Primary cilia and the regulation of hedgehog signaling: link to cardiac development

RESUMO: As células eucarióticas evoluíram um sistema de sinalização complexo que lhes permite responder aos sinais extracelulares e intracelulares. Desta forma, as vias de sinalização são essenciais para a sobrevivência da célula e do organismo, uma vez que regulam processos fundamentais, tais como o desenvolvimento, o crescimento, a imunidade, e a homeostase dos tecidos. A via de transdução de sinal Hedgehog (Hh) envolve o receptor Patched1 (Ptch1), que tem um efeito inibidor sobre a proteína Smoothened (Smo) na ausência dos seus ligandos, as proteínas Sonic hedgehog (Shh). Estas proteínas são reguladores fundamentais do desenvolvimento embrionário, como ilustrado pelas malformações drásticas observadas em embriões humanos e de murganho com perturbações da transdução de sinal da via Hh e que incluem polidactilia, defeitos craniofaciais e malformações ósseas. Igualmente importantes são as consequências da ativação inapropriada da via de sinalização Hh na formação de tumores. Curiosamente, os componentes desta via localizam-se nos cílios primários. Além disso, demonstrou-se que esta localização é crucial para a sinalização através da via Hh. Na presença dos ligandos, Ptch1 é internalizado e destinado a degradação ou sequestrado num compartimento da célula de onde não pode desempenhar o seu papel inibitório. A proteína Arl13b é uma pequena GTPase pertencente à família Arf/Arl da superfamília Ras de pequenas GTPases e foi implicada no síndrome de Joubert, uma ciliopatia caracterizada por ataxia congénita cerebelar, hipotonia, atrso mental e cardiopatia congénita. Murganhos deficientes para Arl13b, chamado hennin (hnn) morrem morrem prematuramente ao dia 13,5 de gestação (E13,5) e exibem anomalias morfológicas nos cílios que levam à interrupção da sinalização Hh. Além disso, a Arl13b está diretamente envolvida na regulação da via Hh, controlando a localização de vários componentes desta via nos cílios primários. Neste trabalho, mostramos que a Arl13b se localiza em circular dorsal ruffles (CDRs), que são estruturas de actina envolvidas em macropinocitose e internalização de recetores, e que regula a sua formação. Além disso, aprofundámos o conhecimento do processo de ativação da via de sinalização Hh, mostrando que as CDRs sequestram seletivamente e internalizam o recetor Ptch1. As CDRs formam-se minutos após ativação da via por ligandos Shh ou pelo agonista de Smo SAG e continuam a ser formadas a partir daí, sugerindo uma indução contínua da reorganização do citoesqueleto de actina quando a via está ativada. Observámos ainda que a inibição da formação de CDRs através do silenciamento de WAVE1, uma proteína necessária para a formação destas estruturas, resulta na diminuição da ativação da via de sinalização Hh. Além disso, o bloqueio da macropinocitose, que se segue ao fecho das CDRs, através do silenciamento de uma proteína necessária para a cisão de macropinossomas, nomeadamente a proteína BARS, tem um efeito semelhante. Estes resultados sugerem que as CDRs e a macropinocitose são necessárias para a ativação da via de sinalização Hh e indicam que esta via de internalização controla os níveis de sinal Hh. Durante o desenvolvimento, as células proliferativas dependem do cílio primário para a transdução de várias vias de sinalização. A via Hh induz a diferenciação do músculo cardíaco. Por conseguinte, os murganhos deficientes na via de sinalização Hh exibem uma variedade de defeitos de lateralidade, incluindo alteração do looping do coração, como pode ser visto em murganhos deficientes para Arl13b. Por conseguinte, investigámos o papel da Arl13b no desenvolvimento do coração. Mostramos que a Arl13b é altamente expressa no coração de embriões de murganho e de murganhos adultos ao nível do mRNA e da proteína. Além disso, o perfil de distribuição da Arl13b no coração segue o dos cílios primários, que são essenciais para o desenvolvimento cardíaco. Corações de murganhos hnn no estadio E12,5 mostram um canal átrio-ventricular aberto, espessamento da camada compacta ventricular e aumento do índice mitótico no ventrículo esquerdo. Além disso, um atraso de 1 a 2 dias no desenvolvimento é observado em corações de murganhos hnn, quando comparados com controlos selvagens no estadio E13,5. Assim, estes resultados sugerem que a Arl13b é necessária para o desenvolvimento embrionário do coração e que defeitos cardíacos podem contribuir para a letalidade embrionária de murganhos hnn. Em suma, foi estabelecido um novo mecanismo para a regulação dos níveis de superfície do recetor Ptch1, que envolve a remodelação do citoesqueleto de actina e a formação de CDRs após a ativação da via de sinalização Hh. Este mecanismo permite um feedback negativo que evita a repressão excessiva da via através da remoção de Ptch1 da superfície da célula. Além disso, determinou-se que uma mutação de perda de função na Arl13b causa defeitos cardíacos durante o desenvolvimento, possivelmente relacionados com a associação dos defeitos em cílios primários e na sinalização Hh, existentes em murganhos deficientes para Arl13b. A via de sinalização Hh tem tido um papel central entre as vias de sinalização, uma vez que a sua regulação é crucial para o