1’s mode of action to further explore its medical application. Vibrant communication between cancer cells regulates their growth, development, and progression. This communication can be mediated by various signalling pathways enabling the contact between cells and extracellular matrix (ECM). One such pathway is tumour-derived exosomes (TDE). These are nano-sized vesicles that originated from the endosomal membrane and secreted by cells. As TDE can transmit genetic material and proteins they play a regulatory function in intracellular crosstalk. Moreover, they have been found to alleviate cancer survival mechanisms, such as multi-drug resistance. Hence, the development of the anticancer agent that could target TDE would envision an interesting strategy for tumour eradication. This first part of the project aimed to target the production of exosomes by breast cancer cells with natural PvD1 peptide. Here we focused on the modulatory effect of PvD1 on the expression level of CD63 and CD9 tetraspanin proteins in TDE. These proteins play an important role in controlling the formation of exosomes and are enriched in exosomal membranes. Additionally, the interaction of PvD1 with various biological membranes was followed by the combination of tailored biophysical techniques including dynamic light scattering (DLS), atomic force microscopy (AFM), and surface plasmon resonance (SPR). The innovative approach of immobilizing exosomes on the surface of the SPR sensor chip revealed that PvD1 can bind to these vesicles. Furthermore, a semi-quantitative analysis of the peptide-membrane interaction in TDE confirmed that, after binding PvD1, remains inserted and resides in the exosomal membrane. In contrast to exosomes, PvD1 does not cause perturbations of the membrane of cancer cells, presumably translocating inside the cell and targeting intracellular functions. Within this work, we contributed to broadening the understanding of the anticancer action of PvD1 and envision an innovative strategy for treating cancer. The advent of solid-phase chemical synthesis (SPPS) has enabled the easy production of pharmaceutical peptides. In addition, SPPS provides the tools for endless sequence manipulation to improve peptides’ properties or facilitate research. These comprise terminal modifications such as methylation or lipidation, introduction/deletion of amino acids, or labelling with fluorophores. Therefore, the goal of the second part of this work was to reproduce synthetically natural peptide PvD1. Analytical and structural analyses were employed in order to compare natural peptide with a synthetically obtained replica. Besides, solution-phase nuclear magnetic resonance (NMR) allowed elucidation of PvD1's structure, yet, predicted only by homology with defensin VrD2. Overlapping spectra of these two peptides and equal RP-HPLC elution time, provide strong evidence suggesting an identical structure. Moreover, to verify biological activity assays were performed on a collection of Candida species. It has been shown by in vitro and in vivo tests that both natural and synthetic PvD1 have expected antifungal activity, determined by the presence of glucosylceramide (GlcCer) in fungal membranes. Additionally, thanks to multiple disulfide bridges, tightly stabilizing the 3D structure, PvD1 preserves high resistance to proteolytic degradation. Overall, this work elaborates on the multifunctional activity of PvD1 peptide revealing a novel mechanism of anticancer action of plant defensin as well as strong antifungal properties. Adding susceptibility of this peptide to the chemical synthesis production method poses a valuable strategy for medical application either as a therapy alone or as a co-adjuvant in conventional treatments.

