Repositório RCAAP

γδ T cells home to tissues and peripheral lymphoid organs where they strongly contribute to the immune response through the production of IFN-γ or IL-17. IL-17 producing γδ (γδ17) T cells, particularly, can be found in almost all barrier tissues in the body, being critical to protect their hosts against infections while being mostly pathogenic in autoimmune settings. More recently γδ17 T cells have been found to contribute to normal physiological functions of the body. Recently, the notion of “immune privilege” of the central nervous system (CNS) has been revised to integrate its infiltration, at steady state, by lymphatic vessels and immune cells that participate in normal neurophysiology. For instance, murine CD4+ alpha-beta (ab) T cells regulate social behaviour and long-term memory. This notwithstanding, evidence for the role of other immune populations in both physiological and pathological contexts is still scarce. In this thesis, we aimed at understanding whether and how γδ T cell subsets may impact on short-term memory under physiological condition or in a neurodegenerative setting, more specifically in Alzheimer’s disease (AD). We found that foetal-derived Vγ6+ γδ T cell accumulate, at steady state, in the meningeal spaces in the first week of life, persisting throughout the mouse life. Murine γδ T cells are the main source of interleukin (IL)-17 in the meninges, while IFN-γ is mostly provided by their ab counterparts. Meningeal γδ17 T cells did not rely on typical inflammatory signals, shown to be important for their homeostasis and maintenance in other tissues. When tested for memory, mice lacking γδ T cells or IL-17 showed deficits in short-term memory (STM) while retaining a normal long-term memory when compared to littermate controls. In the absence of γδ T cells or IL-17, hippocampal basal transmission and long-term potentiation (LTP) are impaired, possibly due to alterations in the AMPA/NMDA ratio of glutamatergic neurons of these mice. Additionally, IL-17 promotes brain-derived neurotrophic factor (BDNF), whose supplementation is sufficient to recues STM deficits and synaptic impairments observed in mice lacking γδ T cells or IL-17. We added γδ17 T cells to the portfolio of the immune populations infiltrating the central nervous system, at steady state. We suggest a co-evolution of the immune and nervous systems where γδ17 T cells regulate short-term by promoting neuronal synaptic plasticity in the hippocampus. On the other hand, γδ17 T cells have been pointed as critical initial players in CNS disease progression, namely in models of multiple sclerosis, by contributing to a local immune amplification within the meningeal spaces and altering the stromal microenvironment of the inflamed brain. We therefore questioned whether this cytokine could be implicated in AD progression, where memory loss is one of the main hallmarks. We observed that the tripletransgenic progressive AD-mouse model (3xTg-AD) display a significant increase of γδ17 T cells infiltrating the meninges. This increase was observed at the onset of memory deficits, before the establishment of amyloid-beta (Ab) or Tau pathologies in 3xTg-AD animals. Interestingly, animals treated with anti-IL-17 mAbs did not develop STM deficits observed in the 3xTg-AD animals treated with isotype control. Furthermore, our data suggests that the anti-IL-17 treatment is able to prevent the basal synaptic transmission and LTP impairments that contribute to the early cognitive deficits observed in the 3xTg-AD mice. We postulate that a dysregulation of steady state meningeal IL-17 may influence the onset of AD. This suggests a hypothetic threshold of IL-17 levels, above which CNS inflammation and neurodegeneration can be promoted. This work highlights the potential value of IL-17 as an early biomarker of disease and as a therapeutic target in AD. Additionally, we questioned whether γδ17 T cells could populate other tissues. As the CNS, the testis was described as “immune privilege” and its immune landscape has been neglected. We uncovered that fetal-derived Vγ6+ γδ17 T cells populate the testis of naive mice, expand at puberty and persist throughout life. γδ T cells were the major source of IL-17 in situ and contrary to the meninges, their homeostasis was seemingly dependent on microbial cues and IL-1α/IL-23 signals. Our data indicates that γδ17 T cells contribute to tissue surveillance upon intratesticular bacterial infection. Altogether, this thesis expanded the knowledge in the γδ field by bringing to light two novel locations where Vγ6+ γδ17 T cells reside and contribute to local pathophysiology. We added γδ T cells and IL-17 as immune players to the field of neuroimmunology, by showing they regulate short-term memory and neuronal connectivity. Additionally, we suggest a dual role for IL-17 in memory. Finding a balance between the protective and pathological levels of IL-17 might open new immunotherapeutic strategies for neurodegenerative diseases, namely, AD.

Ground Surface Temperature (GST) is especially relevant in permafrost regions, such as the ice-free areas of the Antarctic Peninsula, to the understanding of environmental changes, where a long-term warming trend has been detected since 1950. To better understand GST regimes and the topoclimatic controlling factors, 20 iButtons were installed at sites according to elevation, exposure, curvature, and proximity to permanent snow, recording temperatures at 3-hour intervals from March 2019 to February 2020. Multiple Factorial Analysis (MFA) was used to evaluate the influence of these factors on GST parameters and to group the sensors based on their similarities. An analysis of daily temperatures was conducted to classify types of daily ground temperature regimes for use in a spatial model, developed using Discriminant Analysis (DA). As was predicted elevation was identified as the main controlling factor, with a negative correlation to the Mean Annual Ground Surface Temperature, ranging from 0.6 ◦C at 16 m a.s.l. to − 2 ◦C at 254 m a.s.l., and a positive correlation with Freezing Degree Days, ranging from 438 (at 16 m a.s.l.) to 1042 (at 254 m a.s.l.). Snow cover duration is the second control factor highlighted, determining the duration of the freezing season, which was prolonged where snow cover persisted longer, resulting in a more pronounced insulating effect. The diversity of conditions was reinforced with the identification of seven types of daily GST regimes (three frozen, two unfrozen, and two with freeze–thaw), leading to the categorization of four annual distribution types. These were spatialized for Barton Peninsula using the DA model (90 % accuracy). The spatialization revealed a long frost season in proximity to snow patches, moderate frost season in areas above 160 m a.s.l., a short frost season with slow warming in areas ranging from 90 to 160 m a.s. l., and a short frost season with rapid warming in areas below 90 m a.s.l.

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Apresentação e estudo do manuscrito "Breves noções sobre a Medicina Cafreal do Districto de Sofala" da autoria de Guilherme Hermenegildo Ezequiel da Silva.