funcionamento apropriada da célula. Assim, a descoberta de um novo mecanismo de tráfego através de macropinocitose e CDRs que controla a ativação e repressão da via de sinalização Hh traz novas perspetivas de como esta via pode ser regulada e pode ainda conduzir à identificação de novos alvos e estratégias terapêuticas. --------------------ABSTRACT: Eukaryotic cells have evolved a complex signaling system that allows them to respond to extracellular and intracellular cues. Signaling pathways are essential for cell and organism survival, since they regulate fundamental processes such as development, growth, immunity, and tissue homeostasis. The Hedgehog (Hh) pathway of signal transduction involves the receptor Patched1 (Ptch1), which has an inhibitory effect on Smoothened (Smo) in the absence of its ligands, the Sonic hedgehog (Shh) proteins. These proteins are fundamental regulators of embryonic development, as illustrated by the dramatic malformations seen in human and mouse embryos with perturbed Hh signal transduction that include polydactyly, craniofacial defects and skeletal malformations. Equally important are the consequences of inappropriate activation of the Hh signaling response in tumor formation. Interestingly, the components of this pathway localize to primary cilia. Moreover, it has been shown that this localization is crucial for Hh signaling. However, in the presence of the ligands, Ptch1 is internalized and destined for degradation or sequestered in a cell compartment where it no longer can play its inhibitory role. ADP-ribosylation factor-like (Arl) 13b, a small GTPase belonging to Arf/Arl family of the Ras superfamily of small GTPases has been implicated in Joubert syndrome, a ciliopathy characterized by congenital cerebellar ataxia, hypotonia, intellectual disability and congenital heart disease. Arl13b-deficient mice, called hennin (hnn) die at embryonic day 13.5 (E13.5) and display morphological abnormalities in primary cilia that lead to the disruption of Hh signaling. Furthermore, Arl13b is directly involved in the regulation of Hh signaling by controlling the localization of several components of this pathway to primary cilia. Here, we show that Arl13b localizes to and regulates the formation of circular dorsal rufles (CDRs), which are actin-basedstructures known to be involved in macropinocytosis and receptor internalization. Additionally, we extended the knowledge of the Hh signaling activation process by showing that CDRs selectively sequester and internalize Ptch1 receptors. CDRs are formed minutes after Hh activation by Shh ligands or the Smo agonist SAG and keep being formed thereafter, suggesting a continuous induction of actin reorganization when the pathway is switched on. Importantly, we observed that disruption of CDRs by silencing WAVE1, a protein required for CDR formation, results in down-regulation of Hh signaling activation. Moreover, the blockade of macropinocytosis, which follows CDR closure, through silencing of a protein necessary for the fission of macropinosomes, namely BARS has a similar effect. These results suggest that CDRs and macropinocytosis are necessary for activation of Hh signaling and indicate that this pathway of internalization controls Hh signal levels. During development, proliferating cells rely on the primary cilium for the transduction of several signaling pathways. Hh induces the differentiation of cardiac muscle. Accordingly, Hh-deficient mice display a variety of laterality defects, including alteration of heart looping, as seen in Arl13b-deficient mice. Therefore, we investigated the role of Arl13b in heart development. We show that Arl13b is highly expressed in the heart of both embryonic and adult mice at mRNA and protein levels. Also, Arl13b localization profile mimics that of primary cilia, which have been shown to be essential to early heart development. E12.5 hnn hearts show an open atrioventricular channel, increased thickening of the ventricular compact layer and increased mitotic index in the left ventricle. Moreover, a delay of 1 to 2 days in development is observed in hnn hearts, when compared to wild-type controls at E13.5. Hence, these results suggest that Arl13b is necessary for embryonic heart development and that cardiac defects might contribute to the embryonic lethality of hnn mice. Altogether, we established a novel mechanism for the regulation of Ptch1 surface levels, involving cytoskeleton remodeling and CDR formation upon Hh signaling activation. This mechanism allows a negative feedback loop that prevents excessive repression of the pathway by removing Ptch1 from the cell surface. Additionally, we determined that the Arl13b loss-offunction mutation causes cardiac defects during development, possibly related to the associated ciliary and Hh signaling defects found in Arl13b-deficient mice. Hh signaling has taken a center stage among the signaling pathways since its regulation is crucial for the appropriate output and function of the cell. Hence, the finding of a novel trafficking mechanism through CDRs and macropinocytosis that controls Hh signaling activation and repression brings new insights to how this pathway can be regulated and can lead to the discovery of novel therapeutic targets and strategies.

