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Breast cancer is the most frequent malignant disease among women, being responsible for a considerable percentage of fatalities and comorbidities every year. Despite advances in early detection and therapy, evidence shows that breast cancer survivors are at increased risk of developing other chronic conditions, such as cardiovascular diseases. Autonomic dysfunction is an emerging, but poorly understood topic that has been suggested as a risk factor for cardiovascular disease in breast cancer patients. It clinically manifests through persistently elevated heart rates and abnormal heart rate variability, even before any signs of cardiovascular dysfunction appear. Since changes in the left ventricular ejection fraction only manifest when myocardial injury has already occurred, it has been hypothesized that autonomic dysfunction can constitute an early biomarker of cardiovascular impairment in breast cancer patients. This review focuses on the direct and indirect effects of cancer and its treatment on the autonomic nervous system in breast cancer patients. We highlight the mechanisms potentially involved in cancer and antineoplastic therapy-related autonomic imbalance and review the potential strategies to prevent and/or attenuate autonomic dysfunction. There are gaps in the current knowledge; more research in this area is needed to identify the relevance of autonomic dysfunction and define beneficial interventions to prevent cardiovascular disease in breast cancer patients.

Doxorubicin (DOX) is a chemotherapeutic agent that improves the survival and quality of life of patients with solid tumours. However, these improvements are counterbalanced by various dose-dependent side effects including cardiac dysfunction, which may occur in a subset of patients leading to treatment discontinuation. In addition, despite overall insufficiently studied, available data clearly show that women are more susceptible to drugs toxicity due to sex differences in drug metabolism or sensitivity. Regarding doxorubicin, apart its acute toxicity, women are also more vulnerable to late cardiotoxicity. Currently, there are no robust clinical imaging techniques or biomarkers available to detect DOX-cardiotoxicity before functional decline and, despite treatment personalization, subclinical signs of toxicity are not yet well identified. Thus, in the present work, we sought to characterize the toxic effects of a sub-therapeutic dose of doxorubicin upon cardiovascular and autonomic function. For that, adult healthy female wistar rats (n=14), aged >3 months, were intraperitoneally treated with doxorubicin (2 mg/kg) at weekly intervals for up to 4 weeks. A control (Ctrl, n=7) group received saline solution 0,9% NaCl as a vehicle. Rats underwent an echocardiographic evaluation at 4 weeks. Blood pressure, heart rate and respiratory frequency were assessed in an acute experiment. Cardiovascular variability studies in the time-frequency domain (LF, HF and LF/HF calculation) and cardiorespiratory reflexes assessment were performed. T-Student test for paired observations was applied (level significance p DOX-treated animals showed a severe bradypnea and hypotension, significant decrease in ejection fraction and fractional shortening, sympatho-inhibition and reduced baroreceptor reflex gain and chemoreflex sensitivity. Our functional results might be due to cellular respiratory dysfunction and vascular adaptations induced by doxorubicin via TNF alpha actions at central nervous system (CNS) and peripheral level. In fact, peripheral TNF alpha release elicits NO production, which through vasodilation will induce hypotension and baroreceptor reflex adjustments. Reinforcing these actions, and despite doxorubicin low ability to pass BBB, TNF alpha acting at CNS induces neuronal apoptosis and impairment of mitochondrial function which might also contribute to affect autonomic and respiratory function, expressed by a decreased chemoreflex sensitivity and sympathetic tone. In conclusion, sub-therapeutic doses of doxorubicin are able to produce per se the impairment of autonomic and cardiac functions. Thus, a tight monitoring of these functions in patients treated with therapeutic doses of doxorubicin is highly recommended. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fundação para a Ciência e a Tecnologia

© 2020 Springer Nature Switzerland AG. Part of Springer Nature., Long-term lead (Pb) exposure alters the normal development of the nervous system and physiology. It affects multiple organ systems, causing hypertension, cardiorespiratory dysfunction, being a well-known neurotoxin, inducing changes in neurogenesis, neurodegeneration, and glial cells. However, studies of the developmental effects of lead and its outcomes throughout life are lacking. Determine morphofunctional, behavioral, and cognitive developmental effects of long-term lead exposure at three different ages. Wistar rats were exposed to a Pb-acetate solution from fetal period until adulthood and compared to a non-exposed control group. General behavior and cognitive skills were evaluated by behavioral tests and physiological data and cardiorespiratory reflexes measured. Neurodegeneration, neuroinflammation, and synaptic activity were assessed by immunohistochemistry. Lead exposure caused long-lasting anxiety-like behavior and strong long-term memory impairment without changes in locomotor and exploratory activity. Hypertension was observed at all time points, concomitant with baroreflex impairment and increased chemoreflex sensitivity. Persistent neuroinflammation, transient synaptic overexcitation without neurodegeneration was observed. Long-term Pb exposure, since fetal period, causes long-lasting anxiety-like behavior, concomitant with hypertension, without general motor skills impairment. Synaptic overexcitation, reactive astrogliosis, and microgliosis could underlie behavioral and long-term memory changes, which might have been caused during developmental phases and consolidated during adulthood. Also, alterations observed in the cardiorespiratory reflexes can explain persistent hypertension. This longitudinal study identifies and characterizes lead toxicity nature and magnitude, important to devise and test potential interventions to attenuate the long-term harmful effects of lead on the nervous and cardiovascular systems., Vera Geraldes received the post-doctoral fellowship given by the Fundação para a Ciência e Tecnologia (FCT) (Ref: SFRH/BPD/119315/2016).

© 2017 Sociedade Portuguesa de Cardiologia. Published by Elsevier España, S.L.U. All rights reserved, Knowledge of the physiology underlying the autonomic nervous system is pivotal for understanding autonomic dysfunction in clinical practice. Autonomic dysfunction may result from primary modifications of the autonomic nervous system or be secondary to a wide range of diseases that cause severe morbidity and mortality. Together with a detailed history and physical examination, laboratory assessment of autonomic function is essential for the analysis of various clinical conditions and the establishment of effective, personalized and precise therapeutic schemes. This review summarizes the main aspects of autonomic medicine that constitute the background of cardiovascular autonomic dysfunction., O conhecimento subjacente à fisiologia do sistema nervoso autónomo é fundamental para se entender a disfunção autonómica na prática clinica. A disfunção autonómica pode resultar primariamente de modificações do sistema ou, secundariamente, a uma série de patologias conducentes a morbilidade ou mortalidade. Juntamente com a colheita detalhada da história clínica e do exame objetivo, a avaliac¸ão autonómica laboratorial torna-se essencial na análise de algumas condições clínicas e no estabelecimento de esquemas terapêuticos mais eficazes, refinados e personalizados. Assim, nesta revisão sumarizam-se os aspetos mais relevantes da fisiologia autonómica subjacente a disfunção autonómica cardiovascular.