Breast conservative surgery (BCS) combined with radiotherapy has become the treatment of choice for the majority of early breast cancer patients. With an identical overall survival rate when compared to mastectomy, BCS is usually associated with a better cosmetic outcome. BCS aims to achieve optimal long-term local control, thus performing excisions with free margins. However, the wider the excision, and thus the possibility of attaining free margins, the higher the risk of poor cosmetic outcomes and breast deformity. If a positive margin is obtained, a re-excision may be needed, potentially impacting breast shape and cosmesis. At least 30% of cases still result in fair/poor aesthetic outcomes. These fair/poor results depend on several factors that have been thoroughly studied. Among these, breast volume, tumor size and location are the most important with a major impact on aesthetic outcome in multivariate analysis. But a single, easy and reproducible method to estimate breast volume is not available that could ultimately help in surgical planning for selecting the optimal BCS technique. Moreover, tumor size and location interpretation rely on the treating physician’s cognitive spatial interpretation of radiology images. New tools and methodologies are needed to improve breast cancer surgical planning and oncological outcomes. In this work, a new quantitative and an inexpensive method to accurately calculate breast volume using a three-dimensional (3D) technology was developed and validated with a depth sensor low-cost surface device (Microsoft Kinect®). Standard methods like breast MRI and mastectomy specimen volumes were used as ground truth, but differences between all methods need further developments to reach clinical applicability. Imaging fusion technology was attempted to harmonize anatomic limits and to improve volume delimitation between breast MRI and 3D surface scan methods. Furthermore, a methodology for breast MRI to 3D surface scan fusion was investigated. A patient-specific digital breast model integrating the real breast torso and the tumor location was created and validated with breast MRI to 3D surface scan fusion algorithm in 16 breast cancer patients. This protocol was used to quantify breast shape differences between different modalities and measure the target registration error of several MRI/3D scan fusion algorithm variants. The fusion of single breasts with a free form deformation model of pose transformation had acceptable registration errors and accurate tumor locations. The performance of the fusion algorithm was not affected by breast volume. Breast spatial interpretation and visualization by the treating physician can thus be augmented with a digital 3D breast model that integrates radiological images, allowing immediate image interpretation of breast cancer lesions. Using previous data, a clinical surgical use case was designed and attempted after successfully production of a 3D digital breast with breast MRI to 3D surface scan fusion. An experimental test with a digital non-invasive method for intraoperative breast cancer localization using augmented reality to guide breast conservative surgery was attempted. A successful overlap of the previous standard pre-operative localization technique (carbon tattooing) and tumor visualization inside the patient’s breast with augmented reality was obtained during surgery. Along the pathway and through computer vision as artificial intelligence, various methods were investigated to produce a unified view of a patient-specific 3D digital breast model, with tumor included. A clinical use case was developed to create an innovative methodology and a proof of concept for a digital non-invasive medical device for intraoperative breast cancer localization.

No summary/description provided

Adaptive immune responses are supported by lymphocytes that are broadly divided into B and T cells, which provide antibody and cell-mediated immune responses, respectively. Adaptive immunity is characterized by a specific pathogen recognition, generation of memory, and regulation of homeostasis. Lymphocytes develop and are activated in the lymphoid organs and can also re-circulate within the tissues. Intestinal Intraepithelial lymphocytes (IELs) occupy the top layers of epithelial barriers and are broadly composed of natural IELs (CD8ααTCRγδ) and induced IELs (CD8αβTCRαβ and CD4TCRαβ). IELs are kept in a heightened but controlled state of activation, have reciprocal interactions with the intestinal epithelial cells (IECs) and microbiota. There is evidence supporting that IELs contribute to the pathogenesis of gut disorders such as celiac disease and inflammatory bowel disease (IBD). Yet, the stimuli that control IELs activity and the molecular pathways involved remain unclear. In the context of a parasite infection, caused by Eimeria vermiformis that infects mouse IECs, induced