João Seixas

CORM-3 Reactivity toward Proteins: The Crystal Structure of a Ru(II) Dicarbonyl-Lysozyme Complex

CORM-3, [fac-Ru(CO)(3)Cl(κ(2)-H(2)NCH(2)CO(2))], is a well-known carbon monoxide releasing molecule (CORM) capable of delivering CO in vivo. Herein we show for the first time that the interactions of CORM-3 with proteins result in the loss of a chloride ion, glycinate, and one CO ligand. The rapid formation of stable adducts between the protein and the remaining cis-Ru(II)(CO)(2) fragments was confirmed by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES), Liquid-Chromatography Mass Spectrometry (LC-MS), Infrared Spectroscopy (IR), and X-ray crystallography. Three Ru coordination sites are observed in the structure of hen egg white lysozyme crystals soaked with CORM-3. The site with highest Ru occupancy (80%) shows a fac-[(His15)Ru(CO)(2)(H(2)O)(3)] structure.

Reactive Oxygen Species Mediate Bactericidal Killing Elicited by Carbon Monoxide-releasing Molecules

CO-releasing molecules (CO-RMs) were previously shown by us to be more potent bactericides than CO gas. This suggests a mechanism of action for CO-RM, which either potentiates the activity of CO or uses another CO-RM-specific effect. We have also reported that CORM-2 induces the expression of genes related to oxidative stress. In the present study we intend to establish whether the generation of reactive oxygen species by CO-RMs may indeed result in the inhibition of bacterial cellular function. We now report that two CO-RMs (CORM-2 and ALF062) stimulate the production of ROS in Escherichia coli, an effect that is abolished by addition of antioxidants. Furthermore, deletion of genes encoding E. coli systems involved in reactive oxygen species scavenging, namely catalases and superoxide dismutases, potentiates the lethality of CORM-2 due to an increase of intracellular ROS content. CORM-2 also induces the expression of the E. coli DNA repair/SOS system recA, and its inactivation enhances toxicity of CORM-2. Moreover, fluorescence microscopy images reveal that CORM-2 causes DNA lesions to bacterial cells. We also demonstrate that cells treated with CORM-2 contain higher levels of free iron arising from destruction of iron-sulfur proteins. Importantly, we show that CO-RMs generate hydroxyl radicals in a cell-free solution, a process that is abolished by scavenging CO. Altogether, we provide a novel insight into the molecular basis of CO-RMs action by showing that their bactericidal properties are linked to cell damage inflicted by the oxidative stress that they are able to generate.

A novel carbon monoxide-releasing molecule fully protects mice from severe malaria.

ABSTRACT Severe forms of malaria infection, such as cerebral malaria (CM) and acute lung injury (ALI), are mainly caused by the apicomplexan parasite Plasmodium falciparum. Primary therapy with quinine or artemisinin derivatives is generally effective in controlling P. falciparum parasitemia, but mortality from CM and other forms of severe malaria remains unacceptably high. Herein, we report the design and synthesis of a novel carbon monoxide-releasing molecule (CO-RM; ALF492) that fully protects mice against experimental CM (ECM) and ALI. ALF492 enables controlled CO delivery in vivo without affecting oxygen transport by hemoglobin, the major limitation in CO inhalation therapy. The protective effect is CO dependent and induces the expression of heme oxygenase-1, which contributes to the observed protection. Importantly, when used in combination with the antimalarial drug artesunate, ALF492 is an effective adjunctive and adjuvant treatment for ECM, conferring protection after the onset of severe disease. This study paves the way for the potential use of CO-RMs, such as ALF492, as adjunctive/adjuvant treatment in severe forms of malaria infection.

Inhibition of nitric oxide-stimulated vasorelaxation by carbon monoxide-releasing molecules

Objective—Carbon monoxide (CO) is a weak soluble guanylyl cyclase stimulator, leading to transient increases in cGMP and vasodilation. The aim of the present work was to measure the effect of CO-releasing molecules (CORMs) on the cGMP/nitric oxide (NO) pathway and to evaluate how selected CORMs affect NO-induced vasorelaxation. Methods and Results—Incubation of smooth muscle cells with some but not all of the CORMs caused a minor increase in cGMP levels. Concentration-response curves were bell-shaped, with higher CORMs concentrations producing lower increases in cGMP levels. Although exposure of cells to CORM-2 enhanced cGMP formation, we observed that the compound inhibited NO-stimulated cGMP accumulation in cells and NO-stimulated soluble guanylyl cyclase activity that could be reversed by superoxide anion scavengers. Reactive oxygen species generation from CORMs was confirmed using luminol-induced chemiluminescence and electron spin resonance. Furthermore, we observed that NO is scavenged by CORM-2. When used alone CORM-2 relaxed vessels through a cGMP-mediated pathway but attenuated NO donor-stimulated vasorelaxation. Conclusion—We conclude that the CORMs examined have context-dependent effects on vessel tone, as they can directly dilate blood vessels, but also block NO-induced vasorelaxation.

Dinâmicas sociogeográficas e políticas na Área Metropolitana de Lisboa em tempos de crise e de austeridade

Este artigo, reconhecendo a relevância das dimensoes territoriais e urbanas para uma melhor interpretacao da atual crise e seus principais impulsionadores, bem como das suas consequencias e reaccoes sociopoliticas, analisa a evolucao recente da Area Metropolitana de Lisboa, perante tempos de crise economica e de politicas de austeridade. O artigo propoe duas conclusoes, a merecer estudo adicional com o objectivo de uma sua possivel teorizacao mais alargada: 1) podem distinguir-se fases distintas da crise e uma correlacao entre estas, as politicas de austeridade implementadas e a correspondente ampliacao dos efeitos da crise nos tecidos socioeconomicos territoriais; 2) verifica-se o aumento de um confronto de natureza estruturante entre politicas top-down de escalas europeia e nacionais e as dinâmicas sociopoliticas de escalas mais locais e mesmo bottom-up, redesenhando-se os quadros geopoliticos territoriais por formas crescentemente distintas e mais complexas; de divergencias, aproximacoes e interseccoes de base multi-escalar.