Introduction: Parkinson’s disease (PD) is a common late life neurodegenerative disorder. It affects 1-2 individuals per 1000 persons at any time and its prevalence increases with age, affecting 1% of the population above 60 years old. Another important group of diseases that is more frequent in older patients is cardiovascular disease (CVD). Indeed, CVD is the number one cause of death worldwide, but it can be prevented by lifestyle change and/or medicines. These two diseases share some risk factors but also protective factors. Older age and male sex are associated with increased risk of both diseases and coffee consumption and physical activity are associated with a lower risk. However, cardiovascular risk in PD remains uncertain and controversial, with studies pointing to opposite directions. We hypothesized that PD patients could be protected from cardiovascular disorders due to lower blood pressure profile associated with dysautonomia. Aim: The present thesis aims: (1) To quantify the risk of cardiovascular events in PD patients; (2) To assess the frequency and typology of cardiovascular events in PD patients; (3) To compare PD patients to age- and sex-matched controls regarding subclinical cardiovascular disease; (4) To explore the brain-heart link in PD patients and matched controls. Methods: To achieve the aims purposed, four research projects were planned. Research project 1: Systematic review and meta-analysis of observational studies available in MEDLINE, Web of Science and Cochrane Central Register of Controlled Trials from inception to July 2019. The outcomes of interest were myocardial infarction, stroke, and cardiovascular mortality in PD patients compared to age- and sex-matched controls. Pooled estimates of odds ratios (OR) and 95% confidence intervals (CI) were derived by random effects meta-analysis. The study protocol was registered at PROSPERO: CRD42017076527. Research project 2: Systematic review and meta-analysis of randomized placebo- controlled trials with PD patients, available in MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials from inception to February 2017. The primary outcome was the proportion of major cardiovascular adverse events, defined as myocardial infarction, stroke, peripheral artery disease, and sudden death. The secondary outcome was the proportion and typology of all cardiovascular adverse events in placebo arm. A random- effects meta-analysis was performed to derive pooled estimates of the proportion of adverse events and corresponding 95% CIs. Freeman-Turkey transformation (double arcsine transformation) was used to adjust the dataset to estimate the proportion of the events, limiting the CI among 0-100%. Research project 3: Case-control study compared PD patients in the first 10 years of diagnosis with age- and sex-matched community controls. The primary objective was to evaluate the common carotid intima-media thickness (CIMT), as a marker of cardiovascular risk. The secondary objectives were to evaluate the presence of other subclinical organ damage, namely carotid plaques, intracranial stenosis, high pulse pressure, left ventricular hypertrophy, ankle brachial index, and renal dysfunction. An enriched subgroup of patients, with at least a moderate cardiovascular mortality risk based on a Systematic COronary Risk Evaluation (SCORE), was invited to perform a neuroimaging study, focusing on white matter hyperintensities and other markers of neuroimaging cerebrovascular biomarkers. Research project 4: Exploratory sub-study of case-control study population, to assess (a) the risk of atrial fibrillation, based on clinical scores (CHARGE-AF, HAVOC, HATCH) and electrocardiographic measurements (interatrial block/p-wave duration); and (b) the level of serum NTpro-BNP in both groups and its relationship with orthostatic hypotension. Results: Aim 1: Eleven studies were included in the systematic review of observational studies (9 cohort studies and 2 case-control studies). PD was associated with a significantly increased risk of stroke (9 studies: OR 1.66, 95% CI 1.19, 2.34; I2 50%). No significant differences were detected regarding myocardial infarction (8 studies: OR 1.15, 95% CI 0.72, 1.83; I2 76%) nor cardiovascular mortality (7 studies: OR 1.11, 95% CI 0.85, 1.45; I2 47%) risks in PD patients. Aim 2: To systematically review cardiovascular adverse events reported in randomized controlled trials (RCT), we included 236 trials, 80% (n=189; 14704 patients) of which reported data on cardiovascular adverse events. The pooled proportion of major cardiovascular events ranged from 0.00% to 0.06%. The proportion of all cardiovascular adverse events was 3.33% (95% CI 2.14, 4.70%), and ranged from 1.71% in de novo PD patients to 4.56% in patients receiving levodopa as their only antiparkinsonian medication. The most common adverse events were hypertension and orthostatic hypotension. Aim 3: Focusing on the case-control study, 102 participants were included in each arm. There was no difference in CIMT among the groups. Carotid plaques were more frequent in PD patients (OR 1.90, 95% CI 1.02, 3.55), although lipid profile was more favorable in this group. Nocturnal systolic blood pressure was significantly higher in PD patients and more than half were non dippers or reverse dippers. The enriched subgroup of patients included in the sub-study of neuroimaging consisted of 24 patients with PD and 23 controls. The median SCORE was 5% in both groups. No significant difference regarding white matter hyperintensities (OR 4.84, 95% CI 0.50, 47.06), lacunes (OR 0.43, 95% CI 0.07, 2.63), microbleeds (OR 0.64, 95% CI 0.13, 3.26), or infarcts (0.95, 95% CI 0.12, 7.41) was found. Aim 4: In the exploratory analysis regarding atrial fibrillation risk, from the potential 194 participants, 3 (from the control group) were excluded due to previous diagnosis of atrial fibrillation. Comparing PD patients (n = 97) with controls (n = 95), there was no difference regarding mean p-wave duration (121 ms vs. 122 ms, p = 0.64), nor the proportion of advanced interatrial block (OR 1.4, 95% CI 0.37, 5.80). All patients had low or medium risk of developing atrial fibrillation, based on clinical scores. There were not differences between PD patients and controls regarding the mean values of CHARGE-AF, HATCH and HAVOC. NT-proBNP was non-significantly higher in PD patients (158 +/- 214 ng/L vs 143 +/- 272 ng/L; p = 0.65), but it was statistically increased in PD patients with orthostatic hypotension (254 ng/L vs 135 ng/L; p = 0.028). Conclusions: The best evidence available in observational studies showed an association between PD and increased risk of stroke. The risk of myocardial infarction and cardiovascular mortality was not different among PD and controls. On the other hand, data from placebo-arm of RCTs suggests that the proportion of major cardiovascular adverse events is low and that blood pressure abnormalities are the most frequent cardiovascular adverse events in PD patients. Our case-control study allows us to refute the hypothesis that PD could be protected from cardiovascular disease, based on null results of CIMT and neuroimaging cerebrovascular risk factors, and higher frequency of carotid plaques and abnormal dipper profile in PD patients. their only antiparkinsonian medication. The most common adverse events were hypertension and orthostatic hypotension. Aim 3: Focusing on the case-control study, 102 participants were included in each arm. There was no difference in CIMT among the groups. Carotid plaques were more frequent in PD patients (OR 1.90, 95% CI 1.02, 3.55), although lipid profile was more favorable in this group. Nocturnal systolic blood pressure was significantly higher in PD patients and more than half were non dippers or reverse dippers. The enriched subgroup of patients included in the sub-study of neuroimaging consisted of 24 patients with PD and 23 controls. The median SCORE was 5% in both groups. No significant difference regarding white matter hyperintensities (OR 4.84, 95% CI 0.50, 47.06), lacunes (OR 0.43, 95% CI 0.07, 2.63), microbleeds (OR 0.64, 95% CI 0.13, 3.26), or infarcts (0.95, 95% CI 0.12, 7.41) was found. Aim 4: In the exploratory analysis regarding atrial fibrillation risk, from the potential 194 participants, 3 (from the control group) were excluded due to previous diagnosis of atrial fibrillation. Comparing PD patients (n = 97) with controls (n = 95), there was no difference regarding mean p-wave duration (121 ms vs. 122 ms, p = 0.64), nor the proportion of advanced interatrial block (OR 1.4, 95% CI 0.37, 5.80). All patients had low or medium risk of developing atrial fibrillation, based on clinical scores. There were not differences between PD patients and controls regarding the mean values of CHARGE-AF, HATCH and HAVOC. NT-proBNP was non-significantly higher in PD patients (158 +/- 214 ng/L vs 143 +/- 272 ng/L; p = 0.65), but it was statistically increased in PD patients with orthostatic hypotension (254 ng/L vs 135 ng/L; p = 0.028). Conclusions: The best evidence available in observational studies showed an association between PD and increased risk of stroke. The risk of myocardial infarction and cardiovascular mortality was not different among PD and controls. On the other hand, data from placebo-arm of RCTs suggests that the proportion of major cardiovascular adverse events is low and that blood pressure abnormalities are the most frequent cardiovascular adverse events in PD patients. Our case-control study allows us to refute the hypothesis that PD could be protected from cardiovascular disease, based on null results of CIMT and neuroimaging cerebrovascular risk factors, and higher frequency of carotid plaques and abnormal dipper profile in PD patients.