IELs but not natural IELs are strongly activated. Interestingly, upon treatment with broad-spectrum antibiotics, followed by E.vermiformis infection, natural IELs proliferation is boosted. This proliferation boost is traced to Grampositive bacteria, the major producers of short-chain fatty acids (SCFA) and lactate. The hyperproliferative phenotype was also observed in the context of antibiotics and Dextran Sulphate Sodium (DSS) treatment. However, here just natural IELs got activated and induced IELs were not affected by the treatment. Importantly, the IEL proliferation boost is reversed by faecal microbiota transplantation (FMT) under antibiotics treatment and infection compared with no FMT. IELs do not express SCFA receptors but glucagon-like peptide-1 receptor (GLP-1R) is highly expressed by them. Fatty acid and lactate receptors are expressed by intestinal enteroendocrine L cells that produce GLP-1. Interestingly, we found GLP-1 mRNA and serum levels increased upon antibiotics treatment and DSS or E.vermiformis infection. Moreover, administration of Ex-9, a GLP-1 antagonist, reduced natural IELs proliferation. A diverse microbiota composition is important to sustain IELs and maintain their semi-activated state without effector function. Upon bacterial dysbiosis, especially reduced Gram-positives, natural IELs proliferation is released, and GLP-1 levels are increased. Currently, we characterise the microbiota complexity via 16S rRNA sequencing to understand the community that is responsible for keeping the natural IELs at the quiet state of activation. In the absence of this community, natural IELs proliferation is released, which could lead to gut inflammation, such as seen in IBD. Therefore, by manipulating the microbiota composition, the importance of specific bacteria and/or bacterial products that influence natural IELs activation and function will be addressed. This way, it will be possible to control the proliferation and activation of these cells to restore gut homeostasis. On December 2019 a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified from an outbreak in Wuhan, China, being responsible for the COVID-19 pandemic. In Portugal, the first case was reported in March 2020. In April 2020, we started working to understand the spreading of the virus, to identify those who are and were infected, and to follow the immune response longitudinally. We created a reliable and robust assay for SARS-CoV-2 detection and immunological monitoring. We designed an Enzyme linked immunosorbent assay (ELISA) assay to quantify IgM, IgG, and IgA antibodies against SARS-CoV-2 receptor-binding domain (RBD) or the Spike (S) protein. We described the meticulous setup to monitor the humoral immune response of over 300 COVID-19 hospital patients and healthcare workers, 2500 University staff, and 198 post-COVID-19 volunteers. SARS-CoV-2 infection induced a classic pattern of antibody responses with a rapid increase within the first three weeks after symptoms. Anti-SARS-CoV-2 IgG antibodies reduced in titres, although remaining robust with confirmed neutralization activity for up to 6 months in a large proportion of previously virus-positive screened individuals. Our work provides detailed information for the assays used, facilitating longitudinal analysis of protective immunity to SARS-CoV-2. Importantly, it highlights a continued level of circulating neutralising antibodies in most people with confirmed SARS-CoV-2.

γδ T cells have been proposed as a first line of immune defense, acting in response to a variety of stress-inducible or pathogen-associated metabolites. Their functions include a potent cytolytic and inflammatory activity against a wide range of malignant cells. However, γδ T cell-based clinical trials in cancer patients have only achieved objective responses between 10-33%. Therefore, a better understanding of the mechanisms involved in regulating γδ T cell activation and functional differentiation may be critical for their improving their outcomes in clinical settings. Data from the host laboratory has previously shown γδ T cell anti-tumor properties to be selectively acquired upon stimulation with interleukin (IL)-2 – but not with IL-7 – which induced the de novo expression of the transcription factors (TFs) T-bet and eomesodermin, required for the cytotoxic type 1 program. Critically, while the transcriptional key-players behind γδ T cell acquisition of cytotoxic / type 1 effector functions have been extensively explored, the post-transcriptional mechanisms that modulate γδ T cell anti-tumoral functions are still poorly understood. This PhD study aimed to explore an additional layer in the regulation of γδ T cell type 1 differentiation, by characterizing the post-transcriptional mechanisms mediated by microRNAs (miRs) involved in this process. We identified miR-181a(-5p and -2-3p) and miR-135b-5p to be either downregulated