©Springer Science+Business Media, LLC, part of Springer Nature 2018, Hypertension is a prevalent and major health problem, involving a complex integration of different organ systems, including the central nervous system (CNS). The CNS and the hypothalamus in particular are intricately involved in the pathogenesis of hypertension. In fact, evidence supports altered hypothalamic neuronal activity as a major factor contributing to increased sympathetic drive and increased blood pressure. Several mechanisms have been proposed to contribute to hypothalamic-driven sympathetic activity, including altered ion channel function. Ion channels are critical regulators of neuronal excitability and synaptic function in the brain and, thus, important for blood pressure homeostasis regulation. These include sodium channels, voltage-gated calcium channels, and potassium channels being some of them already identified in hypothalamic neurons. This brief review summarizes the hypothalamic ion channels that may be involved in hypertension, highlighting recent findings that suggest that hypothalamic ion channel modulation can affect the central control of blood pressure and, therefore, suggesting future development of interventional strategies designed to treat hypertension., Dr. Rocha reports a grant from Portuguese Foundation for Science and Technology [2010–2013–FCT/PTDC/SAUOSM/109081/2008].

© 2018 Elsevier B.V. All rights reserved., Background: Exposures to lead (Pb) during developmental phases can alter the normal course of development, with lifelong health consequences. Permanent Pb exposure leads to behavioral changes, cognitive impairment, sympathoexcitation, tachycardia, hypertension and autonomic dysfunction. However, the effects of an intermittent lead exposure are not yet studied. This pattern of exposure has been recently increasing due to migrations, implementation of school exchange programs and/or residential changes. Objective: To determine and compare lead effects on mammal’s behavior and physiology, using a rat model of intermittent and permanent Pb exposures. Methods: Fetuses were intermittently (PbI) or permanently (PbP) exposed to water containing lead acetate (0.2% w/v) throughout life until adulthood (28 weeks of age). A control group (CTL) without any exposure to lead was also used. Anxiety was assessed by elevated plus maze (EPM) and locomotor activity and exploration by open field test (OFT). Blood pressure (BP), electrocardiogram (ECG), heart rate (HR), respiratory frequency (RF), sympathetic and parasympathetic activity and baro- and chemoreceptor reflex profiles were evaluated. Immunohistochemistry protocol for the assessment of neuroinflammation, neuronal loss (NeuN), gliosis and synaptic alterations (Iba-1, GFAP, Syn), were performed at the hippocampus. One-way ANOVA with Tukey’s multiple comparison between means were used (significance p < 0.05) for statistical analysis. Results: The intermittent lead exposure produced a significant increase in diastolic and mean BP values, concomitant with a tendency to sympathetic overactivity (estimated by increased low-frequency power) and without significant changes in systolic BP, HR and RF. A chemoreceptor hypersensitivity and a baroreflex impairment were also observed, however, less pronounced when compared to the permanent exposure. Regarding behavioral changes, both lead exposure profiles showed an anxiety-like behavior without changes in locomotor and exploratory activity. Increase in GFAP and Iba-1 positive cells, without changes in NeuN positive cells were found in both exposed groups. Syn staining suffered a significant decrease in PbI group and a significant increase in PbP group. Conclusion: This study is the first to show that developmental Pb exposure since fetal period can cause lasting impairments in physiological parameters. The intermittent lead exposure causes adverse health effects, i.e, hypertension, increased respiratory frequency and chemoreflex sensitivity, baroreflex impairment, anxiety, decreased synaptic activity, neuroinflammation and reactive gliosis, in some ways similar to a permanent exposure, however some are lower-grade, due to the shorter duration of exposure. This study brings new insights on the environmental factors that influence autonomic and cardiovascular systems during development, which can help in creating public policy strategies to prevent and control the adverse effects of Pb toxicity., Vera Geraldes acknowledges the post-doctoral fellow given by the Fundação para a Ciência e Tecnologia (FCT) (Ref: SFRH/BPD/119315/ 2016).

Myocardial infarction (MI) is a high prevalence cardiovascular disease with a tendency to increase. Although MI therapy has increased its effectiveness, the loss of myocardial function in consequence of MI is still responsible for major subsequent complications as rhythm changes and heart failure. In human hearts, MI is characterized by cardiomyocyte death and fibrin deposition, which is later replaced by scar tissue. In opposition, zebrafish heart undergoes minimal scaring and cardiac tissue can undergo full functionally regeneration in 60 to 120 days. Therefore, this animal model has been used in cardiovascular research as a model for studying mechanisms of cardiac regeneration. The hearts of zebrafish is anatomically different from the human heart, though the electrocardiogram (ECG) pattern and the critical conduction pathways are comparable to the human's. The objective of our full project is the implementation of an ECG recording system with 3 orthogonal electrodes in 2 planes, in order to create a 4D recording of the zebrafish cardiac electrical activity. Here we present our preliminary work related to a non-invasive ECG monitoring with two electrodes.