Criatividade e governança na cidade: a conjugação de dois conceitos poliédricos e complementares

This text was based on a research project that observed and interpreted forms and flows of socio-political and cultural governance associated to urban creative dynamics. It results from a theoretical, critical reflection focused on basic concepts – namely, creativity, vitality and governance in the city – and from an empirical projection of such perspectives in three metropolitan territories – Lisbon, Sao Paulo and Barcelona. Different perspectives are identified regarding the concepts and respective dynamics offs complementarity and connectivity among these; and also the structuring and metabolic conditions for sustained development of creativity in the contemporary city, whether with regard to spatial/geographical configurations, or to associated socio-cultural and economic spheres. Furthermore, forms of public and private promotion and support for urban creativity are raised, leading to discussion of political strategies and governance processes for its potentiation.

Political evolution in the Lisbon of the digital era. Fast urban changes, slow institutional restructuring and growing civic pressures

ABSTRACT This article analyses the current outlook for governance and the structuring of urban political spaces in the city of Lisbon, in the face of a combination of transformative and significantly disruptive dynamics: from the far-reaching and dizzyingly fast changes underway in the city’s urban structure and its regional system, through the gradual reorganisation of its institutional and governmental structure, to the growing pressure from emerging urban movements. The article concludes with a critical analysis of the equally rapid redefinition of the main elements of disjunction as well as of the possible recomposition of the city’s political spaces.

Study of the interactions of bovine serum albumin with a molybdenum(II) carbonyl complex by spectroscopic and molecular simulation methods

Therapy with inhaled carbon monoxide (CO) is being tested in human clinical trials, yet the alternative use of prodrugs, CO-Releasing Molecules (CORMs), is conceptually advantageous. These molecules are designed to release carbon monoxide in specific tissues, in response to some locally expressed stimulus, where CO can trigger a cytoprotective response. The design of such prodrugs, mostly metal carbonyl complexes, must consider their ADMET profiles, including their interaction with transport plasma proteins. However, the molecular details of this interaction remain elusive. To shed light into this matter, we focused on the CORM prototype [Mo(η5-Cp)(CH2COOH)(CO)3] (ALF414) and performed a detailed molecular characterization of its interaction with bovine serum albumin (BSA), using spectroscopic and computational methods. The experimental results show that ALF414 partially quenches the intrinsic fluorescence of BSA without changing its secondary structure. The interaction between BSA and ALF414 follows a dynamic quenching mechanism, indicating that no stable complex is formed between the protein Trp residues and ALF414. The molecular dynamics simulations are in good agreement with the experimental results and confirm the dynamic and unspecific character of the interaction between ALF414 and BSA. The simulations also provide important insights into the nature of the interactions of this CORM prototype with BSA, which are dominated by hydrophobic contacts, with a contribution from hydrogen bonding. This kind of information is useful for future CORM design.

Mudar de casa em Lisboa: perfis socioeconômicos da recente mobilidade residencial

As mudancas que tem ocorrido recentemente na cidade de Lisboa estao associadas, em grande medida, as dinâmicas de mobilidade residencial, suas causas e consequencias. A escolha da area de residencia, determinada por fatores economicos, sociais, culturais e psicologicos, resulta numa alteracao gradual da estrutura social da cidade e dos padroes de ocupacao do territorio. Este artigo, partindo dos dados de um inquerito realizado aos municipes de Lisboa entre 2016 e 2017, procura contribuir para melhor compreender essas mudancas, analisando as dimensoes territorial e temporal da mobilidade, assim como os perfis socioeconomicos dos residentes mais antigos e dos novos. Perante a diversidade de experiencias de mobilidade residencial, recorreu-se a analise multivariada para definir cinco perfis com caracteristicas sociais distintas.

Contemporary City and Plural Knowledge: Reframing Urban Planning

Urbanization stands amongst the most significant global trends of the 21st century, with cities accounting already for 70% of global wealth creation, 60% of total energy consumption, 70% of greenhouse gas emissions and 70% of global waste (UN Habitat 2015). These numbers are telling of both the vigor of cities and the challenges they face. The unprecedented levels of urbanization worldwide over the last decades have fuelled global economic growth while raising a number of social, environmental and spatial concerns that are increasingly shared by all cities, rather than unique to particular sites, thanks to globalization. Sustainability, socio-territorial cohesion and sustainable urban development became mots d’ordre and the city emerged as the critical building block for a more sustainable future. In this chapter, we consider the imperatives of sustainability and how they impact the urban planning field. In light of the United Nations declaration of the Decade of Education for Sustainable Development (DESD 2004–2014), we look into the requirements of higher education for sustainable urban planning and development (SUPD) and examine how sustainability concepts and principles are shaping the curricula of academic programs with a focus on the built environment. Supported by topical literature on sustainability and urban planning, the content analysis of a sample of 10 top world academic programs dealing with the built environment—covering the areas of architecture, urban design, urbanism and city planning—helped to identify the topical themes/fields for conceptualization and action, as well as the key skills which urban professionals need to master, in order to advance SUPD. Findings support the idea that, over the last decades, the imperatives of sustainability for urban planning have contributed to a growing convergence between the social sciences and the spatial planning field.