or upregulated, respectively, in differentiated (IL-2 cultured) γδ thymocytes, when compared with their immature (IL-7 cultured) counterparts. Interestingly, we observed a significant inverse correlation between miR-181a(-5p and -2-3p) expression and the protein levels of three important hallmarks of γδ T cell acquisition of type 1 effector program, namely tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and natural killer group 2 member D (NKG2D), suggesting a miR-181a-mediated suppression of γδ T cell differentiation. On the other hand, miR-135b-5p was positively correlated with these differentiation hallmarks, thus suggesting that this miR could promote γδ T cell acquisition of type 1 effector functions. Of note, while the miR candidates levels correlated with both NKG2D and type 1 cytokine production in in vitro-differentiated γδ thymocytes, these associations were no longer sustained for the type 1 cytokine levels in mature γδ peripheral blood lymphocytes (PBLs) ex vivo, indicating that miR-181a(-5p and -2-3p) and miR-135b-5p regulatory effect likely depends on γδ T cell maturation status. In line with this, while miR-181a overexpression significantly decreased TNF-α and NKG2D protein levels in artificially (in vitro-)differentiated γδ T cells, it only impaired the expression of NKG2D in naturally differentiated γδ PBLs, whereas type 1 cytokine production remained unaffected. Also noteworthy was the fact that miR-135b-5p overexpression in γδ PBLs elicited a mild decrease in TNF-α and NKG2D messenger (m)RNA levels, while it did not affect TNF-α, IFN-γ, or NKG2D protein levels. In silico data coupled with a Gene Set Enrichment Analysis (GSEA) identified two potential miR-181a-5p and -2-3p targets, Map3k2 and Notch2, which are linked with T cell development and differentiation processes; and two miR-135b-5p targets, Il10 and Cd39, associated with the establishment of an immunosuppressive phenotype in T cells. Importantly, Map3k2 and Notch2 levels were downregulated in γδ T cells overexpressing miR-181a; while miR-135b-5p overexpression in γδ PBLs impaired Cd39 (but not Il10) levels. Consistently, our luciferase reporter assays validated the direct binding between miR-181a and its two targets, as well as between miR-135b-5p and its Cd39 target. Finally, by assessing these miRs species profile in mature γδ PBLs isolated from metastatic cancer patients versus healthy donors, we found miR-181a(-5p and -2-3p) to be highly expressed in prostate cancer patients, and this pattern associated with lower NKG2D levels. On the other hand, we consistently observed a significant decrease in miR-135b-5p expression for all the cancer patients cohorts, which also associated with a decrease in NKG2D levels in these patients. Furthermore, we found CD39 and IL-10 expression to be increased in γδ PBLs from metastatic cancer patients, and both these molecules expression negatively correlated with NKG2D levels in these patients. Thus, we believe our study constitutes an important addition to the molecular mechanisms that govern the acquisition of type 1 effector functions of human γδ T cells, especially at the post-transcriptional level, which is less explored. Altogether our work highlights the critical role of miR-mediated mechanisms in modulating human γδ T cell type 1 functional differentiation / anti-tumor responses and open new avenues for the design of new (or improved) γδ T cell-based clinical protocols, towards their effective application in cancer immunotherapy.

We review the electrophysiological studies concerning the effects of caffeine on muscle, lower and upper motor neuron excitability and cognition. Several different methods have been used, such as electromyography, recruitment analysis, H-reflex, transcranial magnetic stimulation (TMS), electroencephalography and event-related potentials. The positive effect of caffeine on vigilance, attention, speed of reaction, information processing and arousal is supported by a number of electrophysiological studies. The evidence in favor of an increased muscle fiber resistance is not definitive, but higher or lower motor neuron excitability can occur as a consequence of a greater excitation of the descending input from the brainstem and upper motor neurons. TMS can address the influence of caffeine on the upper motor neuron. Previous studies showed that cortico-motor threshold and intracortical excitatory and inhibitory pathways are not influenced by caffeine. Nonetheless, our results indicate that cortical silent period (CSP) is reduced in resting muscles after caffeine consumption, when stimulating the motor cortex with intensities slightly above threshold. We present new data demonstrating that this effect is also observed in fatigued muscle. We conclude that CSP can be considered a surrogate marker of the effect of caffeine in the brain, in particular of its central ergogenic effect.