A proposta de regulamento relativo a um direito europeu comum da compra e venda e as suas implicações no direito português

We have witnessed in recent years, an obvious effort by the competent European institutions, towards the harmonization of general law applicable to all Member States (MS's). Many developments have been registered in several areas of law, a europeanization process that aims to add value to cross-border transactions and, consequently, the internal market and european trade. This trend manifests itself in general to the private law level, and particularly in contract law. The extension of the field in which market participants - whether professionals or consumers - can act, must imperatively be articulated with a consequent wider protection. After all, the consumer is also a leading European purposes and its level should not be called into question for the sake of promoting trade. The link between the positions of two opposing parties, professionals and consumers, requires commitment and work reinforced by the institutions but only on that basis is consistent legislative production. The proposed Regulation on a Common European Sales Law of the sale, the European Commission, set focus to European contract law and raises questions about the relevance and necessity of such uniformity. An instrument for purposes of harmonization of European contract law, that can be applied to all cross-border consumer contracts, similar in all MS's certainly bring many benefits. However, its applicability and usefulness would depend on the level of protection that would provide, compared to the existing national rights. Would an optional instrument ensure the designs of a common law? Moreover, would a binding instrument be the best alternative in that sense? Keywords:

Da intercepção de comunicações, acções encobertas e serviços de informações

The Portuguese Intelligence Services have their operational skills limited due to the grievances caused by the Dictatorship and, in particular, by its political police. With the help of historical elements, and by analyzing current legislation, we demonstrate that such grievances are today unjustified and misplaced, mainly taking into account the Risk Society’s multifaceted threats. Also part of our analysis is the impugnment of the Constitutional Court’s decision nº 413/2015, which pronounced unconstitutional the norm contained in Decree nº 426/XII, of the Republic’s Assembly, article nº 78, nº2, which intended to allow Intelligence Services access to the so-called “metadata”, as well as to tax and banking information. It is our understanding, and we demonstrate it in our dissertation, that should be allowed the access of, not only the above mentioned information, but also the means known as communications interception and undercover operations to the Intelligence Services, as long as properly supervised and inspected.

A presunção de inocência nas fases preliminares do processo penal

This study analyses the principle of presumption of innocence in the preliminary stages of the Portuguese criminal process, its procedural aspect related with the principle of in dubio pro reo and its material aspect concerning the treatment of the defendant during the proceedings. The consequences and manifestations of the principle of presumption of innocence are analysed in the decisions of the closing stages of the preliminary criminal procedure and the application of the principle of in dubio pro reo is analysed in the judgement of sufficiency of evidence for the procedure to continue. It addresses the question of circumstantial evidence, its particular relevance in economic and financial crime, highly organized crime, the grounds for the indictment in general and when the sufficiency of evidence criteria is based on that evidence. It analyses the scope of the principle of presumption of innocence in the application of coercive measures, with reference to the arrest, first interrogation of the accused under detention and reasons for the subsequent dispatch about the measures. The asset assurance measures of preventive seizure and the preventive seizure to ensure confiscation are analysed and principle of presumption of innocence is considered non applicable to those measures.

Has the time for renewables finally arrived? – An event study on EPA’s Clean Power Plan

The recent proposals presented by EPA aimed to reduce the dependency of fossil fuels and to lower current emissions levels, hoping to gradually shift electric generation units to renewable energy sources. Actually, the Final Rule Proposal announcement day exhibited a negative Abnormal Return on Fossil Fuels but the following days had positive Abnormal Returns, mostly due to legislative change perceived by financial markets which eased up implementation periods of the proposed measures in the Final Rule when compared to the Draft Rule. Oppositely, Renewables and Solar Portfolios exhibited negative Cumulative Abnormal Returns over the period surrounding the Final Rule.