New Findings • What is the central question of this study?Will a chronic reduction of neuronal excitability within the paraventricular nucleus of the hypothalamus reduce arterial blood pressure and sympathetic activity in the long term in an animal model of neurogenic hypertension? • What is the main finding and its importance?We show, for the first time, that overexpression of an inwardly rectifying potassium channel in the paraventricular nucleus provided a long-term (>60 days) antihypertensive response in conscious spontaneously hypertensive rats that was associated with a reduction in neurohumorally mediated vasoconstriction, enhanced baroreflex sensitivity and reduced peripheral chemosensitivity; no such response was observed in normotensive rats. Our results support the paraventricular nucleus as a therapeutic target for the chronic control of blood pressure in neurogenic hypertension. Changes in the sympathetic nervous system are responsible for the initiation, development and maintenance of hypertension. An important central sympathoexcitatory region is the paraventricular nucleus (PVN) of the hypothalamus, which may become more active in hypertensive conditions, as shown in acute studies previously. Our objective was to depress PVN neuronal activity chronically by the overexpression of an inwardly rectifying potassium channel (hKir2.1), while evaluating the consequences on blood pressure (BP) and its reflex regulation. In spontaneously hypertensive rats (SHRs) and Wistar rats (WKY) lentiviral vectors (LVV-hKir2.1; LV-TREtight-Kir-cIRES-GFP5 4 × 109 IU and LV-Syn-Eff-G4BS-Syn-Tetoff 6.2 × 109 IU in a ratio 1:4) were stereotaxically microinjected bilaterally into the PVN. Sham-treated SHRs and WKY received bilateral PVN microinjections of LVV-eGFP (LV-Syn-Eff-G4BS-Syn-Tetoff 6.2 × 109 IU and LV-TREtight-GFP 5.7 × 109 IU in a ratio 1:4). Blood pressure was monitored continuously by radio-telemetry and evaluated over 75 days. Baroreflex gain was evaluated using phenylephrine (25 μg ml−1, i.v.), whereas lobeline (25 μg ml−1, i.v.) was used to stimulate peripheral chemoreceptors. In SHRs but not normotensive WKY rats, LVV-hKir2.1 expression in the PVN produced time-dependent and significant decreases in systolic (from 158 ± 3 to 132 ± 6 mmHg; P < 0.05) and diastolic BP (from 135 ± 4 to 113 ± 5 mmHg; P < 0.05). The systolic BP low-frequency band was reduced (from 0.79 ± 0.13 to 0.42 ± 0.09 mmHg2; P < 0.05), suggesting reduced sympathetic vasomotor tone. Baroreflex gain was increased and peripheral chemoreflex depressed after PVN microinjection of LVV-hKir2.1. We conclude that the PVN plays a major role in long-term control of BP and sympathetic nervous system activity in SHRs. This is associated with reductions in both peripheral chemosensitivity and respiratory-induced sympathetic modulation and an improvement in baroreflex sensitivity. Our results support the PVN as a powerful site to control BP in neurogenic hypertension.

Copyright © 2016 Elsevier Inc. All rights reserved., Mortality and morbidity by toxic metals is an important issue of occupational health. Lead is an ubiquitous heavy metal in our environment despite having no physiological role in biological systems. Being an homeostatic controller is expected that the autonomic nervous system would show a degree of impairment in lead toxicity. In fact, sympathoexcitation associated to high blood pressure and tachypnea has been described together with baroreflex dysfunction. However, the mechanisms underlying the autonomic dysfunction and the interplay between baro- and chemoreflex are not yet fully clarified. The angiotensinogenic PVN-NTS axis (paraventricular nucleus of the hypothalamus – nucleus tractus solitarius axis) is a particularly important neuronal pathway that could be responsible for the autonomic dysfunction and the cardiorespiratory impairment in lead toxicity. Within the current work, we addressed in vivo, baro- and chemoreceptor reflex behaviour, before and after central angiotensin inhibition, in order to better understand the cardiorespiratory autonomic mechanisms underlying the toxic effects of long-term lead exposure. For that, arterial pressure, heart rate, respiratory rate, sympathetic and parasympathetic activity and baro- and chemoreceptor reflex profiles of anaesthetized young adult rats exposed to lead, from foetal period to adulthood, were evaluated. Results showed increased chemosensitivity together with baroreceptor reflex impairment, sympathetic over-excitation, hypertension and tachypnea. Chemosensitivity and sympathetic overexcitation were reversed towards normality values by NTS treatment with A-779, an angiotensin (1–7) antagonist. No parasympathetic changes were observed before and after A-799 treatment. In conclusion, angiotensin (1–7) at NTS level is involved in the autonomic dysfunction observed in lead toxicity. The increased sensitivity of chemoreceptor reflex expresses the clear impairment of autonomic outflow to the cardiovascular and respiratory systems induced by putative persistent, long duration, alert reaction evoked by the long term exposure to lead toxic effects. The present study brings new insights on the central mechanisms implicated in the autonomic dysfunction induced by lead exposure which are relevant for the development of additional therapeutic options to tackle lead toxicity symptoms., This work was supported by Sorin Portugal grants to Isabel Rocha.

© 2016 Elsevier B.V. All rights reserved., Background: Chronic overexpression of an inwardly rectifying potassium channel (hKir2.1) in the paraventricular nucleus of the hypothalamus (PVN) and in the rostral ventrolateral medulla (RVLM) to suppress neuronal excitability, resulted in a long term decrease of blood pressure and sympathetic output in spontaneously hypertensive rats (SHR). Objective: Evaluate gene expression in end-organs of SHR after a chronic overexpression of hKir2.1 channels in either the PVN or RVLM. Methods: mRNA levels of 16 genes known to be involved with blood pressure regulation were evaluated using RT-PCR in tissues from the heart, common carotid artery and kidney of SHR submitted to chronic depression of PVN and RVLM excitability using a lentiviral vector (LVhKir2.1). Results: In SHR hearts in which either the PVN or RVLM were injected with LVhKir2.1, there was a downregulation of angiotensin II receptor 1b (AT1), ATPase, Ca(2+)-transporter, troponin T2 and tropomyosin2 (only in RVLM) relative to the sham group. In the kidney of SHR with LVhKir2.1 injections in PVN and RVLM, angiotensinogen, angiotensin II receptor2 (AT2) and endothelin1 were all upregulated compared to sham. In the carotid artery, endothelin2, endothelin receptor A and B were up-regulated following LVhKir2.1 in to either the PVN or RVLM relative to sham. Conclusion: Chronic overexpression of hKir2.1 channels in PVN and RVLM, promoted a BP decrease with up-regulation of angiotensinogen and AT2 genes expression in the kidney and down-regulation of AT1 in the heart of SHR. Thus, we demonstrate the potential efficacy of central manipulation to protect against end-organ damage in essential hypertension., This work was supported by Fundação para a Ciência e Tecnologia (PTDC/SAU-OSM/109081/2008) and Sorin Portugal grants to Isabel Rocha; British Heart Foundation (RG/12/6/29670) and National Institutes of Health (RO1 NS069220). The authors want to acknowledge the technical contributions of Raquel Laires and technical advices of Mafalda Carvalho.