Caffeine, an adenosine receptors antagonist, and marijuana, a cannabinoid receptor agonist, both affect human cognition. Their effects mostly result from their interference with the endogenous purinergic system and endocannabinoids (eCBs) system in the brain. Neuromodulators such as adenosine and endocannabinoids are important regulators of central nervous system neuronal activity. They are also able to modulate synaptic transmission individually. In the hippocampus, adenosine receptor type 1 (A₁R) and cannabinoid receptor type 1 (CB₁R) are the main receptors for adenosine and endocannabinoids, respectively. Both receptors are G-protein-coupled receptors that activate Gi/o. They are expressed in both inhibitory and excitatory neurons, and also in glial cells such as astrocytes. Chronic caffeine administration exacerbates spatial memory impairment caused by acute tetrahydrocannabinol (THC), which is the main psychoactive molecule of cannabis. In the hippocampus, A₁R and CB₁R are involved in memory impairment caused by CB₁R activation. It has also been shown that CB₁R activation decreases GABA and glutamate release in the hippocampus. These effects are partially reduced by co-activation of A₁R, suggesting an interaction between these modulatory pathways at the level of G-protein activation. The main sources of extracellular adenosine are 1) the extracellular production from the hydrolysis of adenine nucleotides and 2) transport from intracellular adenosine sources. eCB synthesis mostly results from the cleavage of postsynaptic membrane lipids. The activation of postsynaptic G-coupled glutamate metabotropic receptors induces the cleavage of postsynaptic membrane lipids, which is predominantly activated due to high neuronal firing. Thus, eCBs travel in a retrograde manner to activate astrocytic and nerve-terminal CB₁R, which inhibit neurotransmitter release and lead to several forms of short-term synaptic plasticity. CB₁Rs are endogenously activated by eCBs, mainly the fatty acids 2-arachidonoyl-sn-glycerol (2- AG) and anandamide. CB₁R agonists impair cognition and prevent long-term potentiation (LTP), synaptic plasticity induced by brief high-frequency neuronal firing, and synaptic transmission. Still, the influence of endogenously formed cannabinoids on hippocampal LTP remains ambiguous. In this study, it was possible to evaluate the influence of endocannabinoids on synaptic plasticity. I also wanted to determine if adenosine, through A₁R, could affect eCB signalling. The deletion or blockade of A₁R did not significantly change the modulatory effect of eCBs on LTP. I did not find evidence of a cross-talk mechanism at the synaptic plasticity level involving the two neuromodulators (A₁R and CB₁R). LTP induced by a weak θ-burst stimulation (wTBS) protocol was facilitated when blocking the action of eCB on CB₁R. In contrast, LTP induced by a strong θ-burst stimulation (sTBS) protocol was inhibited when the endogenous activation of CB₁R was blocked. This suggests that eCB inhibit weak LTP and facilitate strong LTP. The dual effect is mediated by 2-AG, suggesting that it acts as a high-pass filter that likely reduces the signal-to-noise-ratio of synaptic strengthening. The facilitatory effect of eCBs upon strong LTP depends on the activity of GABAergic interneurons. It was also described a modulatory role for A₁R on the CB₁R activation effect on inhibitory postsynaptic potential currents (IPSCs). When A₁R was blocked, the inhibitory effect of CB₁R activation on pyramidal cells was abolished, suggesting that an interaction between A₁R and CB₁R is at play. Overall, this work provides a better understanding of the neuromodulation of synaptic transmission and plasticity in the hippocampal CA1 region. It was able to show that CB₁R plays an important homeostatic role in synaptic plasticity phenomena through an A₁R independent process. However, A₁R seems to play a modulatory role in the action of eCB on inhibitory synaptic transmission.

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This study presents a theoretical model regarding the entrepreneurial potential construct, and the main psychosocial aspects that contribute towards an individual’s preparedness to engage in activities typically associated with entrepreneurship. The general question addressed in this study is: How to explain the entrepreneurial potential construct theoretically, and how to assess it empirically? This study seeks to contribute by creating an instrument (the EPAI – Entrepreneurial Potential Assessment Inventory) that can be used to measure the entrepreneurial potential construct. In this paper we present four studies on its empirical validation. The results suggest reliable scale characteristics, convergent and discriminant validity. The EPAI can be used by an entrepreneur for self-assessment, for training, and for professional development.

Regulatory T cells (Tregs) are essential elements of a healthy immune system. They comprise 5-10% of the peripheral blood CD4 T cell compartment in healthy individuals and play a critical role in protecting their host against immunopathological damage following inflammatory or immunological challenges. Tregs are composed of heterogeneous subsets that together can suppress a wide range of effector cell types through several mechanisms. Humans have since birth Treg maturation and phenotypic heterogeneity, which increases during our childhood. The Treg capacity to suppress effector cells allows transplantation tolerance, and when this careful balance is disturbed, it can lead to graft-versus-host disease (GVHD). Treg heterogeneity also rapidly increases after hematopoietic stem cell transplantation (HSCT). However, a precise and coordinated balance between several immune cells is required to prevent transplant rejection. Patients with GVHD not only have fewer Treg cells but also fewer Treg subpopulations and higher activation of effector cells. Two newer possible ways to treat GVHD, are with low-dose interleukin 2 (IL-2) or with antibodies conjugated to an immunotoxin (CD3/CD7-ricin A). Low-dose IL-2 therapy selectively induces the expansion of Tregs, improving the clinical manifestations of chronic GVHD. It promotes Treg homeostasis without activating effector cells. The anti-CD3/CD7-ricin A therapy, which mostly depletes T and natural killer cells, has an overall response rate of 60%, with 50% achieving a complete response. The 6-month overall survival rate is 60%. After the profound depletion caused by the treatment, the immune system recovers with a diverse T cell repertoire leading to the transplant tolerance. The profound but highly selective immunologic effects of both therapies in promoting immune tolerance may be used in a wide variety of clinical settings. Furthermore, the translational research methodology developed can be reapplied in future clinical studies, unveiling the defects that contribute to other immune dysfunctions. Chapter 1 contains a general introduction. We start by defining Treg cells and explaining how these cells are essential for maintaining tolerance and preserving the immune system's homeostasis. Then, we analyzed the relationship between Treg cells and HSCT. Tregs have the ability to mediate transplant tolerance and, when this careful balance is disturbed, it can trigger GVHD. The imbalance between effector and regulatory T cells that occurs during GVHD is explored in detail. In addition, we highlight the relevance of low-dose IL-2 therapy for patients with GVHD and how the therapy positively recovers the Treg population. Finally, we briefly describe the main objectives of this thesis. In chapter 2 we specify the aims and outline of this thesis. In chapter 3, we investigated the diversity of the Treg cell compartment in newborns, healthy adults, during the 2-year period after HSCT and in patients suffering from chronic GVHD. We have shown that adults have significantly higher amounts of Tregs in their blood than those present in umbilical cord blood (CB), but they also have additional functional and memory subpopulations of Tregs that are not present in CB. The number of subpopulations of functional and memory Tregs expands from birth to adulthood. As for patients after HSCT, the memory Treg subpopulation repertoire is already present after transplantation, while the diversity of Treg cells still expands within the 2-year post-transplant. GVHD patients present significantly fewer distinct subpopulations of functional Tregs, proposing a correlation between the lack of heterogeneity of Tregs and chronic GVHD. In chapter 4, we demonstrated that low-dose IL-2 therapy selectively induces the expansion of CD4 Tregs and improves the clinical manifestations of chronic GVHD. Using mass cytometry, we demonstrated in chapter 4 that low concentrations of IL-2 selectively induce STAT5 phosphorylation in Helios CD4 Tregs cells and CD56bright CD16– NK cells in vitro and in vivo. The effects of low-dose IL-2 therapy on conventional CD4 T cells and CD8 T cells were limited to increased expression of PD-1 in memory effector T cells. The selective effects of low-dose IL-2 therapy on Helios CD4 Tregs cells and NK CD56bright cells induce the constitutive expression of high-affinity IL-2 receptors. Recognizing that we urgently need more effective therapies for the treatment of patients with steroid-refractory acute graft-versus-host-disease (SR-aGVHD), we describe, in chapter 5, a phase I / II clinical trial that examines the safety and effectiveness of a new biological therapy for (SR-aGVHD). A combination of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7–1T). This induces in vivo depletion of T cells and NK cells, and suppresses the activation of the cellular receptor in T cells. On the 28th day after the initiation of therapy with CD3/CD7-IT, the overall response rate was 60%, with 50% reaching a complete response. The overall 6-month survival rate was 60%. The treatment, which was administered for 1 week, caused profound but transient depletion of T cells and NK cells, followed by rapid recovery of the immune system with a diverse repertoire of T cells and preservation of specific T cells capable of identifying Epstein-Barr virus and cytomegalovirus. Treatment with CD3/CD7- IT was safe and well tolerated by patients, with a relatively low prevalence of adverse events, such as hypoalbuminemia, microangiopathy and thrombocytopenia, which were controllable and reversible. Finally, we were interested in testing the same mass cytometry panel used in chapter 3, to study Treg cells in other circumstances of immune disorder, unrelated to HSCT. In chapter 6, we evaluated idelalisib as a first-line therapy for the treatment of chronic lymphocytic leukemia (CLL) relapse. After a follow-up period of 14.7 months (on average), hepatotoxicity was considered a frequent and often serious adverse event. Several lines of evidence suggest that this hepatotoxicity was immune-mediated. A lymphocytic infiltrate was seen in liver biopsy samples taken from 2 individuals with transaminitis, and the levels of pro-inflammatory cytokines CCL-3 and CCL-4 were higher in individuals with hepatotoxicity. A decrease in peripheral blood regulatory T cells was observed in patients who suffered from hepatotoxicity during therapy, which is consistent with an imune-mediated mechanism. In chapter 7 we conclude this thesis by summarizing and discussing the results.