Introdução e objetivos: O estímulo de valores educativos em contexto escolar, no âmbito da Psicologia Positiva (“Values In Action”/VIA), pode não só facilitar o interesse pela aprendizagem numa perspetiva construtiva em relação ao futuro, mas também incentivar a uma mudança comportamental nos jovens, no sentido de prevenir o aparecimento de condutas agressivas. O objetivo deste trabalho consistiu em analisar os efeitos de uma intervenção através de técnicas psicoterapêuticas de imaginação guiada, na promoção de uma educação de valores junto de alunos do 2º Ciclo, com benefícios para a saúde mental. Pais e professores participaram na escolha destes valores (estudo observacional) e na avaliação do seu progresso ao longo do tempo durante o estudo experimental. Métodos: Participaram no total 83 alunos de quatro turmas do 2º Ciclo, tendo-se distribuído aleatoriamente duasturmas pelo grupo experimental (46 alunos) e duasturmas pelo grupo controlo (37 alunos). As turmas do grupo experimental foram sujeitas a técnicas de imaginação guiada, sob relaxamento, com leitura de histórias enquadradas em contexto positivo, incidindo sobre seis valores educativos previamente escolhidos por pais e professores (“Amizade”, “Cidadania”, “Aprendizagem”, “Esperança”, “Comportamento” e “Autenticidade”). A intervenção psicológica decorreu ao longo de um ano letivo, em paralelo com o leccionamento da disciplina de Formação Cívica incidindo sobre os mesmos valores, nos alunos do grupo controlo. A pontuação obtida para cada um dos seis valores foi avaliada nos dois grupos de alunos, mediante a aplicação de uma escala visual analógica (EVA) em três momentos: um pré-teste (M0) e dois pós-teste (M1, M2). Resultados: Devido às diferenças de pontuação encontradas entre as quatro turmas no M0, partiu-se de uma comparação entre turmas controlo-experimental com uma “baseline” semelhante de valores educativos (p > 0.05), por forma a evitar qualquer viés. Segundo pais e professores, verificou-se uma melhoria significativa (p < 0.05) na aquisição dos 6 valores educativos nas duas turmas experimentais, em relação às respetivas turmas controlo, principalmente no “Comportamento”, “Autenticidade” e “Esperança”. As turmas sem intervenção, registaram uma melhoria significativa (p < 0.05) apenas na “Amizade” e “Autenticidade”, chegando mesmo a registar-se um decréscimo significativo na pontuação dos outros valores. Conclusão: A intervenção através de imaginação guiada usada sob relaxamento, demonstrou uma melhoria significativa (p < 0.05) na aquisição dos valores educativos em estudo, nos alunos das turmas experimentais, em relação aos alunos das turmas da disciplina de Formação Cívica. Os alunos que registaram um aumento mais significativo na pontuação, foram aqueles que partiram de uma “baseline” de valores educativos mais baixa.

Transcription is an inherently mutagenic process that requires tight surveillance mechanisms to guarantee the preservation of genomic integrity. During transcription, the nascent RNA molecule can hybridize with the template DNA and form a DNA:RNA hybrid, displacing the single-stranded DNA (ssDNA). Although these triple-stranded structures, called R-loops, are physiologically relevant intermediates of several cellular processes, such as immunoglobulin class-switch recombination and gene expression, non-scheduled or persistent R-loops constitute an important source of DNA damage, namely DNA double-strand breaks (DSBs). To preserve genome integrity, cells possess diverse mechanisms to prevent the formation of R-loops or to resolve them. R-loop formation is restricted by RNA-binding proteins and topoisomerases, whereas R-loops are removed by helicases and ribonucleases, such as ribonuclease H enzymes RNase H1 and RNase H2, which degrade R-loops by digesting the RNA strand of the DNA:RNA hybrid. The concerted action of several R-loop resolving enzymes at different stages of the transcription cycle and in distinct physiological contexts is extremely important to maintain gene expression homeostasis and to prevent transcription-dependent DNA damage. Intrinsic features of the transcribed DNA influence the propensity to form R-loops. An asymmetrical distribution of guanines (G) and cytosines (C) nucleotides in the DNA duplex, with an excess of Cs in the template DNA strand (positive G:C skew), favors R-loop formation, which is further stabilized by the establishment of G quadruplexes in the G-rich coding strand. Additionally, the negative DNA supercoiling accumulating upstream of a transcribing RNA Polymerase II (RNA Pol II) creates a local DNA unwinding that provides a window of opportunity for nascent RNA to hybridize with the template DNA, hence promoting R-loop formation. R-loops can drive chromatin modifications that are critical for transcription regulation. Promoter-proximal R-loops enhance the recruitment of the Tip60–p400 histone acetyltransferase complex and inhibit the binding of polycomb repressive complex 2 and histone H3 lysine-27 methylation. Also, R-loops formed over G-rich terminator elements promote histone H3 lysine-9 dimethylation, a repressive mark that reinforces RNA Pol II pausing during transcription termination. Besides affecting histone modifications, R-loops act as barriers against DNA methylation spreading into active genes. DNA methylation, namely 5-methylcytosine (5mC), results