The margin of appreciation doctrine and the right to life : the article 2 of the ECHR

The margin of appreciation is proving to be an increasingly fundamental mechanism when it comes to human rights since the protection given to such rights varies from state to state, and from time to time, depending on their beliefs and traditions. The purpose of this thesis is not only to explain the concept of the margin of appreciation and its evolution throughout time but also to establish the relationship of this doctrine with the right to life, enshrined in Article 2 of the European Convention on Human Rights. Thus, and because it becomes interesting from a comparative perspective, the legal regime of three Member States of the Council of Europe, at the time of writing, will be indicated.A doutrina da margem nacional de apreciação revela-se um mecanismo cada vez mais fundamental no que toca aos direitos humanos, uma vez que a proteção dada a tais direitos varia de Estado para Estado, e de tempo em tempo, consoante as crenças e as tradições daqueles. Esta tese tem como objetivo não apenas dar a conhecer o conceito de margem nacional de apreciação e a sua evolução, mas também estabelecer a relação de tal doutrina com o direito à vida, previsto no artigo 2.º da Convenção Europeia dos Direitos Humanos. Deste modo, e por se afigurar interessante do ponto de vista comparativo, indicar-se-á o regime legal, à data da escrita da dissertação, de três Estados membros do Conselho da Europa

Perfis psicológicos de crianças e jovens em acolhimento residencial

A investigação que aborda as necessidades psicológicas de crianças em AcolhimentoResidencial (AR) tem sido consistente em identificar um padrão de desajustamentopsicológico e sintomatologia de saúde mental. A evidência de padrões de funcionamentopositivo tem sido mais escassa e centrou-se em variáveis especificas, como bem-estarsubjetivo e satisfação com a vida. O presente estudo pretende identificar grupos decrianças/jovens em AR com diferentes perfis de funcionamento psicológico (FP),atendendo, simultaneamente, a características positivas e a sinais específicos dedesajustamento psicológico; explorar a associação entre estes grupos e variáveisindividuais das crianças e contextuais das casas de acolhimento. Participaram nesteestudo 545 crianças/jovens, de ambos os sexos (54.9% são raparigas), com uma idademédia 15.38 anos. Os participantes responderam ao SDQ, YSR, IBP, ESCV, EFS,PANAS, e RSES. Através de uma análise de clusters foram identificados quatro gruposde crianças/jovens em AR com diferentes perfis de FP: Perfil de FP Positivo, Perfil de FPBorderline, Perfil de FP Borderline e Clínico, e Perfil de FP Clínico. Verificaram-se maisrapazes no Perfil de FP Positivo e de FP Clínico, existindo uma tendência para as criançasdo Perfil de FP Positivo estarem acolhidas em casas mistas. Revelaram-se mais raparigasno Perfil de FP Borderline e Clínico, com tendência a estarem acolhidas em casassegregadas. Os resultados do estudo oferecem conhecimento acerca dos diferentes perfispsicológicos das crianças em AR e reforçam ainda a relevância do acolhimento em casasmistas, bem como a importância de adequar melhor o AR às necessidades das raparigas