O fenómeno da criminalidade itinerante

In an increasingly globalized society, the crime appears as a reality that crosses borders. Globalization has potentiated the emergence of new forms of crime, which have been the subject of more interventional, particularly in terms of political, judicial and police authorities as well as civil society approaches. The media allow rapid expansion of criminal methodologies, which aggregate to the ease of movement of itinerant criminal groups, increases the opportunities for the continuation of the practice of criminal offenses, threatening, increasingly, the tranquility and safety of populations. Criminal organizations are characterized by their complexity, thus contributing to the difficulty in combat, by police and judicial authorities, forcing rapid adaptation to new political and criminal reality, particularly at the level of institutional cooperation, national and international, as exemplified by the creation of the "European Area of Freedom, Security and Justice" and new agencies in the field of police cooperation. It was intended with this paper to answer the central question: Is it possible to define a concept of Itinerant Crime in the European regulatory framework (Police and Judiciary)? To fulfill this aim, we performed the characterization of the concept of itinerant crime including itinerant criminal group, we analyzed the work that is being done by the authorities, police and judiciary, in order to contain the phenomenon. Finally, we studied type of existing cooperation at European level between the Member States and the authorities with responsibilities in this area. At the end, we conclude that efforts are being made towards the enhancement of operational, police and judicial cooperation, between the competent authorities of the European Union by combating this phenomenon. Define, and also proposed, a unique concept of Itinerant Crime, in order to be included in the legal standards, in order to facilitate research, in particular to better fit the itinerant crime and assist the prosecution of offenders.

Relatório de estágio curricular no Centro de Arbitragem Comercial da Câmara de Comércio e Indústria portuguesa

This report is the outcome of an internship that took place in Centro de Arbitragem Comercial da Câmara de Comércio e Indústria Portuguesa and its completion is an essential part of the path towards obtaining the Master’s Degree in Faculdade de Direito da Universidade Nova de Lisboa. This report has been structured in two stages – firstly, the presentation of the Centro de Arbitragem Comercial, focusing on its field of expertise, organic structure, principles and advantages. Then, the description of the activities developed within the Secretariat over the several stages of the arbitration procedure – since the reception of the arbitration requirement in institutional proceedings, terms of reference in ad hoc procedures, through the monitoring of the arbitral tribunal sessions (preliminary hearings, submission of evidence and final allegations) and the notification of the arbitration award. The second stage of this report is related to the description of the functions and powers of the President of Centro de Arbitragem Comercial. Firstly, it defines those powers by analyzing the statutes and rules of proceedings of the Centro de Arbitragem, drawing comparisons between the above mention and the rules of proceedings of others arbitral institutional centres, some of them are international references. The report assesses and describes the presidential powers, such as: configuration and composition of the arbitral tribunal (including arbitrator’s replacements, excuses and refusals); deadline extensions; determination of procedural rules and decision-making on any procedural incidents which arise before the constitution of the arbitral tribunal; definition of arbitration costs and fees; joinder of parties and consolidation of proceedings admission; and appointment of an emergency arbitrator. Lastly, this report analyzes some decisions delivered by the President in the respective institutional procedures which took place in the Centre.

O princípio da confidencialidade na mediação em matéria civil e comercial

The purpose of the following study is to analyze the relevance of the principle of confidentiality concerning mediation on civil and commercial matters developed in Portugal. We will, essentially, try to determine just how pivotal is this principle and how it affects the effectiveness of that method of alternative dispute resolution. We believe it is fundamental to understand the true extent of this principle and its goals, emphasizing the protection given to those who decide to resort to mediation and its impact on this process. For this dissertation, we have based our analysis on the interpretation of the set rules assembled by Law nr 29/2013, April 19th, while combining it with data gathered from other laws and regulations that had also addressed mediation. Furthermore, given the fact that this subject has been regulated by Directive 2008/52/EC, we deem pertinent to include references to other European mediation regulations, namely from Germany, Spain and France. With this study, we have established that, even though the Portuguese mediation law is based on a European Directive, we have determined a more restrictive regulation for the principle of confidentiality. We have concluded that the rules regarding this principle try to preserve, above all, the trust and honesty established during the course of the mediation, while restricting the possibility of using the information disclosed during these sessions on other cases. Additionally, we believe confidentiality is such a distinctive and relevant feature that its legal framework leads us to deem it as a true obstacle to the parties’ private autonomy and their power to determine how the mediation should be carried out.