from the covalent addition of a methyl group to the carbon 5 of a C attached to a G through a phosphodiester bond (CpG). The activity of DNA methyltransferase (DNMT) enzymes makes 5mC widespread across the mammalian genome, where it plays major roles in imprinting, retrotransposon silencing, and gene expression regulation. More than 70% of all human gene promoters contain stretches of CpG dinucleotides, termed CpG islands (CGIs), whose transcriptional activity is repressed by CpG methylation. R-loops positioned near promoters of active genes maintain CGIs in an unmethylated state, likely by reducing the affinity of DNMT1 binding to DNA, or by recruiting active DNA demethylation machinery: the ten-eleven translocation (TET) methylcytosine dioxygenases. In mammals, TET family comprises TET1, TET2 and TET3, which share the ability to oxidize 5mC to 5-hydroxymethylcytosine (5hmC), a relatively rare DNA modification found across the genome much less frequently than 5mC. TETs can further oxidize 5hmC to 5-formylcytosine (5fC) and to 5-carboxylcytosine (5caC), which engage in different pathways that lead to their replacement by native cytosine. Genome-wide, 5hmC is more abundant at regulatory regions such as promoters and exons, consistent with its role in gene expression regulation. The levels of 5hmC are enriched at active promoter regions, as observed upon activation of neuronal function-related genes in neural progenitors and neurons. Interestingly, 5hmC has the potential to modify the DNA helix structure by favoring DNA-end breathing motion, a dynamic feature of the protein–DNA complexes thought to control DNA accessibility. Moreover, 5hmC weakens the interaction between DNA and nucleosomal H2A-H2B dimers, facilitating transient nucleosome unfolding to accommodate the passage of RNA Pol II during transcription elongation. Also, 5hmC diminishes the thermodynamic stability of the DNA duplex: while 5mC increases the melting temperature, 5hmC reduces the amount of energy needed to separate the two strands of the DNA double helix. Molecular dynamics simulations revealed that the highest amplitude of GC DNA base-pair fluctuations is observed in the presence of 5hmC, whereas 5mC yielded GC base-pairs with the lower amplitude values. 5hmC destabilizes GC pairing by alleviating steric constraints through an increase in molecular polarity, rendering the DNA more flexible and less rigid. Because features that destabilize the DNA duplex, such as supercoiling or G-quadruplexes, are known to facilitate nascent RNA annealing with the template DNA strand, we reasoned that 5hmC may favor R-loop formation. We used an in vitro transcription model to show that the presence of 5hmC in the transcribed DNA promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. This hypothesis was further tested in vivo by editing 5hmC density and assessing the consequent impact on R-loops. Indeed, depletion of the three TET enzymes, which significantly reduced cellular 5hmC levels, caused a pronounced decrease in endogenous R-loops in mouse embryonic stem (ES) and fibroblast cells. Interestingly, the results obtained with individual depletion of each single TET were much milder, suggesting that there is a partial redundancy in the activity of the three TET enzymes. Additionally, CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The above described effects occurred independently of changes in transcription rate, firmly pointing towards a direct impact of 5hmC on DNA:RNA annealing. Collectively, these data suggest that editing 5hmC density by changing the expression levels or the genomic distribution of TET enzymes influences R-loop formation in cells. The interplay between 5hmC and R-loops was further strengthened by genome-wide analysis revealing a strong overlap, detected in half of all active genes, between both structures, which was validated through single-molecule co-localization techniques. Metagene plots of 5hmC and R-loops density show that overlapping of 5hmC and R-loops peaks at the transcription termination site (TTS), suggesting a dedicated role of TET activity in transcription termination by guiding the formation of R-loops. Strikingly, TET abrogation leads to significantly higher levels of readthrough transcripts genome-wide, a characteristic of defective termination. These data support a model whereby TET enzymes act upstream of R-loop formation during efficient transcription termination. Owing to the effect of 5hmC as R-loop facilitator, we also described a positive correlation between 5hmC-rich regions and DNA damage response markers, positioning 5hmC decorated loci as genomic instability hotspots. Finally, we wanted to explore the functional impact of R-loops formed in 5hmC-rich regions. Since TETs drive the developmental DNA methylome reprogramming, the role of 5hmC on stem cell differentiation and development has been widely acknowledged. As such, we used transcriptomic data from ES cells with global R-loop suppression. Gene expression analysis revealed that depletion of R-loops specifically positioned in 5hmC-rich loci impinges most significantly on pathways that control the proliferation/ dormancy balance in ES cells. Therefore, this study discloses a putative role for R-loops, instructed by controlled 5hmC deposition, as mediators in the activation of ES cells gene expression programs.