Giant cell arteritis (GCA) is the most common form of primary systemic vasculitis in patients aged ≥50 years, causing chronic inflammation of the large- and medium-sized arteries. If left untreated, GCA can result in permanent visual loss or other ischaemic complications. However, its established treatment with glucocorticoids (GCs) can lead to significant toxicity. Therefore, it is crucial to establish a correct diagnosis in patients with suspected GCA. For many years, temporal artery biopsy (TAB) has been considered the diagnostic ‘gold standard’ for GCA, but it has many limitations, including its low sensitivity. Ultrasound has proven to be effective for diagnosing GCA and can reliably replace temporal artery biopsy in particular clinical settings. Moreover, in cases of suspected large-vessel GCA (LV GCA), other imaging modalities can also be used (e.g., positron emission tomography). However, good diagnostic algorithms that take into account the different manifestations of the disease and the uneven accessibility to all diagnostic tests between centres are yet lacking. In addition, potential diagnostic composite scores, with a strong emphasis on different imaging findings, are still an unmet need. The widespread use of vascular imaging in clinical practice has expanded the clinical spectrum of large-vessel vasculitis (LVV) and exposed the limitations of the 1990 American College of Rheumatology (ACR) classification criteria for GCA, which are biased to identify patients with disease confined to the cranial arteries. Patients with GCA may have vasculitic involvement of the aorta and primary branches in a pattern that resembles angiographic findings in Takayasu arteritis (TAK). Hence, updated classification criteria that include vascular imaging findings are needed in LVV. Not all patients with GCA follow the same course, but there are no valid biomarkers to assess therapy response. Changes in the traditional acute phase reactants - erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) - do not consistently reflect disease activity, are not GCA-specific, and have limited value in assessing patients treated with IL 6 inhibitors such as tocilizumab. The role of ultrasound as an alternative monitoring tool for these patients is still poorly understood and needs further investigation. This doctoral thesis sought to provide new evidence on the use of ultrasound to diagnose, classify and monitor patients with GCA. Its specific aims were: 1) to investigate the diferente diagnostic assessment strategies for GCA worldwide; 2) to evaluate the impact of disease extent and severity detected by ultrasound in the diagnosis of GCA; 3) to develop new classification criteria for large vessel vasculitis incorporating imaging; 4) to explore the use of ultrasound in monitoring disease activity and response to treatment in patients with GCA Encompassed by the first aim of this dissertation, we developed a Portuguese registry for patients with vasculitis – Reuma.pt/vasculitis. By July 2018, 125 patients with the diagnosis of GCA had already been registered. The ultrasound of the temporal ± axillary arteries was the diagnostic modality of choice for these patients (n=94), with 82/94 (87%) showing a non-compressible halo sign, followed by TAB (n=72), with 47/72 (65%) showing histologic features compatible with vasculitis. We further evaluated the diagnostic assessment strategies for GCA using data from a large, international cohort (the Diagnostic and Classification Criteria for Vasculitis [DCVAS] cohort). A total of 941 patients recruited into DCVAS had a confirmed diagnosis of GCA: 705 (75%) patients underwent TAB, 446 (66%) considered diagnostic for GCA, and 328 (35%) patients underwent temporal artery ultrasound, 258 (79%) with a presence of halo sign. Overall, these results reflect the current confidence of clinicians in requesting ultrasound for diagnostic proposes, and demonstrate the higher sensitivity of ultrasound to diagnose GCA, when compared to TAB. To address the second aim of this thesis, we developed a composite scoring system, including ultrasound quantitative findings, combined with clinical features, to stratify patients based on the risk of having a positive TAB. We included 135 patients with GCA recruited into a large prospective multicentre study (the Temporal Artery Biopsy vs. ULtrasound [TABUL] study) who had a positive halo sign on the temporal and/or axillary arteries. Four clinical models using “positive TAB” as the outcome of interest were tested. The best-fitting model included a combination of six positive items (maximum temporal artery intima-media thickness [IMT] >0.70 mm, bilateral temporal artery halos, maximum axillary artery IMT >1.30 mm, bilateral axillary artery halos, ischaemic GCA-features, and elevated acute phase reactants) and one negative item (symptoms of polymyalgia rheumatica). Our GCA-ultrasound score was successfully tested in an independent cohort of 72 patients, although no formal validation could be made due to the lack of TAB data in the testing cohort. We then proposed a diagnostic algorithm for patients with suspected cranial-GCA and rapid access to high-quality ultrasound. After a comprehensive literature review, we advocated that ultrasound should be the initial modality of choice and that the need for further testing should be tailored according to the clinical probability of GCA and quality of ultrasound results (e.g., presence of high IMT in various arterial segments). For the third aim, we used the DCVAS cohort to identify different patterns of arterial disease on vascular imaging able to differentiate LV-GCA from TAK. Patients with LV-GCA showed more vasculitic changes of the axillary/subclavian arteries bilaterally, more diffuse disease with vasculitic findings throughout the aorta and the aortic arch branch vessels, and less involvement of the abdominal vasculature than patients with TAK. Using the same DCVAS cohort, we then developed and validated new classification criteria for GCA and TAK, which included these different patterns of arterial involvement. In comparison to the original 1990 ACR Classification Criteria, the new ACR-European League Against Rheumatism (EULAR) Classification Criteria for GCA considers age at diagnosis of ≥50 years as a mandatory requirement to classify patients with GCA, and the new ACR/EULAR Classification Criteria for TAK defines age at diagnosis of ≤60 years as an absolute requirement to classify patients with TAK. Moreover, both criteria were developed based on weighed items with different threshold scores. The final criteria for GCA included: positive TAB or temporal artery halo sign on ultrasound (+5); ESR ≥50 mm/hour or CRP ≥10mg/L (+3); sudden visual loss (+3); and morning stiffness in shoulders or neck, jaw or tongue claudication, new temporal headache, scalp tenderness, temporal artery abnormality on vascular examination, bilateral axillary involvement on imaging, and FDG PET activity throughout the aorta (+2 each). A patient could be classified as GCA with a cumulative score of ≥6 points. The final criteria for TAK included: female sex (+1); angina (+2); limb claudication (+2); arterial bruit (+2); reduced upper extremity pulse (+2); reduced pulse or tenderness of the carotid artery (+2); blood pressure difference ≥20mmHg in the arms (+1); number of affected arterial territories (+1 to +3); paired artery involvement (+1) and abdominal aorta plus renal or mesenteric involvement (+3). A patient could be classified as TAK with a cumulative score of ≥5 points. The new 2022 ACR/EULAR classification criteria for GCA showed a sensitivity of 87.0% (95%CI: 82.0-91.0%) and a specificity of 94.8% (95%CI: 91.0-97.4%) and the new 2022 ACR/EULAR classification criteria for TAK showed a sensitivity of 93.8% (95%CI: 88.6-97.1%) and a specificity of 99.2% (95%CI: 96.7-100.0%). When compared to the original 1990 criteria, the new criteria for GCA and TAK demonstrated superior sensitivity while maintaining similar specificity. For the final aim, we performed a cross-sectional analysis of all patients from the TABUL cohort with GCA who had a temporal and/or axillary artery halo sign on ultrasound in the first 7-days of commencing GCs. A consistent reduction of the halo size with number of days on GCs was observed for the temporal arteries, but not for the axillary arteries. We then conducted a prospective analysis of 49 patients with newly diagnosed GCA looking at the following time-points: baseline and weeks 1,3,6,12 and 24. The halo size of the temporal arteries, as well as the number of temporal artery segments with halo, showed a significant reduction between all time-points and baseline. Moreover, a significant correlation between temporal artery halo features and disease activity markers (CRP, ESR and the Birmingham vasculitis activity score) and GC cumulative dose was found. Only after week 6, a significant reduction in halo size of the axillary arteries was observed, but no association between axillary halo features and disease activity markers or GC treatment was found. These findingssupport the use of ultrasound halo sign as a potential monitoring tool for patients with GCA. In conclusion, this thesis provides new evidence on the use of ultrasound to diagnose, classify and monitor patients with GCA, which will have future implications for clinical trials and research studies.