A type system for value-dependent information flow analysis

Information systems are widespread and used by anyone with computing devices as well as corporations and governments. It is often the case that security leaks are introduced during the development of an application. Reasons for these security bugs are multiple but among them one can easily identify that it is very hard to define and enforce relevant security policies in modern software. This is because modern applications often rely on container sharing and multi-tenancy where, for instance, data can be stored in the same physical space but is logically mapped into different security compartments or data structures. In turn, these security compartments, to which data is classified into in security policies, can also be dynamic and depend on runtime data. In this thesis we introduce and develop the novel notion of dependent information flow types, and focus on the problem of ensuring data confidentiality in data-centric software. Dependent information flow types fit within the standard framework of dependent type theory, but, unlike usual dependent types, crucially allow the security level of a type, rather than just the structural data type itself, to depend on runtime values. Our dependent function and dependent sum information flow types provide a direct, natural and elegant way to express and enforce fine grained security policies on programs. Namely programs that manipulate structured data types in which the security level of a structure field may depend on values dynamically stored in other fields The main contribution of this work is an efficient analysis that allows programmers to verify, during the development phase, whether programs have information leaks, that is, it verifies whether programs protect the confidentiality of the information they manipulate. As such, we also implemented a prototype typechecker that can be found at http://ctp.di.fct.unl.pt/DIFTprototype/.

A indústria de defesa na cooperação Brasil-Portugal

The global dynamics of alliances are strongly determined by the level of cooperation among states. This cooperation can be embodied in various aspects, but the level of defense and security cooperation becomes usually more doctrinal and lasting. By the nature of sovereignty that instills in the bilateral relationship, cooperation at defense and security level can leverages other forms of cooperation. The circumstances and relational balance between Brazil and Portugal seem to evolve towards distancing opportunities, despite they are culturally and institutionally untainted. The economic dynamics, the strategic projection in global sustainability terms, the scale and ambition of Brazilian regional leadership, contrasts with the actual context of Portugal, distancing himself both on the stage where they operate. On the other hand, the historical and cultural roots, the language, the affinity of the peoples of CPLP and some opportunities for economic niches, trend to attract both countries. The condition of Portugal in NATO and Europe, coupled with the ability to export technical and human resources to value-added for Brazil, seems also to become approaching factors. On the balance of these dynamics, there is a set of exogenous factors (economic, external global relations matrix, regional stability, among others), which are not always controlled by any of both countries. These factors call for strong capacity for foresight analysis and decision making, with the inherent risk. There is cooperation vectors that are not apparently penalized by geographic distance, or by the difference of realities. Among these vectors we shall highlight synergies in technological niches, highly tradable goods and, mostly, using the domain of dual technologies. The thirteen niches herein identified are: Monitoring, Navigation, Command and Control, Electronics, Optoelectronics, Communication and remote sensing, Information Technologies, Flight Simulation, Specialized Training, Fiber Optic Sensors, Materials Engineering, Nanotechnology and Communications. Cumulating with identified opportunities in traditional relational framework, both countries are growing (in geography and economic terms) into the Atlantic, making it a central element in the bilateral approach. By being at the same time a growing stage of disputes and which stability tends to be threatened, it will be done an analysis of these synergistic vectors, superimposed on the impact on Atlantic securitization process.

Relatório de estágio curricular realizado na Comissão Nacional de Protecção de Dados

The personal data protection is presented as an indisputably complex and transversal subject and gives an account of this report, a result of curricular internship at the Portuguese Commission for Data Protection. The Commission is the competent authority for the control and supervision of personal data processing. The subject around which this report was prepared is the protection of personal data, analyzed in several aspects. The protection of personal data is, for some time, a topic that raises many concerns, because it is closely linked to fundamental rights constitutionally protected. Fundamental rights inherent in each of us are a result of Article 1 of the Constitution of the Portuguese Republic, in the sense that the dignity of the human person is affirmed as the first value around which the Portuguese legal system will have to be based. In other words, is the dignity of the human person the highest value in the Portuguese legal system. Was the development of societies to the point that we know today that has led to the importance to the personal data of citizens. In modern societies, it is possible to know everything about everyone and the curiosity of others seems not to worry about the injuries that affect the rights of citizens. Where new technologies make excuses for the excessive processing of personal data and where subjects do not seem to bother about their personal data crossing the world, it is important that jurisdictions give value the protection of personal data and the implications of its misuse, in that as these are the mirror of identity each of us and can be used against their owners, causing irreparable damage to the their fundamental rights. Being understood as protection of personal data the possibility of each citizen to decide the use of their data and how they can be used, we can say that its protection depends essentially on each of us, as holders of personal data. Therefore, the protection of our data begins in ourselves.