Regulação de eventos exocitóticos pela via de sinalização do cAMP

Doutoramento em BiologiaPermanece por esclarecer como a via de sinalização do cAMP modula a exocitose regulada. Os principais objetivos deste trabalho foram: i) avaliar o efeito do cAMP nos eventos exocitóticos, nas propriedades dos poros de fusão e na secreção hormonal; ii) perceber o impacto da sinalização por cAMP-HCN na exocitose e nas propriedades do poro de fusão; e iii) estudar as propriedades do poro de fusão na presença de um agente neurotóxico comum, como o alumínio. Lactotrofos, isolados a partir da hipófise anterior de ratos Wistar machos, foram usados como modelo celular. Os eventos unitários de fusão exocitótica e a prolactina (PRL) libertada foram avaliados, respetivamente, em ensaios eletrofisiológicos efectuados segundo a técnica de contacto hermético no modo sobre a célula aderida à pipeta porta-elétrodo e com recurso a métodos imunológicos de deteção. Os níveis intracelulares de cAMP foram aumentados por 3-isobutil-1-metilxantina (IMBX), forscolina e N6,2'-O-dibutiril adenosina- 3',5'-monofosfato cíclico (dbcAMP). A expressão dos canais HCN foi determinada por Western-blot, qRT-PCR e imunocitoquímica em combinação com microscopia confocal. Culturas primárias de lactotrofos foram também transfetadas com DNA plasmídico que codifica HCN2 juntamente com a proteína-verde-fluorescente e um agente farmacológico foi usado para avaliar o efeito de cAMP-HCN na exocitose. Observou-se que os lactotrofos responderam à forscolina e ao dbcAMP libertando PRL de um modo bifásico e dependente da concentração, uma vez que a secreção aumentou e diminuiu, respectivamente, na gama de baixas e altas concentrações. Os compostos que elevaram os níveis de cAMP aumentaram os eventos transientes e impediram a fusão completa. Além disso, o dbcAMP promoveu o aparecimento de eventos exocitóticos transientes de elevada periodicidade, cujos poros de fusão, de maior diâmetro, se mativeram abertos durante mais tempo. A expressão das quatro isoformas de HCN foi confirmada nos lactotrofos ao nível do mRNA e, tal como no coração, rim e hipófise, o mais abundante codifica a isoforma HCN2. Nos lactotrofos com sobre-expressão desta isoforma, o dbcAMP não só aumentou a frequência dos eventos transientes e a condutância dos poros, mas também a frequência dos eventos de fusão completa. Enquanto o bloqueador dos canais HCN, ZD7288, reduziu a frequência dos eventos transientes e de fusão completa desencadeados por dbcAMP e diminuiu o diâmetro dos poros de fusão. A simultânea diminuição da libertação de PRL, da frequência dos eventos transientes e do diâmetro dos poros de fusão representaram as principais alterações observados após pré-tratamento dos lactotrofos com concentração micromolar de alumínio. Em conclusão, os resultados demonstram que elevados níveis de cAMP reduzem a secreção de PRL devido à estabilização dos poros de fusão no estado de maior abertura. Além disso, a via de sinalização cAMP-HCN afecta a actividade exocitótica e modifica as propriedades dos poros de fusão, que parecem ser igualmente importantes na citotoxicidade induzida por alumínio.How the second messenger cAMP modulates regulated exocytosis is still poorly understood. The goals of this work were: i) to evaluate cAMP effect on single exocytotic events, fusion pore properties and hormone release; ii) to assess the impact of cAMP-HCN signaling on exocytosis and fusion pore properties; and iii) to study fusion pore properties in the presence of a common neurotoxic agent, such as aluminium. Anterior pituitary lactotrophs isolated from male Wistar rats were used as the cell model. Electrophysiology in cell attached patch-clamp mode and immuno assays were used to monitor unitary exocytic fusion events and prolactin (PRL) release, respectively. Intracellular cAMP concentration was raised with 3- isobutyl-1-methylxanthine (IBMX), forskolin and N6,2’-O-dibutyryl adenosine- 3′,5′-cyclic monophosphate (dbcAMP). HCN channel expression was determined by using Western-blot, qRT-PCR and immunocytochemistry in combination with confocal microscopy. Primary cell cultures were also transfected with plasmid DNA encoding HCN2 together with enhanced-greenfluorescence protein and a pharmacological agent was applied to evaluate cAMP-HCN effects on exocytosis. We observed that lactotrophs responded to forskolin and dbcAMP by releasing PRL in a biphasic dose-dependent manner, since secretion increased and decreased in the presence of low and high concentration ranges, respectively. cAMP-increasing agents enhanced transient events, while full-fusion events were prevented. Moreover, dbcAMP promoted the appearance of extreme periodic bursts of transient events that exhibited longer fusion pore life-time and wider diameters. The expression of the four HCN isoforms was confirmed at mRNA transcript level in lactotrophs and, like in heart, kidney and pituitary, the most abundant encodes HCN2 isoform. In HCN2 over-expressing lactotrophs, dbcAMP not only enhanced the frequency of transient events and pore conductance but also the frequency of full-fusion events. While, the HCN channel blocker, ZD7288, reduced the frequency of full-fusion and transient events elicited by dbcAMP and narrowed the fusion pore diameters. Finally, the simultaneous reduction on PRL release, transient events frequency and fusion pore diameters represented the main alterations observed after lactotrophs pretreatment with micromolar aluminium concentration. In conclusion, the results show that high cAMP reduces PRL secretion due to the stabilization of the wide fusion pore state. Additionally, cAMP-HCN signaling cascade affects exocytic activity and modifies fusion pore properties, which also appear to be relevant in the aluminium induced cytotoxicity