Neurocardiogenic syncope (NCS) is a common clinical entity resulting from an excessive reflex autonomic response, particularly during orthostatism. Treatment options are controversial and of limited effectiveness. Tilt training (TT) is a promising option to treat these patients. However, its mechanism of action and clinical impact remain unclear. Objective: To characterize hemodynamic and autonomic responses during a TT program in patients with NCS refractory to conventional measures. Methods: We studied 28 patients (50% male, mean age 41 ± 14 years) without structural heart disease, with NCS documented by tilt testing. The TT program included nine tilt sessions (three times a week, 30 min) (60° – 6 sessions, 70° – 3 sessions), under ECG and blood pressure monitoring combined with home orthostatic self-training and 10° head-up during sleep. Systolic volume, cardiac output, total peripheral resistance, baroreflex sensitivity and heart-rate variability were computed. Patients were reassessed at 1 month and every 6 months for a maximum of 36 months (24 ± 12 months). Results: Over the course of the TT program there was a significant increase in total peripheral resistance (1485 ± 225 vs. 1591 ± 187 dyn s cm−5, p

Introduction: Atrial fibrillation (AF) is a complex disease with multiple mechanisms, including the interaction with the autonomic nervous system (ANS), electrophysiological properties of the atria and pulmonary veins (PVs), and vulnerability for AF. We assessed the effects of acute vagal (vagus_stim) and sympathetic stimulation (symp_stim) on atrial conduction, atrial and PV refractoriness and inducibility of AF in an in vivo rabbit model with preserved autonomic innervation. Methods: An open-epicardial approach was used in 17 anesthetized and artificially ventilated New Zealand white rabbits. The ECG was recorded with bipolar subcutaneous electrodes placed in the four limbs. Electrograms were obtained with four monopolar electrodes placed epicardially along the atria, and an electrode adapted to the proximal PVs. The cervical vagus nerve and thoracic sympathetic trunk were stimulated with bipolar electrodes. Epicardial activation was recorded in sinus rhythm, and effective refractory periods (ERPs) and conduction times from the high-lateral right atrium (RA) to the high-lateral left atrium (LA) and PVs were quantified at baseline and during vagus_stim, symp_stim, or combined vagal and sympathetic stimulation (dual_stim). Burst pacing (50 Hz, 10 s), alone or combined with vagus_stim, symp_stim or dual_stim, was performed in the right (RAA) and left atrial appendage (LAA) and PVs to test for AF inducibility. Results: At baseline, ERPs were higher in the LAA and there was a delay in the conduction time from RA to PV, compared to the mean activation time from RA to LA. During vagus_stim or dual_stim, ERP decreased significantly at all sites, and baseline interatrial activation times changed from 20±4 ms to 30±10 ms and from 21±5 ms to 31±11 ms, respectively (p10 s a arritmia terminou imediatamente após interrupção da estim_vagal. Conclusões: No coração de coelho inervado in vivo, a estimulação autonómica aguda encurta a refractariedade auricular e das VP, e modifica a velocidade de condução auricular, potenciando a indução e duração de FA. Os resultados sugerem que as variações agudas e a interacção da actividade autonómica podem desempenhar um papel importante na fisiopatologia da FA. Keywords: Animal model, Vagus, Sympathetic, Refractoriness, Conduction velocity, Atrial fibrillation, Palavras-chave: Modelo animal, Vago, Simpático, Refractariedade, Velocidade de condução, Fibrilhação auricular

Resumo: A influência do sistema nervoso autónomo (SNA) na génese da fibrilhação auricular (FA) envolve múltiplos mecanismos complexos com impacto nas propriedades eletrofisiológicas cardíacas. A importância dos efeitos da estimulação autonómica no substrato elétrico auricular e das veias pulmonares (VP) e na vulnerabilidade para FA requer melhor compreensão. Objetivo: Avaliar os efeitos da estimulação vagal (estim_vag) e simpática (estim_simp) aguda na condução e refratariedade das aurículas e VP e na indutibilidade de FA no coração de coelho in vivo com inervação autonómica preservada. Métodos: Estudámos 17 coelhos New Zealand de ambos os sexos. Para abordagem de «toráx-aberto» procedeu-se a anestesia, entubação e ventilação após bloqueio neuro-muscular. O ECG foi obtido a partir de 3 derivações dos membros. Os eletrogramas foram registados com 4 elétrodos monopolares colocados na superfície epicárdica, distribuídos ao longo das aurículas e com um elétrodo circular adaptado à porção proximal das VP. Estimulou-se o nervo vago cervical direito e o tronco simpático torácico com elétrodos bipolares de platina. Estudámos os períodos refratários efetivos (PRE) e a condução elétrica auricular, entre a aurícula direita lateral-alta (AD) e a aurícula esquerda lateral-alta (AE), e entre AD e VP, em condições basais e durante estim_vag, estim_simp e estimulação autonómica combinada (dual_estim). Para indução de FA, procedeu-se a pacing rápido (50 Hz, 10 s, isolado ou com estim_vag, estim_simp ou dual_estim) com elétrodo bipolar no apêndice auricular direito (AAD), apêndice auricular esquerdo (AAE) e VP. Resultados: Em condições basais: os PRE eram maiores no AAE e registou-se um atraso na ativação da AD para as VP, comparando com a condução interauricular. Durante estim_vag ou dual_estim: os PRE encurtaram significativamente em todos os locais, o intervalo de condução interauricular variou de 20 ± 4 ms para 30 ± 10 ms (p 10 s a arritmia terminou imediatamente após interrupção da estim_vagal. Conclusões: No coração de coelho inervado in vivo, a estimulação autonómica aguda encurta a refratariedade auricular e das VP, e modifica a velocidade de condução auricular, potenciando a indução e duração de FA. Os resultados sugerem que as variações agudas e a interação da atividade autonómica podem desempenhar um papel importante na fisiopatologia da FA. Abstract: Atrial fibrillation (AF) is a complex disease with multiple mechanisms, involving the interaction between the autonomic nervous system (ANS), electrophysiological properties of the atria and pulmonary veins (PVs), and vulnerability for AF. Aim: We assessed the effects of acute vagal (vagus_stim) and sympathetic stimulation (symp_stim) on atrial conduction, atrial and PV refractoriness and inducibility of AF in an in vivo rabbit model with preserved autonomic innervation. Methods: An open-epicardial approach was used in 17 anesthetized and artificially ventilated New Zealand white rabbits. The ECG was recorded with bipolar subcutaneous electrodes placed in the four limbs. Electrograms were obtained with four monopolar electrodes placed epicardially along the atria, and a circular electrode adapted to the proximal PVs. The cervical vagus nerve and thoracic sympathetic trunk were stimulated with bipolar electrodes. Epicardial activation was recorded in sinus rhythm, and effective refractory periods (ERPs) and conduction times from the high-lateral right atrium (RA) to the high-lateral left atrium (LA) and PVs were quantified at baseline and during vagus_stim, symp_stim, or combined vagal and sympathetic stimulation (dual_stim). Burst pacing (50 Hz, 10 s), alone or combined with vagus_stim, symp_stim or dual_stim, was performed in the right (RAA) and left atrial appendage (LAA) and PVs to test for AF inducibility. Results: At baseline, ERPs were higher in the LAA and there was a delay in the conduction time from RA to PV, compared to the mean activation time from RA to LA. During vagus_stim or dual_stim, ERP decreased significantly at all sites, and baseline interatrial activation times changed from 20 ± 4 ms to 30 ± 10 ms and 31 ± 11 ms, respectively (p