For emergency response teams such as SWAT (special weapons and tactics) or police tactical teams, team performance comes with life or death consequences. Nevertheless, research gives little attention to the dynamics particular to teams performing in extreme, dangerous, and stressful situations. In teams like police tactical teams the ability to coordinate members’ actions and expertise, while adapting to evolving circumstances, is paramount. This study examines the combined effects of team implicit coordination and transactive memory systems on team adaptive behaviors and performance in a sample of 42 real police tactical teams. Contrary to predictions in the literature, our findings suggest that team implicit coordination can benefit performance even for teams performing nonroutine tasks. Moreover, we found that the relationship between team implicit coordination and team adaptive behaviors is strengthened by transactive memory systems. In the end, we discuss the implications of these findings and point new directions for future research.

Em 1853, a Grã-Bretanha e a França, juntaram-se, numa aliança com o Império Otomano contra o Império Russo, ficando esta conhecida como a Guerra da Crimeia (1853-1856). Numa quezília entre a França e a Rússia, sobre a gestão dos Lugares Sagrados, em Jerusalém, pelas Igrejas Católica e Ortodoxa, uma guerra de projeção global tornou-se inevitável, quando esta situação rapidamente escalou para uma disputa estratégica pelo domínio dos Estreitos de Bósforo e Dardanelos. Devido à inexistência de documentos que permitam, atualmente, comprovar os interesses estratégicos russos, face ao Mar Negro, apenas um complexo contexto de expansão territorial secular, em direção à Crimeia permitem reconhecer que a Rússia, de facto tinha uma estratégia naval que, a partir de finais do séc. XVIII ficou centrada em garantir controlo sobre os Estreitos, o que viria a torná-la a potência hegemónica do Mar Negro. Finda a Guerra, ao Czar Alexandre II assumiu uma humilhante derrota política, aceitando um conjunto de limitações estratégicas, para prosseguir com a sua política. No entanto, em poucos anos, conseguiu reagir a essas limitações, voltando a ameaçar o status quo na bacia do Mar Negro, em virtude da prossecução dos seus objetivos estratégicos.

O presente estudo sobre o trabalho desenvolvido pelo Centro Novas Oportunidades da Escola Profissional Cândido Guerreiro (Alte) tem como objectivo realçar a sua abordagem territorial constituindo deste modo um relato inspirador, baseado no percurso da investigadora e logo, na sua interpretação. Com o intuito de explorar alternativas ao cenário actual em Portugal, foi feita uma contextualização sobre a Educação de Adultos. A par da explicação da metodologia RVCC colocada em prática, com recurso às parcerias e itinerâncias locais, realçou-se a implementação de um Plano de Actividades. Este constituiu o grande factor de inovação, na medida em que representa uma intervenção integrada (por ser sustentável e participada) e ecológica (por ser suscitada pelos locais e beneficiários últimos) da Educação de Adultos, através da animação sócio-cultural assente em dinâmicas locais. A metodologia adoptada consistiu na análise qualitativa de diversos documentos e registos, bem como na descrição dos factos pela investigadora (através de observação participativa).