A criatividade, em contexto organizacional, tem sido definida enquanto a produção de ideias originais, úteis e apropriadas (Zhou & Su, 2010) e constitui um passo necessário para a inovação (Amabile, 1988). No entanto, existem questões sobre as condições convenientes que estimulam a criatividade que não estão esclarecidas (Mumford, Hester & Robledo, 2012), nomeadamente o impacto de recompensas extrínsecas (Oldham & Baer, 2012) e o impacto do feedback negativo (Zhou, 2008). O presente estudo examina o efeito destes dois fatores anteriores na geração de ideias criativas. Foi realizado um estudo experimental com quatro condições (2x2 inter-individual), com 80 enfermeiros de um hospital central. Esta investigação contribui para obter mais conhecimento sobre as condições apropriadas para que a criatividade e a inovação prosperem nas organizações.

A autossuficiência alimentar é um conceito fundamental para responder aos desafios presentes na sociedade global atual. Nesta dissertação são propostos dois modelos que podem ser utilizados integradamente para medir a autossuficiência potencial agroalimentar da Bacia Alimentar de Lisboa. Com a utilização de um método de inteligência artificial, sobre 45 variáveis explicativas relacionadas com condições socioeconómicas, biofísicas e bioclimáticas, procura-se entender quais as variáveis que podem influenciar a produtividade dos produtos agrícolas em estudo. Partindo deste conhecimento, numa segunda fase são aplicados três cenários dietéticos (dieta atual praticada pela generalidade da população, dieta de acordo com as recomendações da Direção Geral da Saúde e dieta vegetariana) com o intuito de entender, por um lado, qual a autossuficiência potencial da Bacia Alimentar de Lisboa e, por outro, qual deve ser o incremento em termos de área utilizada para que exista maior autossuficiência alimentar. Conclui-se que, apesar do desempenho do modelo ser abaixo dos valores de referência, as variáveis que têm um maior impacto na explicação da variável dependente (produtividade medida em calorias) vão ao encontro de outros modelos explorados anteriormente na bibliografia consultada. Conclui-se também que a Bacia Alimentar de Lisboa não é totalmente autossuficiente em nenhum dos cenários. No entanto, no cenário alimentar da dieta vegetariana a área de estudo tem valores mais próximos desse objetivo.

ENQUADRAMENTO: A biologia molecular representa um método promissor para diagnóstico e screening in vivo de condições neuropsiquiátricas, particularmente devido aos recentes avanços metodológicos de análise das ciências ómicas. Neste trabalho de tese, é explorado a neuropsicofisiologia de jovens indivíduos saudáveis sem doença neuropsiquiátrica ou do neurodesenvolvimento. Para tal, desenvolveu-se uma estratégia que combina saúde mental e estratificação molecular, de forma a definir subtipos de funcionamento, strata ou traços fenotípicos, numa amostra neurotípica. A definição de traços fenotípicos de funcionamento é importante para a adequada investigação de biomarcadores de suscetibilidade. Esta tese clássica é publicada como uma dissertação e é estruturada em duas seções fundamentais: Estratificação de Saúde Mental e Estratificação Molecular, compreendendo três subcapítulos cada. Neste trabalho é apresentado um modelo de estratificação para descrever correlatos mistos de saúde mental e funcionamento molecular, e, para esse efeito, são apresentados diferentes conjuntos de resultados para identificar as sucessivas etapas de caracterização.