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Measurements made in feces and urine of Wistar rats exposed to lead acetate (n = 20) in drinking water since the fetal period were compared with those obtained from a control group (n = 20) in order to assess the age influence on Pb excretion. The measurements were made in different collections of rats aging between 1 and 11 months. To determine the Pb content of the samples, total reflection X-ray fluorescence (TXRF) and electrothermal atomic absorption spectrometry (ETAAS) were used for the urine samples and energy dispersive X-ray fluorescence (EDXRF) was used for the feces. The results show high concentrations of Pb being eliminated from the organism by urine and feces in contaminated rats. Values vary from (600 ± 140) µg l−1 to (5 460 ± 115) µg l−1 in urine and from (4 500 ± 300) µg g−1 to (11 400 ± 3 300) µg g−1 in dry feces. The control rats show, in general, low lead concentrations or below detection limits. The fecal/urinary ratio was studied. It was shown to be about three to four orders of magnitude and positively correlated with time. It was verified in feces and urine that excretion decreases with the animal age and that this decrease is made by different levels of excretion. The excretions of Pb in urine and in feces are positively correlated. A good agreement was found between the results obtained with TXRF and ETAAS for urine samples. This work also stresses the suitability of these techniques in the study of Pb intoxication. Copyright © 2011 John Wiley & Sons, Ltd.

An ultrasonic assisted solid–liquid extraction method was developed to determine the level of lead in the brain and urine of rats. Lead was determined by electrothermal atomic absorption spectrometry with longitudinal-Zeeman background correction. Several analytical drawbacks were addressed and overcome, namely small brain sample mass and the formation of precipitate in the urine samples. Utrasonication provided by an ultrasonic probe succeeded in extracting lead from brain samples. Furthermore, it was demonstrated that the formation of a precipitate lowered the lead content in the liquid phase of the urine. Lead was back extracted from the precipitate to the liquid phase with the aid of ultrasonic energy and acidifying the urine with 10% v/v nitric acid. A microwave-assisted acid digestion protocol was used to check the completeness of the lead extraction. The within bath and between bath precision was 5% ( n = 9) and 7% ( n = 3) respectively. The limit of quantification was 1.05 μg g −1 for brain samples and 2.1 μg L −1 for urine samples. A total of 6 samples of urine and 12 samples of brain from control rats and another 6 samples of urine and 12 samples of brain from rats fed with tap water rich in lead acetate were used in this research. Lead levels in brain and urine from exposed rats ranged from1.9 ± 0.2 μg g −1 to 3.5 ± 0.2 μg g −1 and from 752 ± 56 μg L −1 to 60.9 ± 1.2 mg L −1 respectively. Statistically significant differences of levels of lead in brain and urine were found between exposed and non exposed rats.

Vagal activity is thought to influence atrial electrophysiological properties and play a role in the initiation and maintenance of atrial fibrillation (AF). We evaluated the effects of acute vagal stimulation on atrial conduction, refractoriness of atrial and pulmonary veins (PVs) and inducibility of AF. An open-chest epicardial approach was performed in New Zealand White rabbits with preserved autonomic innervation. Atrial electrograms were obtained with four unipolar electrodes placed epicardially along the atria (n = 22) and an electrode adapted to the proximal left PV (n = 10). The cervical vagus nerve was stimulated with bipolar platinum electrodes (20 Hz). Epicardial activation was recorded in sinus rhythm, and effective refractory periods (ERPs), dispersion of refractoriness and conduction times from high-lateral right atrium (RA) to high-lateral left atrium (LA) and PVs assessed at baseline and during vagal stimulation. Burst pacing (50 Hz, 10 s), alone or combined with vagal stimulation, was applied to the right (RAA) and left atrial appendage (LAA) and PVs to induce AF. At baseline, ERPs were lower in PVs than in LA and LAA, but did not differ significantly from RA and RAA, and there was a significant delay in the conduction time from RA to PVs compared with the activation time from RA to LA (P < 0.01). During vagal stimulation, ERP decreased significantly at all sites, without significant differences in the dispersion of refractoriness, and the atrial conduction times changed from 39 ± 19 to 49 ± 9 ms (RA to PVs; n.s.) and from 14 ± 7 to 28 ± 12 ms (RA to LA; P = 0.01). Induction of AF was reproducible in 50% of cases with 50 Hz and in 82% with 50 Hz combined with vagal stimulation (P < 0.05). During vagal stimulation, AF cycle length decreased at all sites, and AF duration changed from 1.0 ± 0.9 to 14.0 ± 10.0 s (P < 0.01), with documentation of PV tachycardia in three cases. In 70% of the animals, AF ceased immediately after interruption of vagal stimulation. We conclude that in the intact rabbit heart, vagal activity prolongs interatrial conduction and shortens atrial and PV ERP, contributing to the vulnerability to the induction and maintenance of AF. This model may be useful in the assessment of the autonomic influence in the mechanisms underlying AF.