Nesta dissertação, fazemos previsões de constrangimentos para dois modelos de Gravidade Teleparalela Simétrica usando a abordagem da Matriz de Fisher. Para estudar estes modelos usamos dados das Supernovas Ia para primeiro nos fornecer a abundância de matéria hoje e construímos catálogos de sirenes padrão com base nas especificações de três observatórios de ondas gravitacionais: LIGO, ET e LISA. O objectivo consiste em prever as suas respectivas capacidades em restringir os parâmetros dos modelos e consequentemente de averiguar se seremos capazes de distingui-los do modelo padrão da cosmologia ΛCDM. O modelo ΛCDM é bem conhecido por ser o modelo mais simples que fornece uma boa descrição das propriedades do Universo, tal como a existência e a estrutura da radiação cósmica de fundo nos micro-ondas (CMB), que é a evidência direta da expansão do Universo; a expansão acelerada do universo descoberta a partir do brilho e desvio para o vermelho de supernovas distantes; e a homogeneidade e isotropia em escalas suficientemente grandes do nosso Universo. Apesar do sucesso do modelo ΛCDM, ele ainda enfrenta algumas inconsistências, tal como a tensão de Hubble (o valor obtido a partir de dados de supernovas é significativamente maior do que o inferido usando a escala angular das flutuações da radiação cósmica de fundo) e as anomalias nas anisotropias do CMB (uma polarização ligeiramente rodada na radiação cósmica de fundo que não é explicada pelo modelo padrão). Durante décadas, os cientistas tentaram propor um modelo alternativo para resolver estas dificuldades modificando a ação de Einstein-Hilbert, onde em vez de se usar a curvatura para descrever a gravidade, consideraram-se grandezas matemáticas tais como não-metricidade ou torção. Esta dissertação baseia-se na não-metricidade, que é uma medida da variação do comprimento de um vetor durante o transporte paralelo. Em 2015, a deteção de ondas gravitacionais abriu uma nova porta para obtermos informações sobre o Universo. Uma das previsões da teoria da Relatividade Geral é que a fusão de sistemas binários como buracos negros ou estrelas de neutrões, resulta numa perda de energia na forma de ondas gravitacionais. Em particular, uma sirene padrão, é um evento que emite ondas eletromagnéticas e ondas gravitacionais, na sequência da fusão, por exemplo, de duas estrelas de neutrões. Um evento deste género fornece-nos a relação direta entre o desvio para o vermelho e distância luminosa ao binário. Esta é uma vantagem enorme em relação aos métodos tradicionais de medição de distâncias pelo método de escada de distâncias cosmológicas, que são susceptíveis a erros de calibração. Infelizmente, até ao momento apenas um evento sirene padrão foi confirmado, GW170817. Isto quer dizer que de momento ainda não somos capazes de colocar constrangimentos aos modelos cosmológicos mas podemos tentar antecipar o nível de precisão desses constrangimentos no futuro. Para fazer essa análise, geramos catálogos de sirenes padrão realistas e usamos o método da Matriz de Fisher (FM) para fazer a inferência estatística dos modelos em estudo. O método FM fornece um aumento extraordinário na velocidade de computação em relação aos métodos tradicioanais (de grelha ou Markov Chain Monte Carlo) quando lidamos com modelos com alguns parâmetros. Em vez de computação por força bruta, FM é uma aproximação analítica de uma função de verossimilhança Gaussiana, dado que a probabilidade máxima é conhecida a partir de informação obtida por observações independentes, como as restrições de parâmetros fornecidas pelos dados de supernovas na nossa dissertação. No primeiro capítulo desta dissertação, apresentamos uma breve história da cosmologia moderna a partir do aparecimento da Relatividade Geral. Em seguida, mencionamos algumas das descobertas mais importantes do último século, tais como a descoberta da radiação cósmica de fundo que indica que o Universo é globalmente plano, velocidade excessiva das galáxias que levou à ideia da matéria escura e a descoberta da expansão acelerada do Universo, que desvendou a existência de uma energia escura. Em seguida, apresentamos os pressupostos estabelecidos pelo modelo ΛCDM e os desafios que este enfrenta. Além disso, explicamos a distância luminosa e apontamos as Sirenes Padrão como ferramentas para a medição de distâncias em cosmologia. No segundo capítulo, apresentamos as ferramentas matemáticas utilizadas na Relatividade Geral e revemos dois conceitos geométricos chamados torção e não-metricidade que recuperam a formulação padrão da GR sem se usar curvatura. Em seguida, identificamos as fontes do tensor Energia-Momento na equação de campo de Einstein e explicamos a métrica FLRW assumindo o universo homogéneo, isotrópico e plano. Em seguida, mostramos os detalhes da modificação da equação de campo de Einstein com base na não-metricidade. O final deste capítulo explica o comportamento das ondas gravitacionais na gauge transversa e sem traço e como se obtém a distância luminosa das ondas gravitacionais para as teorias f(Q). O resultado é semelhante ao da contraparte electromagnetic (EM) mas com um termo multiplicativo adicional. No terceiro capítulo, apresentamos a metodologia para calcular a função de verossimilhança dos parâmetros de um modelo usando dados de supernovas e eventos de sirene padrão. Em seguida, explicamos o método Matriz de Fisher e como ele transforma um teste qui-quadrado dependente de dados numa formulação de Matriz de Fisher independente de dados. Depois, mostramos algumas propriedades essenciais da FM, como a transformação de variáveis, maximização e marginalização da função de verossimilhança. Também mostramos o procedimento de implementação de um código para o método da grelha e para o método Fisher Matrix. No final, este capítulo fornece a especificação dos detalhes de dois observatórios de ondas gravitacionais atualmente em operação, Laser Interferometer Gravitational-Wave Observatory (LIGO)-Virgo e dos dois futuros observatórios Laser Interferometer Space Antenna (LISA), e Einstein Telescope (ET). Esses detalhes consistem nas distribuições populacionais esperadas de eventos SS, as incertezas de observação em termos de desvio para o vermelho e o procedimento para a geração de catálogos de SS. No quarto capítulo, mostramos os principais resultados desta análise aos modelos de f(Q). Demonstramos e comparamos os constrangimentos de parâmetros de diferentes combinações de missões conjuntas. Devido ao rápido aumento da incerteza com o redshift, os dados do LIGO são incapazes de fornecer qualquer constrangimentos aos modelos por si só. Portanto, LIGO é usado como um complemento para outras missões, tirando partido da boa qualidade em eventos de redshift extremamente baixos que não estão disponíveis com os outros observatórios. Enquanto isso, para o LISA, classificamos três conjuntos de catálogos, o pior, o mediano e o melhor catálogo com base em quão bons são os resultados nos constrangimentos aos parâmetros. Por fim, concluímos que o caso dos dados ET+Pantheon, é o caso que nos oferece os melhores constrangimentos. Isso deve-se ao grande número de eventos SS que se espera que sejam observados pelo Einstein Telescope e com altas precisões de observação. Para o modelo quadrático, ao contrário do primeiro modelo, descobrimos que as restrições de ET+Pantheon não são capazes de superar LISA+Pantheon em todos os parâmetros, e chegamos à conclusão de que a análise conjunta de LISA(pior)+ET+Pantheon é suficiente para constranger o modelo. No quinto capítulo, estendemos a matriz de Fisher com termos de ordem mais elevados que nos fornecem um método alternativo para aproximar uma probabilidade não-gaussiana com pelo menos 20 vezes maior velocidade computacional do que o método da grelha. Nós fornecemos o procedimento de implementação do código, e escolhemos dois modelos de Quintessência para demostrar que o método DALI dá constrangimentos adequadas nos casos para os quais o método da matriz de Fisher falha. No último capítulo, damos uma conclusão sobre os principais resultados dos dois modelos de f(Q) e listamos alguns trabalhos futuros que vale a pena explorar.

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The relationship between work characteristics, student well-being and performance was assessed, based upon Karasek and Theorell’s (1990) Job Demands–Control–Support model. A sample of Portuguese university students (N = 825) answered a questionnaire comprising measures of academic work demands and control, peer support, satisfaction with academic life, anxiety/depression and academic performance. Results suggested that, similar to other work contexts, student satisfaction with academic life and anxiety/ depression levels are strongly dependent on their perceptions of work characteristics. Levels of satisfaction have a direct impact on student performance and mediate the relationship between academic work control and performance.

Esta tese toma como objeto a tradução da experiência de um grupo de pessoas cegas na plateia de dois teatros de Lisboa entre dezembro de 2019 e fevereiro de 2022. Na Primeira Parte, o documento apresenta as linhas filosóficas e psicossociais que abordaram a cegueira e delineiam “Os mundos da cegueira” onde a experiência da pessoa cega se desenvolve. A análise da cegueira desde uma perspetiva normovisual e não normativa, revela uma condição existencial de liminaridade entre dois mundos, condicionados pelo poder do ato de ver, que é similar à vivência estética. Com o modelo social da deficiência, a cegueira é apresentada como uma diferença existencial em condição de equidade com outras diferenças. Para uma equidade também no teatro, propõe-se identificar a pessoa cega com o neologismo “normocego”. A Segunda Parte, “Os mundos do teatro”, apresenta características de acontecimentos teatrais nos quais o “normocego” pode participar em comunidade. A experiência do público “normocego” narrada em primeira pessoa, é analisada com os estudos de receção liderados por Erika Fischer-Lichte, a relação cena-público sob abordagem dos afetos realizados por Ana Pais e com a abordagens filosóficas do teatro produtor de invisibilidades. O neologismo “hapsorvedor” é apresentado para traduzir a ação do público “normocego” que está num teatro como num templo de fazer previsões, ao reter e sustentar uma paisagem como num theasthai. A Terceira Parte apresenta três diários do trabalho de campo: num espetáculo, num encontro entre artistas com e sem deficiência promovido pela investigação e da vivência em espaços de cultura analisada pelo grupo de pesquisa. Como três “ruínas” do trabalho de campo que, em conjunto, são úteis “para um mundo imaginado com a diferença”, a Terceira Parte atende um dos objetivos traçados por esta investigação e oferece recursos práticos à gestão e à criação teatral que queiram assumir um compromisso social com a diferença.