Os mais de 30 anos de investigação sobre as células T γδ desde a sua descoberta mostraram que elas não seguem várias das “regras” ou paradigmas dos linfócitos T ditos “convencionais”, nomeadamente as células CD4+ e CD8+ da linhagem T αβ. Parte da biologia não convencional das células T γδ reside em seu programa de desenvolvimento tímico, que difere drasticamente dos linfócitos T αβ; enquanto estes precisam passar por processos periféricos de ativação e diferenciação, as células T γδ saem do timo com um programa efetor completamente maduro e estável. Dessa forma, os linfócitos T γδ chegam aos diferentes órgãos periféricos onde residem e exibem a sua capacidade de produzir citocinas pró-inflamatórias, nomeadamente IFN-γ ou IL-17 (estas últimas chamadas linfócitos T γδ17). Esse programa de desenvolvimento pouco flexível contrasta com evidências recentes que atribuem características adaptativas às respostas de células T γδ, e sugerem um papel central para essas células na detecção e integração de sinais ambientais. As células T γδ17, em particular, são críticas no controle das respostas de outras populações do sistema imunológico e de diferentes comunidades microbianas comensais, em diferentes tecidos de mucosa; a geração dessas células, no entanto, é altamente dependente do período de maturação no timo, e acredita-se que sua geração seja limitada a um curto período do desenvolvimento embrionário. Portanto, na presente tese, procuramos determinar se precursores encontrados na medula óssea adulta são capazes de originar células T γδ17 em resposta a fatores encontrados no ambiente extra-tímico. Mostrámos que, de fato, as células T γδ17 podem ser geradas de novo em linfonodos periféricos em resposta a estímulos inatos. Através do uso de abordagens in vitro e in vivo, conseguimos identificar a citocina IL-23 como o principal regulador desse processo. Por fim, descobrimos que as células T γδ17 induzidas na periferia representam uma fração substancial do total de linfócitos T γδ produtores de IL-17 durante o curso da encefalomielite autoimune experimental, modelo animal da esclerose múltipla, destacando a importância biomédica dessas descobertas. Além de estudar o papel de fatores provenientes de tecidos periféricos na homeostase das células T γδ17, outro objetivo importante desta tese foi estabelecer os mecanismos pelos quais as células T γδ regulam a homeostase do sistema imune nos tecidos de mucosa, concentrando-se nos pulmões. Curiosamente, descobrimos que, embora murganhos deficientes em células T γδ tenham uma distribuição normal de células inatas e adaptativas no tecido pulmonar ao longo do desenvolvimento, animais nascidos de mães deficientes em células T γδ um viés na produção de citocinas do tipo 2 no pulmão logo após o nascimento. Tais efeitos parecem ser independentes da transferência de anticorpos maternos. Contudo, em concordância com diferenças encontradas na microbiota tanto das mães quanto de suas proles, o uso de um coquetel de antibióticos anula essas diferenças; sugerindo que a seleção de comunidades microbianas por células T γδ maternas é importante no estabelecimento da colonização microbiana da prole durante os primeiros dias de vida e, consequentemente, no desenvolvimento do sistema imunológico pulmonar. Em conjunto, os resultados obtidos nesta tese lançam nova luz sobre o processo de geração periférica de células T γδ17 no contexto de doenças inflamatórias. Além disso, revelam uma contribuição inesperada das células T γδ maternas no desenvolvimento do sistema imunológico pulmonar das suas crias, abrindo uma nova avenida na investigação do papel das células T γδ na imunidade nos tecidos de mucosa.