The physiological signals are a challenge in terms of identification and processing due to its lack of stationarity and linearity. The autonomic output to the cardiovascular system is classically quantified by analysis of heart rate variability. The Fast Fourier Transform (FFT) and Discrete Wavelet Transform applied to the cardiac signals showed limitations in terms of time and frequency resolutions. With this work we propose the application of a Modified Hilbert-Huang Transform for tachogram and systogram evaluation with the introduction of a new method to smooth fluctuations endpoint. This work presents a comparative study using FFT and Hilbert-Huang methodologies. It is known that patients and animal models with diseases involving sympathoexcitation, eg.: Metabolic Syndrome (MetS), show increases in low frequencies band. Therefore, these mathematical tools are evaluated by analyzing experimental data from two animal species: MetS-rabbits (n=6) and MetS-rats (n=6) and their respective lean age and sex-matched controls (n=6 for each group). Modified Hilbert-Huang Transform analysis showed a sympathoexcitatory condition in MetS comparing to controls both in a rabbit and in a rat model of MetS. These significant differences on sympathetic tone were also confirmed by FFT but, the parasympathetic activity, was similar between normal and MetS animals. Results and its physiological interpretation clearly show the suitability of the modified Hilbert-Huang Transform as an alternative methodology for the dynamic assessment of the autonomic nervous system in MetS.

Recent clinical research directs up to provide personalized health care, adapted to each patient and to his clinical situation. With this work, we showed that a wearable device with heart rate (HR), respiratory and temperature sensors could be used, under certain technical and environmental requirements, for a reliable autonomic evaluation outside the confinement of an autonomic laboratory.

© 2014 Elsevier B.V. All rights reserved., Background: It is well established that sympathetic nervous system is responsible for the onset, development and maintenance of neurogenic hypertension. The rostroventrolateral medulla (RVLM) and medullo-cervical pressor area (MCPA) are important central sympathoexcitatory regions whose role on neurogenic hypertension remains unknown. Objective: To establish RVLM and MCPA roles in the long-term regulation of blood pressure by depressing their neuron activity through the over-expression of hKir2.1-potassium channel in conscious spontaneously hypertensive rats (SHR). Methods: In SHR, a lentiviral vector LVV-hKir2.1 was microinjected into RVLM or MCPA areas. A sham group was injected with LVV-eGFP. Blood pressure (BP) and heart rate (HR) were continuously monitored for 75 days. Baroreflex and chemoreflex functions were evaluated. Baroreflex gain, chemoreflex sensitivity, BP and HR variability were calculated. Results: LVV-hKir2.1 expression in RVLM, but not in MCPA, produced a significant time-dependent decrease in systolic, diastolic, mean-BP and LF of systolic BP at 60-days post-injection. No significant changes were seen in LVV-eGFP RVLM injected SHR. Conclusion: Data show that chronic expression of Kir2.1 in the RVLM of conscious SHR caused a marked and sustained decrease in BP without changes in the baro- and peripheral chemoreceptor reflex evoked responses. This decrease was mostly due to a reduction in sympathetic output revealed indirectly by a decrease in the power density of the SBP-LF band. Our data are amongst the firsts to demonstrate the role of the RVLM in maintaining BP levels in hypertension in conscious SHR. We suggest that a decrease in RVLM neuronal activity is an effective anti-hypertensive treatment strategy., This work was supported by Fundação para a Ciência e Tecnologia (PTDC/SAU-OSM/109081/2008), British Heart Foundation (RG/12/6/29670) and National Institutes of Health (RO1 NS069220).

Changes in the sympathetic nervous system are responsible for the initiation, development and maintenance of hypertension. An important central sympathoexcitatory region is the paraventricular nucleus (PVN) of the hypothalamus, which may become more active in hypertensive conditions, as shown in acute studies previously. Our objective was to depress PVN neuronal activity chronically by the overexpression of an inwardly rectifying potassium channel (hKir2.1), while evaluating the consequences on blood pressure (BP) and its reflex regulation. In spontaneously hypertensive rats (SHRs) and Wistar rats (WKY) lentiviral vectors (LVV-hKir2.1; LV-TREtight-Kir-cIRES-GFP5 4 × 10(9) IU and LV-Syn-Eff-G4BS-Syn-Tetoff 6.2 × 10(9) IU in a ratio 1:4) were stereotaxically microinjected bilaterally into the PVN. Sham-treated SHRs and WKY received bilateral PVN microinjections of LVV-eGFP (LV-Syn-Eff-G4BS-Syn-Tetoff 6.2 × 10(9) IU and LV-TREtight-GFP 5.7 × 10(9) IU in a ratio 1:4). Blood pressure was monitored continuously by radio-telemetry and evaluated over 75 days. Baroreflex gain was evaluated using phenylephrine (25 μg ml(-1), i.v.), whereas lobeline (25 μg ml(-1), i.v.) was used to stimulate peripheral chemoreceptors. In SHRs but not normotensive WKY rats, LVV-hKir2.1 expression in the PVN produced time-dependent and significant decreases in systolic (from 158 ± 3 to 132 ± 6 mmHg; P0.05) and diastolic BP (from 135 ± 4 to 113 ± 5 mmHg; P0.05). The systolic BP low-frequency band was reduced (from 0.79 ± 0.13 to 0.42 ± 0.09 mmHg(2); P0.05), suggesting reduced sympathetic vasomotor tone. Baroreflex gain was increased and peripheral chemoreflex depressed after PVN microinjection of LVV-hKir2.1. We conclude that the PVN plays a major role in long-term control of BP and sympathetic nervous system activity in SHRs. This is associated with reductions in both peripheral chemosensitivity and respiratory-induced sympathetic modulation and an improvement in baroreflex sensitivity. Our results support the PVN as a powerful site to control BP in neurogenic hypertension.