A single cell study of aluminium effect on rat anterior pituitary

Mestrado em Biologia Molecular e CelularO alumínio foi pela primeira vez reconhecido como um agente neurotóxico para o Homem em 1886. Uma vasta gama de efeitos tóxicos do alumínio a nível celular foi já demonstrada, no entanto permanece desconhecido o modo como o alumínio afecta as células da hipófise anterior. A exposição ocupacional a alumínio pode causar redução dos níveis séricos de prolactina, uma hormona peptídica sintetizada e secretada principalmente pelas células lactotropas hipofisárias. O principal objectivo deste trabalho foi estudar o efeito do alumínio na viabilidade celular e na actividade exocitótica em culturas de células da hipófise anterior do rato enriquecidas em lactotrofos. A quantificação das células vivas e mortas foi realizada por citometria de fluxo após dupla marcação com sondas fluorescentes. Observámos uma diminuição significativa na viabilidade celular após 30 min de exposição a 300 μM de AlCl3. Uma concentração dez vezes menor causou perda significativa de células apenas após 4 dias de exposição. Para exposições prolongadas a concentrações inferiores a 30 μM, a viabilidade celular manteve-se elevada. A morte celular induzida por alumínio parece ser precedida pela sua acumulação nas células, visto que a análise por espectrometria de absorção atómica relevou um aumento significativo de Al3 + nos lactotrofos após 1h de exposição à menor concentração com efeito na viabilidade (30 μM AlCl3). Os efeitos subletais do AlCl3 na exocitose regulada foram avaliados com recurso a métodos de electrofisiologia celular. O registo com elevada resolução das alterações de capacitância e condutância da membrana celular permitiu diferenciar eventos de fusão singelos entre vesículas de secreção e a membrana plasmática em condições espontâneas e durante estimulação. Após 24 h de exposição a 30 μM AlCl3, a resposta exocitótica à estimulação foi reprimida e verificaram-se diferenças nas propriedades dos poros de fusão. Actualmente considera-se que durante a exocitose regulada e reversível ocorre secreção do conteúdo vesicular através do poro de fusão, o qual é formado pelas membranas vesicular e plasmática. Após a exposição ao alumínio os poros de fusão, observados durante o processo de fusão reversível (“kiss-and-run”) revelaram-se mais estreitos, visto que as suas condutâncias eram inferiores às registadas durante os eventos espontâneos ocorridos em condições fisiológicas normais. Estes resultados levam os autores a sugerir que (1) concentrações subletais de alumínio tornam a exocitose regulada improdutiva, de tal modo que grandes moléculas, como a prolactina, não conseguem ser secretadas e que (2) a perda de células lactotropas hipofisárias poderá ocorrer devido a exposição a este agente neurotóxico. ABSTRACT: Aluminium was first recognized as a human neurotoxic agent in 1886. Besides a wide range of toxic effects of aluminium have been demonstrated at cellular level, how it affects anterior pituitary cells remains unknown. It was shown that workers exposed to aluminium presented lower levels of serum prolactin, a peptide hormone mainly synthesised and secreted by anterior pituitary lactotrophs. The aim of this work was to study the aluminium effect on cell viability and regulated exocytotic activity in rat pituitary lactotrophs-enriched cultures. Dual-labeling flow cytometric assays were performed to quantify live and dead cells simultaneously. We observed a significant decrease of cell viability following 30 min-exposure of cell cultures to 300 μM AlCl3. At a ten-fold lower concentration, overt cell lost was only achieved by extending exposure time to 4 days. For continuous exposure to AlCl3 concentrations lower than 30 μM, even for prolonged periods of up to 4 days, enriched cultures of pituitary lactotrophs maintained high levels of viability. Aluminium-induced cell lost seems to be headed by its cellular accumulation, given that atomic absorption spectrometry analyses revealed a significant increase of Al3+ retention by lactotrophs after 1h-exposure to the lowest concentration with effect in viability (30 μM AlCl3). The sublethal effect of AlCl3 on the regulated exocytosis by rat pituitary lactotrophs was assessed by patch-clamp cell-attached technique. We performed high-resolution membrane capacitance and conductance measurements to resolve unitary fusion events of large dense-core vesicle with the plasma membrane under spontaneous and stimulated conditions. After 24-h exposure to 30 μM AlCl3, exocytotic response to stimulation was repressed and differences in elementary fusion pore properties were detected. The transient fusion pore of regulated exocytosis is believed to be an aqueous channel that connects and spans the vesicle and the plasma membrane, through which the content of secretory vesicle may be released. Following exposure to aluminium fusion pores, seen during reversible fusion process (kiss-and-run), appeared narrower, since they had lower conductance than that of resting values under normal physiological conditions. These results lead the authors to suggest that (1) sublethal aluminium concentrations shift the mode of regulated exocytosis to unproductive such that large cargo molecules like prolactin cannot be secreted and (2) lactotroph lost at the anterior pituitary might occur due to exposure to this neurotoxic agent