Estuários, tal como outras regiões marinhas biologicamente e economicamente importantes, estão mundialmente sob grandes pressões antropogénicas. Entre as principais pressões está a poluição da água, através de múltiplas fontes, incluindo a indústria, a dispersão de águas agrícolas ou urbanas, a aquicultura, a pecuária ou a descarga de estações de tratamento de águas residuais. Estas descargas são o principal ponto de entrada para uma vasta gama de poluentes, incluindo nutrientes, compostos orgânicos e outros contaminantes químicos emergentes. Apesar de se estar a introduzir uma quantidade relativamente baixa por composto (ca. ng/L), a descarga contínua pode levar a concentrações crescentes dos mesmos originando por sua vez efeitos desconhecidos a longo prazo. Apesar dos fármacos serem principalmente concebidos para uso humano ou veterinário – através de vias metabólicas conhecidas e em doses baixas –, espécies não-alvo com elevado número de vias metabólicas evolutivamente conservadas podem sofrer efeitos desconhecidos ou imprevistos. Dado que o fitoplâncton é uma das principais fontes de energia primária para a maioria dos ecossistemas aquáticos e é sensível a poluentes, estes organismos, e em particular a diatomácea Phaeodactylum tricornutum, têm sido objeto de vários estudos ecotoxicológicos. A sensibilidade da espécie às alterações físicas e químicas torna- -a um excelente bioindicador para avaliar a qualidade da água. A imobilização de células de P. tricornutum em esferas de alginato foi escolhida como uma abordagem potencialmente valiosa para a monitorização ambiental, dada a sua relação custo-eficácia na deteção de poluentes. Já a capacidade desta abordagem para detetar fármacos em concentrações ambientais ainda não foi avaliada. Dado que a fotoquímica celular sofre alterações rápidas sob stresse, métodos óticos podem ser usados para detetar as mesmas, permitindo medições diárias in situ e, portanto, reduzindo a carga de trabalho laboratorial necessária para a obtenção de dados. O principal objetivo deste projeto foi avaliar a eficácia de células imobilizadas de P. tricornutum como biomonitores de contaminação de água em amostras ambientais através da aplicação de medidas bio-óticas não invasivas sobre os principais parâmetros fotofisiológicos e compará-los com a pressão antropogénica que contaminantes clássicos (metais) e emergentes (fármacos) exercem. Foram selecionados seis pontos de amostragem, abrangendo quatro estuários ao longo da costa Portuguesa (Douro, Tejo, Sado, Mira), juntamente com um local costeiro adjacente ao estuário do Tejo (Cabo Raso). O “Polution Load index” (PLI) foi usado como modelo matemático para resumir as concentrações de metais. O potencial risco ecológico das amostras de água foi analisado através do quociente de risco (RQ), utilizando as concentrações ambientais medidas (MEC) e as concentrações sem efeito previsto (PNEC). A avaliação da toxicidade de fármacos em mistura foi baseada na metodologia proposta por Backhaus e Faust, 2012 onde os quocientes de risco (RQ) são obtidos através do ratio entre concentrações detetadas e a concentração máxima sem efeitos previstos. Por sua vez a utilização de dados relativos à toxicidade de cada composto consiste na soma dos quocientes de risco (RQ) para fármacos com base na concentração mais baixa capaz de induzir efeitos tóxicos. A análise de componentes principais (PCA) foi aplicada aos dados de caracterização química ambiental das águas recolhidas, para avaliar padrões de contaminação espacial e visualizar possíveis relações entre contaminantes. A análise canônica de coordenadas principais (CAP), foi realizada a partir dos dados fotobiológicos e utilizada para identificar dissimilaridades entre parâmetros fisiológicos e determinar o ponto temporal com maior separação de grupos para caracterizar o modelo fotobiológico e avaliar as respostas. Os testes Kruskal-Wallis foram utilizados para determinar diferenças significativas entre variáveis bióticas e abióticas e os coeficientes de correlação de Spearman para determinar possíveis correlações entre variáveis fisiológicas e concentrações ambientais. A presença de poluentes emergentes foi observada em todas as 44 amostras, e dos 69 compostos testados, foram detetados 23. Dois grupos terapêuticos tiveram frequência de deteção acima de 95%: β-bloqueadores e antibióticos. Os antibióticos foram o grupo terapêutico com a maior variedade de compostos detetados, sendo que a maioria em concentrações relativamente baixas (<8 ng/L). Os antihipertensivos, β-bloqueadores e os antidepressivos foram as classes com maiores concentrações em todas as amostras. As amostras do estuário do Tejo (Tj1 e Tj2) foram as amostras com concentrações medianas de fármacos mais elevadas (1898,88 ng/L e 1198,2 ng/L, respetivamente). Quatro compostos – Bisoprolol, Irbesartan, Losartan e Venlafaxina – foram detetados em níveis consideravelmente superiores aos relatados em 2021. As concentrações de metais detetadas foram baixas (<0,55 mg/L) e foi obtido um PLI = 0 para todas as amostras. Os dois primeiros componentes da PCA explicaram 62.6% da variação nos dados ambientais (metais e fármacos). Dos 23 fármacos detetados tres antidepressivos e dois antihipertensivos, foram categorizados como risco moderado, sete compostos foram classificados como baixo risco e onze indicam nenhum risco ambiental. Avaliando por pontos de amostragem, apenas os pontos dentro do tejo (Tj1 e Tj2) atingiram a categoria de elevado risco ecológico requerendo implementação de medidas de regulação. Os RQ obtidos diferem de outros estudos que consideram concentrações em toda a Europa. Diclofenac, Carbamazepin e Venlafaxina são considerados compostos de alto risco na Europa, no entanto as concentrações detetadas pertencem a uma faixa inferior (risco moderado a baixo).As curvas de Kautsky obtidas da exposição das diatomáceas demonstram decréscimo em certas fases durante o período de incubação. A análise CAP determinou que às 24h de exposição das amostras foi obtida a eficiência de classificação máxima de 91,1%. Os parâmetros fotofisiológicos derivados dos testes de fluorescência de clorofila (OJIP) realizados nas diatomaceas apresentaram variações distintas e únicas entre as diatomaceas expostas às diferentes amostras. Existem algumas correlações entre os fármacos e as características fotoquímicas das diatomáceas, sendo que a maioria, fluxo de energia de transporte relativo e absoluto (ET/RC e ET/(%)), o índice estrutural e funcional (SFI), e o índice de desempenho (PI) estão negativamente correlacionadas com o aumento da concentração de fármacos. O índice estrutural e funcional para as reações não fotoquímicas (SFI(NPQ)) é o único que está positivamente correlacionado. Apenas os metais, chumbo e titânio tiveram alguma correlação com a fotofisiologia. Embora as amostras recolhidas no estuário do Tejo (Tj1 e Tj2) demonstrem níveis de poluição semelhantes com um RQ superior a 1, estas apresentam características fotoquímicas significativamente diferentes entre si. Uma vez que é difícil identificar qual o composto ou combinação dos mesmos que é responsável por uma determinada resposta metabólica é importante realçar que estes poluentes exercem claros sinais de stresse às diatomaceas, tal como fariam em condições ambientais reais. Os resultados evidenciam que é possível realizar uma análise diária custo-efetiva contínua recorrendo a esta metodologia. Para manter um ecossistema estuarino ou costeiro estável e biodiverso, monitorizar e compreender o efeito que poluentes tais como fármacos ou metais podem ter na fauna e flora é fundamental. A desestabilização das populações de fitoplâncton através da contaminação da água pode ter efeitos em cascata em toda a rede trófica. Além disso, a biomagnificação de fármacos ou de metabólitos pode afetar negativamente a biodiversidade local. A sustentabilidade a longo prazo está intrinsecamente relacionada com a qualidade da água pelo que a sua monitorização é crucial para a deteção de novas tendências de poluição. Só acompanhando a qualidade da água será possível a rápida implementação de medidas ambientais adequadas, por forma a garantir a capacidade natural do habitat.