© 2011 Sociedade Portuguesa de Cardiologia. Publicado por Elsevier España, S.L. Todos os direitos reservados., Neurocardiogenic syncope (NCS) is a common clinical entity resulting from an excessive reflex autonomic response, particularly during orthostatism. Treatment options are controversial and of limited effectiveness. Tilt training (TT) is a promising option to treat these patients. However, its mechanism of action and clinical impact remain unclear. Objective: To characterize hemodynamic and autonomic responses during a TT program in patients with NCS refractory to conventional measures. Methods: We studied 28 patients (50% male, mean age 41±14 years) without structural heart disease, with NCS documented by tilt testing. The TT program included 9 tilt sessions (3 times a week, 30 min) (60° - 6 sessions, 70° - 3 sessions), under ECG and blood pressure monitoring combined with home orthostatic self-training and 10° head-up during sleep. Systolic volume, cardiac output, total peripheral resistance, baroreflex sensitivity and heart-rate variability were computed. Patients were reassessed at 1 month and every 6 months for a maximum of 36 months (24±12 months). Results: Over the course of the TT program there was a significant increase in total peripheral resistance (1485±225 vs. 1591±187 dyn·s·cm(-5), p, A síncope neurocardiogénica (SNc) é uma entidade clínica comum, resultante de uma resposta autonómica reflexa excessiva durante o stress ortostático. As diferentes opções terapêuticas são controversas e de eficácia limitada. O treino de ortostatismo (TTr) tem-se mostrado uma alternativa prometedora no tratamento destes doentes (D). No entanto, permanece por esclarecer o seu mecanismo de ação e o impacto clínico numa população com SNc recorrente. Objectivo: Caracterizar a resposta hemodinâmica e autonómica durante um programa de TTr em doentes com SNc refratária às medidas convencionais. População e métodos: Foram estudados 28D (50% do sexo masculino, 41 ± 14 anos), sem evidência de cardiopatia, com SNc documentada em teste de ortostatismo passivo. O TTr incluiu 9 sessões hospitalares (3x/semana, 30 minutos) com monitorização contínua de pressão arterial e frequência cardíaca (60◦ - 6 sessões - 70◦ - 3 sessões), complementadas com treino diário no domicílio e elevação da cabeceira a 10◦ durante o sono. O volume sistólico, o débito cardíaco, a resistência vascular periférica, a sensibilidade do barorreflexo e a variabilidade da frequência cardíaca foram calculados. Todos os doentes foram reavaliados no fim do 1.◦ mês e no final de cada 6 meses num período máximo de 36 meses (follow-up 24 ± 12 meses). Resultados: Ao longo das sessões de TTr verificou-se um aumento significativo e consistente da resistência total periférica (1485 ± 225 vs. 1591 ± 187 dyne*s/cm−5 , p < 0,05) associado a uma diminuição do seu desvio-padrão (206 ± 60 vs. 150 ± 42, p < 0,05). Durante o período de follow-up, houve recorrência de síncope em 5D (19%), com redução significativa do número de síncopes (4,0 ± 3,2/D nos 12 meses pre-TTr vs. 1,4 ± 0,8/D pos-TTr, p < 0,05). Conclusão: Em doentes com SNc refratária, o TTr mostrou ser uma opção terapêutica eficaz, com beneficio a longo prazo. A melhor tolerância ao ortostatismo parece resultar do aumento da reserva vasoconstritora e da sua menor variabilidade.

© The Royal Society of Chemistry 2012., The accumulation of lead in several bones of Wistar rats with time was determined and compared for the different types of bones. Two groups were studied: a control group (n = 20), not exposed to lead and a contaminated group (n = 30), exposed to lead from birth, first indirectly through mother's milk, and then directly through a diet containing lead acetate in drinking water (0.2%). Rats age ranged from 1 to 11 months, with approximately 1 month intervals and each of the collections had 3 contaminated rats and 2 control rats. Iliac, femur, tibia-fibula and skull have been analysed by Energy Dispersive X-ray Fluorescence Technique (EDXRF). Samples of formaldehyde used to preserve the bone tissues were also analysed by Electrothermal Atomic Absorption (ETAAS), showing that there was no significant loss of lead from the tissue to the preservative. The bones mean lead concentration of exposed rats range from 100 to 300 μg g(-1) while control rats never exceeded 10 μg g(-1). Mean bone lead concentrations were compared and the concentrations were higher in iliac, femur and tibia-fibula and after that skull. However, of all the concentrations in the different collections, only those in the skull were statistically significantly different (p < 0.05) from the other types of bones. Analysis of a radar chart also allowed us to say that these differences tend to diminish with age. The Spearman correlation test applied to mean lead concentrations showed strong and very strong positive correlations between all different types of bones. This test also showed that mean lead concentrations in bones are negatively correlated with the age of the animals. This correlation is strong in iliac and femur and very strong in tibia-fibula and skull. It was also shown that the decrease of lead accumulation with age is made by three plateaus of accumulation, which coincide, in all analysed bones, between 2nd-3rd and 9th-10th months., This research was supported in part by FCT––Fundação para a Ciência e a Tecnologia (Portugal) Project No. PEstOE/FIS/UI0303/2011, financed by the European Community Fund FEDER through the COMPETE–––Competitiveness Factors Operational Programme. D. Guimarães acknowledges Fundação para a Ciência e Tecnologia (FCT) for the Ph.D. Grant (SFRH/BD/38788/2007).

© 2012 Elsevier GmbH. All rights reserved., The concentration of lead in liver and kidneys of Wistar rats, fed with lead since fetal period in relation to their age and to a control group, was determined. A group of rats was exposed to lead acetate (n=30) in drinking water and the other group was exposed to normal water (n=20). Samples were collected from rats aging between 1 and 11 months and were analyzed by Energy Dispersive X-ray Fluorescence (EDXRF) without any chemical preparation. The EDXRF results were assessed by the PIXE (Proton Induced X-ray Emission) technique. The formaldehyde used to preserve the samples was also analyzed by ETAAS (Electro-Thermal Atomic Absorption Spectrometry) in order to verify if there was any loss of lead from the samples to the formaldehyde. We found that the loss was not significant (, J.P. Santos, D. Guimarães and M.L. Carvalho acknowledge the support by FCT – Fundação para a Ciência e a Tecnologia (Portugal) under the Project No. PEstOE/FIS/UI0303/2011, financed by the European Community Fund FEDER through the COMPETE – Competitiveness Factors Operational Programme.

Vagal activity is thought to influence atrial electrophysiological properties and play a role in the initiation and maintenance of atrial fibrillation (AF). We evaluated the effects of acute vagal stimulation on atrial conduction, refractoriness of atrial and pulmonary veins (PVs) and inducibility of AF. An open-chest epicardial approach was performed in New Zealand White rabbits with preserved autonomic innervation. Atrial electrograms were obtained with four unipolar electrodes placed epicardially along the atria (n = 22) and an electrode adapted to the proximal left PV (n = 10). The cervical vagus nerve was stimulated with bipolar platinum electrodes (20 Hz). Epicardial activation was recorded in sinus rhythm, and effective refractory periods (ERPs), dispersion of refractoriness and conduction times from high-lateral right atrium (RA) to high-lateral left atrium (LA) and PVs assessed at baseline and during vagal stimulation. Burst pacing (50 Hz, 10 s), alone or combined with vagal stimulation, was applied to the right (RAA) and left atrial appendage (LAA) and PVs to induce AF. At baseline, ERPs were lower in PVs than in LA and LAA, but did not differ significantly from RA and RAA, and there was a significant delay in the conduction time from RA to PVs compared with the activation time from RA to LA (P0.01). During vagal stimulation, ERP decreased significantly at all sites, without significant differences in the dispersion of refractoriness, and the atrial conduction times changed from 39 ± 19 to 49 ± 9 ms (RA to PVs; n.s.) and from 14 ± 7 to 28 ± 12 ms (RA to LA; P = 0.01). Induction of AF was reproducible in 50% of cases with 50 Hz and in 82% with 50 Hz combined with vagal stimulation (P0.05). During vagal stimulation, AF cycle length decreased at all sites, and AF duration changed from 1.0 ± 0.9 to 14.0 ± 10.0 s (P0.01), with documentation of PV tachycardia in three cases. In 70% of the animals, AF ceased immediately after interruption of vagal stimulation. We conclude that in the intact rabbit heart, vagal activity prolongs interatrial conduction and shortens atrial and PV ERP, contributing to the vulnerability to the induction and maintenance of AF. This model may be useful in the assessment of the autonomic influence in the mechanisms underlying AF.