Cytotoxicity of temperature-responsive cationic diblock copolymers in human cancer and non-cancer cells lines

Abstract in proceedings of the Fourth International Congress of CiiEM: Health, Well-Being and Ageing in the 21st Century, held at Egas Moniz’ University Campus in Monte de Caparica, Almada, from 3–5 June 2019.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.info:eu-repo/semantics/publishedVersio

Therapeutic effects of platelet-derived extracellular vesicles in a bioengineered tendon disease model

Tendon injuries represent over 30–50% of musculoskeletal disorders worldwide, yet the available therapies do not provide complete tendon repair/regeneration and full functionality restor-ing. Extracellular vesicles (EVs), membrane-enclosed nanoparticles, have emerged as the next breakthrough in tissue engineering and regenerative medicine to promote endogenous tissue regen-eration. Here, we developed a 3D human in vitro model mimicking the signature of pathological tendon and used it to evaluate the influence that different platelet-derived EVs might have in tendon tissue repair mechanisms. For this, different EV populations isolated from platelets, small EVs (sEVs) and medium EVs (mEVs), were added to the culture media of human tendon-derived cells (hTDCs) cultured on isotropic nanofibrous scaffolds. The platelet-derived EVs increased the expression of tenogenic markers, promoted a healthy extracellular matrix (ECM) remodeling, and the synthesis of anti-inflammatory mediators. These findings suggest that platelet EVs provided relevant biochemical cues that potentiated a recovery of hTDCs phenotype from a diseased to a healthy state. Thus, this study opens new perspectives for the translation of platelet-derived EVs as therapeutics.This research was funded by the ERC CoG MagTendon grant agreement 772817; EC Twinning project Achilles 810850; Fundacao para a Ciencia e a Tecnologia (FCT) for PhD grant PD/59/2013 and PD/BD/135255/2017, individual contracts CEECIND/01375/2017 (M.G.-F.) and 2020.03410.CEECIND (R.M.A.D), and project PTDC/NAN-MAT/30595/2017

Bioluminescence imaging on-chip platforms for non-invasive high-content bioimaging

Incorporating non-invasive biosensing features in organ-on-chip models is of paramount importance for a wider implementation of these advanced in vitro microfluidic platforms. Optical biosensors, based on Bioluminescence Imaging (BLI), enable continuous, non-invasive, and in-situ imaging of cells, tissues or miniaturized organs without the drawbacks of conventional fluorescence imaging. Here, we report the first-of-its-kind integration and optimization of BLI in microfluidic chips, for non-invasive imaging of multiple biological readouts. The cell line HEK293T-GFP was engineered to express NanoLuc® luciferase under the control of a constitutive promoter and were cultured on-chip in 3D, in standard ECM-like hydrogels, to assess optimal cell detection conditions. Using real-time in-vitro dual-color microscopy, Bioluminescence (BL) and fluorescence (FL) were detectable using distinct imaging setups. Detection of the bioluminescent signals were observed at single cell resolution on-chip 20 min post-addition of Furimazine substrate and under perfusion. All hydrogels enabled BLI with higher signal-to-noise ratios as compared to fluorescence. For instance, agarose gels showed a ∼5-fold greater BL signal over background after injection of the substrate as compared to the FL signal. The use of BLI with microfluidic chip technologies opens up the potential for simultaneous in situ detection with continuous monitoring of multicolor cell reporters. Moreover, this can be achieved in a non-invasive manner. BL has great promise as a highly desirable biosensor for studying organ-on-chip platforms.</p

Comparison of unitary exocytic events in pituitary lactotrophs and in astrocytes: modeling the discrete open fusion-pore states