Skeletal muscle is formed by the largest cells in the human body. Muscle cells or myofibers are elongated cells with multiple nuclei evenly distributed in a manner that maximizes the distance between nuclei (Bruusgaard et al., 2003). During development, the nuclei undergo a series of movements until they reach their final position and are anchored in the cell periphery (Roman & Gomes, 2018). It was previously described that nuclear positioning is important for muscle function (Metzger et al., 2012). The spaced distribution of nuclei suggests that nuclear positioning can be relevant for the establishment of the myonuclear domains. According to this hypothesis, each nucleus within the multinucleated myofiber is responsible for a limited volume of cytoplasm. In the neuromuscular and mypotendinous junctions there is compartmentalization of specific messenger ribonucleic acids (mRNAs) and proteins, but the mRNA distribution in non specialized areas of the myofiber remains largely unexplored. In this work, we analyzed the distribution of several mRNAs important for muscle function in highly differentiated myofibers in vitro. We observed that the bulk of mRNA is enriched around the nucleus of origin. The perinuclear enrichment depends on recently transcribed mRNAs. Surprisingly, we found a subset of mRNAs that are differentially distributed. mRNAs encoding large proteins are spread throughout the cell. By exogenous expression of mRNAs with different sizes, we found that transcript size contributes to mRNA spreading in a sequence independent manner. Microtubules are involved in the distribution of normal size and large mRNAs away from the nucleus, but the effect is more evident in the distribution of large mRNAs. Additionally, we observed that the differential distribution is independent of the nuclear positioning and mRNA expression and stability. Considering our findings, we propose that mRNA distribution in non-specialized regions of skeletal muscle is size selective to ensure the distribution of mRNAs with special characteristics, such as large size, while maintaining cellular compartmentalization around each nucleus.

Circadian rhythms (from the Latin circa diem meaning “about a day”) are biological cycles endogenously generated. These rhythms are self-sustained, have a period of approximately 24h and are generated by an internal biological master clock, which entrains (synchronizes) to environmental cues. The 24h light–dark cycle, resulting from the Earth's rotation on its own axis, is the major synchronizer shaping several aspects of physiology and behavior of species from all phyla. Individual differences in how the human biological clock entrains to the 24h day – earlier or later, (e.g., in reference to dawn) – are expressions of different chronobiological traits (i.e., chronotypes). Chronotype ranges from extreme early/morning-types to extreme late/evening-types (the colloquial larks and owls), with the majority of people standing in between, presenting an approximately Gaussian distribution in the population. The Munich ChronoType Questionnaire (MCTQ) estimates chronotype through individuals’ sleep phase, assessing current sleep–wake behavior separately for work and work-free days. An MCTQ-derived chronotype is a continuous variable expressed in local time, the Midpoint of Sleep on Free days, corrected for sleep debt accumulated through weekdays (MSFsc). In addition to chronotype, MCTQ estimates the amount of strain on the biological circadian system exerted by social commitments, measured as the Social JetLag (SJL). SJL is a continuous variable expressed in hours and it is considered a proxy (and a quantitative marker) for the circadian misalignment. Circadian misalignment can emerge as a consequence of social obligations when there is a discrepancy between the internal sleep window (mostly driven by chronotype) and the social sleep opportunity (mostly driven by working schedules). Circadian misalignment can be transient, such as in transmeridian travels (i.e., jetlag) but it can be chronic, such as in shift work. Shift work affects more than 20% of the European workforce and has long been associated with an increased risk for chronic diseases and injuries. Major pathophysiological mechanisms that explain this link include circadian misalignment, sleep disturbances, unhealthy behaviors and psychosocial stress. The association between shift work and cardiovascular diseases has been researched for more than two decades and one systematic review showed that shift work was associated with a 23% increased risk of ischemic heart disease and 5% for stroke. Cardiovascular diseases are a major cause of morbidity and mortality in modern societies and guidelines on cardiovascular prevention stress the importance of non-traditional risk factors, such as occupational factors. Therefore, innovative ways to address cardiovascular health, especially among working adults are needed. To date, knowledge about the health impact of circadian misalignment and the role of the chronobiological inter-individual differences in real-life working settings is scarce. This thesis aims to improve knowledge in this field by revising the evidence concerning the cardiovascular consequences of different types of shift work and by exploring the role of social jetlag in the cardiovascular risk of shift workers. To achieve this aim, three studies were performed: Study 1 aimed to review and synthesize the existing scientific literature concerning the impact of different types of shift work both on systolic and diastolic blood pressure values and in the hypertension risk of shift workers, in comparison with day workers. A systematic review and meta-analysis were performed according to the PRISMA guidelines. Forty-five independent studies assessing ~117 thousand workers were included. The most frequent shift work type was “rotational shifts including night work” (n=28), followed by “permanent night work” (n=14) and “rotational shifts without night work” (n=4). Meta analysis pooled results reveal that: 1) permanent night workers had a significant increase in both systolic and diastolic blood pressure values; 2) rotational shift workers, both with and without night work, had a significant increase, only in systolic blood pressure; 3) no shift work type significantly influenced the risk of hypertension. The magnitude of the effect was rather small, ranging from 0.65 to 2.52 mmHg, and the larger upper bound of the pooled confidence intervals was 4.29 mmHg. This might seem not clinically significant, however, it should be considered in susceptible populations continuously exposed over a long period of time to shift work, as a possible contributing factor for the development of hypertension and/or for the need of more intensive drug treatment. Moreover, these results highlight the heterogenous aspect of shift work as a potential risk factor and reinforces the need for a more tailored assessment of the potential risks associated with shift work exposure. Study 2 aimed to validate the European Portuguese variant of the Munich Chronotype Questionnaire (MCTQ) to the adult population living in Portugal, using subjective and objective measures. We made the cross-cultural adaptation of the original MCTQ to develop the Portuguese variant (MCTQPT), and we explore its psychometric properties. A cross-sectional observational study including 80 volunteers (age and gender balanced according to the Portuguese distribution) filled out the MCTQPT and used actimetry for 4 weeks to objectively assess the sleep–wake cycle. Additionally, we compared MCTQPT results to the most widely used chronotyping questionnaire (i.e., MEQ. Results show that MCTQ key variables correlated highly with their counterparts calculated from actimetry (MSW: rho = 0.697; MSF: rho = 0.747; MSFsc: rho = 0.646; SJL: rho = 0.452; all p < 0.001). The MCTQ assessment of the chronotype showed very good test–retest reliability (rho = 0.905; p< 0.001). Therefore, MCTQPT proved to be a simple and valid instrument to assess chronotype and social jetlag among the Portuguese population. Study 3 aimed to investigate the association between social jetlag, as a measure of circadian misalignment, and cardiovascular risk, estimated from modifiable risk factors in a population of blue-collar workers following permanent atypical schedules. A cross-sectional observational study including 301 workers, who either worked morning, evening, or night shifts, filled out the MCTQPT and had major risk factors measured (blood pressure, total cholesterol, and smoking status) to estimate the cardiovascular relative risk SCORE. Our results showed that higher levels of social jetlag were associated with worse cardiovascular parameters. A higher degree of social jetlag was independently associated with greater odds of having a high cardiovascular relative risk, even after adjusting for sociodemographic, lifestyle, sleep, and work variables. This risk increased 31% for each additional hour of social jetlag that the worker is exposed to (odds ratio 1.31, 95% confidence interval 1.02– 1.68). Additionally, higher levels of working productivity were found among workers with less amount of social jetlag. To our knowledge, this was the first study indicating that social jetlag potentially increases cardiovascular risk and suggests that sleep and individual circadian traits (i.e., chronotype) are conceivably useful in preventing the health consequences of shift work. Our results support interventional studies on optimizing working schedules, that is, to test if the allocation of working schedules in accordance with the worker’s chronotype (e.g., early chronotypes are assigned to early shifts) have favorable results in both health and corporate outcomes. We conclude that the health consequences of shift work depend on both the characteristics of the work schedule and the individual chronobiological characteristics of the person who performs it. Effective ways to improve health and performance of shift workers are pressing and the solution might rely on the diversity of human chronotypes.