Vagal activity is thought to influence atrial electrophysiological properties and play a role in the initiation and maintenance of atrial fibrillation (AF). In this study, we assessed the effects of acute vagal stimulation (vagus_stim) on atrial conduction times, atrial and pulmonary vein (PV) refractoriness, and vulnerability to induction of AF in the rabbit heart with intact autonomic innervation.An open-chest epicardial approach was performed in 11 rabbits (New Zealand; 3.9-5.0 kg), anesthetized and artificially ventilated after neuromuscular blockade. A 3-lead ECG was obtained. Atrial electrograms were recorded along the atria, from right to left (four monopolar electrodes), together with a circular electrode adapted for proximal left PV assessment. Acute vagus nerve stimulation was obtained with bipolar electrodes (20 Hz). Epicardial activation was recorded in sinus rhythm, and the conduction time from right (RA) to left atrium (LA), and from RA to PVs, was measured in basal conditions and during vagus_stim. The atrial effective refractory period (ERP) and dispersion of refractoriness (Disp_A) were analyzed. Vulnerability to AF induction was assessed at the right (RAA) and left (LAA) atrial appendages and the PVs. Atrial stimulation (50 Hz) was performed alone or combined with vagus_stim. Heart rate and blood pressure were monitored.In basal conditions, there was a significant delay in conduction from RA to PVs, not influenced by vagus_stim, and the PV ERPs were shorter than those measured in LA and LAA, but without significant differences compared to RA and RAA. During vagus_stim, conduction times between RA and LA increased from 16+8 ms to 27+6 ms (p0.05) and ERPs shortened significantly in RA, LAA and LA (p0.05), but not in RAA. There were no significant differences in Disp_A. AF induction was reproducible in 45% of cases at 50 Hz and in 100% at 50 Hz+vagus_stim (p0.05). The duration of inducible AF increased from 1.0 +/- 0.2 s to 12.0 +/- 4.5 s with 50 Hz+vagus_stim (p0.01). AF lasted10 s in 45.4% of rabbits during vagus_stim, and ceased after vagus_stim in 4 out of these 5 cases. In 3 animals, PV tachycardia, with fibrillatory conduction, induced with 50 Hz PV pacing during vagus_stim.Vagus_stim reduces interatrial conduction velocity and significantly shortens atrial ERP, contributing to the induction and duration of AF episodes in the in vivo rabbit heart. This model may be useful for the assessment of autonomic influence on the pathophysiology of AF

Background: In keeping with the fundamental practice of transparency in the discussion and resolution of ethics conflicts raised by research, a summary of ethics issues raised during Portuguese biomonitoring in health surveillance and research is presented and, where applicable, their resolution is described. Methods: Projects underway aim to promote the surveillance of public health related to the presence of solid waste incinerators or to study associations between human exposure to environmental factors and adverse health effects. The methodological approach involves biomonitoring of heavy metals, dioxins and/or other persistent organic pollutants in tissues including blood, human milk and both scalp and pubic hair in groups such as the general population, children, pregnant women or women attempting pregnancy. As such, the projects entail the recruitment of individuals representing different demographic and health conditions, the collection of body tissues and personal data, and the processing of the data and results. Results: The issue of autonomy is raised during the recruitment of participants and during the collection of samples and data. This right is protected by the requirement for prior written, informed consent from the participant or, in the case of children, from their guardian. Recruitment has been successful, among eligible participants, in spite of incentives rarely being offered. The exception has been in obtaining guardians' consent for children's participation, particularly for blood sampling. In an attempt to mitigate the harm-benefit ratio, current research efforts include alternative less invasive biomarkers. Surveys are currently being conducted under contract as independent biomonitoring actions and as such, must be explicitly disclosed as a potential conflict of interests. Communication of results to participants is in general only practised when a health issue is present and corrective action possible. Concerning human milk a careful approach is taken, considering breast-feeding's proven benefits. Conclusion: No national legislation currently accounts for the surveillance component of biomonitoring as distinct from research. Ethics issues arising within the domain of research are resolved according to available regulations. For issues encountered during surveillance, the same principles are used as guidance, completed by the authors' best judgement and relevant ethics committees' findings., Fundação para a Ciência e a Tecnologia with participation by FEDER, under POCI2010; ref. POCI/ SAU-ESP/58298/2004 (VAEDA) and POCI/SAU-ESP/62115/2004 (FEXHEBIO)

Objective:Evaluate the effects of acute vagal stimulation on atrial conduction, atria and pulmonary veins (PV) refractoriness and AFib inducibility in a MetS rabbit model.Design and method:MetS was induced in male NZW rabbits, 8 weeks, by a high sucrose diet given for 6 months, after which, under an

Tese de doutoramento, Ciências Biomédicas (Fisiologia), Universidade de Lisboa, Faculdade de Medicina, 2014 Submitted by Amelia Janeiro (ajaneiro@reitoria.ul.pt) on 2015-01-27T13:08:11Z No. of bitstreams: 1 ulsd069771_td_Vera_Geraldes.pdf: 10177972 bytes, checksum: df070f6354197d9e2bce9ee4c8566664 (MD5) Made available in DSpace on 2015-02-02T12:20:17Z (GMT). No. of bitstreams: 1 ulsd069771_td_Vera_Geraldes.pdf: 10177972 bytes, checksum: df070f6354197d9e2bce9ee4c8566664 (MD5) Previous issue date: 2014 Fundação para a Ciência e a Tecnologia (FCT)