In regulated exocytosis the merger between the vesicle and the plasma membranes leads to the formation of an aqueous channel (a fusion-pore), through which vesicular secretions exit into the extracellular space. A fusion pore was thought to be a short-lived intermediate preceding full-fusion of the vesicle and the plasma membranes (full-fusion exocytosis). However, transient exocytic events were also observed, where the fusion-pore opens and closes, repetitively. Here we asked whether there are different discrete states of the open fusion-pore. Unitary exocytic events were recorded by the high-resolution cell-attached patch-clamp method in pituitary lactotrophs and brain astrocytes. We monitored reversible unitary exocytic events, characterized by an on-step, which is followed by an off-step in membrane capacitance (C m ), a parameter linearly related to the membrane area. The results revealed three categories of reversible exocytic events (transient fusion-pore openings), which do not end with the complete integration of the vesicle membrane into the plasma membrane. These were categorized according to the observed differences in the amplitude and sign of the change in the real (Re) parts of the admittance signals: in case I events (Re ≈ 0) fusion pores are relatively wide; in case II (Re > 0) and case III (Re < 0) events fusion pores are relatively narrow. We show that case III events are more likely to occur for small vesicles, whereas, case II events are more likely to occur for larger vesicles. Case III events were considerably more frequent in astrocytes than in lactotrophs

Bioengineered 3D living fibers as in vitro human tissue models of tendon physiology and pathology

Clinically relevant in vitro models of human tissue's health and disease are urgently needed for a better understanding of biological mechanisms essential for the development of novel therapies. Herein, physiological (healthy) and pathological (disease) tendon states are bioengineered by coupling the biological signaling of platelet lysate components with controlled 3D architectures of electrospun microfibers to drive the fate of human tendon cells in different composite living fibers (CLFs). In the CLFs-healthy model, tendon cells adopt a high cytoskeleton alignment and elongation, express tendon-related markers (scleraxis, tenomodulin, and mohawk) and deposit a dense tenogenic matrix. In contrast, cell crowding with low preferential orientation, high matrix deposition, and phenotypic drift leading to increased expression of nontendon related and fibrotic markers, are characteristics of the CLFs-diseased model. This diseased-like profile, also reflected in the increase of COL3/COL1 ratio, is further evident by the imbalance between matrix remodeling and degradation effectors, characteristic of tendinopathy. In summary, microengineered 3D in vitro models of human tendon healthy and diseased states are successfully fabricated. Most importantly, these innovative and versatile microphysiological models offer major advantages over currently used systems, holding promise for drugs screening and development of new therapies.Work developed under the framework of the Cooperation Agreement established with the Serviço de Imuno-Hemoterapia do Centro Hospitalar de S. João, EPE. The authors would like to thank the Plastic Surgery Department of Hospital da Prelada (Porto, Portugal) for providing tendon tissue samples. Authors acknowledge the financial support from the ERC Grant CoG MagTendon No. 772817; FCT– Fundação para a Ciência e a Tecnologia for the Ph.D. grant of IC (PD/BD/128088/2016) and CL (PD/BD/150515/2019); for the contract to M.G.F. (CEECIND/01375/2017); and for project SmarTendon (PTDC/NAN-MAT/30595/2017) and Achilles (Grant no. 810850). After ini tial online publication, the present address for D.D. was added to the af filiations section on August 3, 2022

cAMP-Mediated stabilization of fusion pores in cultured rat pituitary lactotrophs

Regulated exocytosis mediates the release of hormones and transmitters. The last step of this process is represented by the merger between the vesicle and the plasma membranes, and the formation of a fusion pore. Once formed, the initially stable and narrow fusion pore may reversibly widen (transient exocytosis) or fully open (full-fusion exocytosis). Exocytosis is typically triggered by an elevation in cytosolic calcium activity. However, other second messengers, such as cAMP, have been reported to modulate secretion. The way in which cAMP influences the transitions between different fusion pore states remains unclear. Here, hormone release studies show that prolactin release from isolated rat lactotrophs stimulated by forskolin, an activator of adenylyl cyclases, and by membrane-permeable cAMP analog (dbcAMP), exhibit a biphasic concentration dependency. Although at lower concentrations (2-10 μm forskolin and 2.5-5 mm dbcAMP) these agents stimulate prolactin release, an inhibition is measured at higher concentrations (50 μm forskolin and 10-15 mm dbcAMP). By using high-resolution capacitance (Cm) measurements, we recorded discrete increases in Cm, which represent elementary exocytic events. An elevation of cAMP leaves the frequency of full-fusion events unchanged while increasing the frequency of transient events. These exhibited a wider fusion pore as measured by increased fusion pore conductance and a prolonged fusion pore dwell time. The probability of observing rhythmic reopening of transient fusion pores was elevated by dbcAMP. In conclusion, cAMP-mediated stabilization of wide fusion pores prevents vesicles from proceeding to the full-fusion stage of exocytosis, which hinders vesicle content discharge at high cAMP concentrations