Follow the red road of triheme cytochromes in Geobacter sulfurreducens

The bacterium Geobacter sulfurreducens (Gs) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membrane in a process designated as extracellular electron transfer. The Gs genome was fully sequenced and a family composed by five periplasmic triheme cytochromes c7 (designated PpcA-E) was identified. These cytochromes play an important role in the reduction of extracellular acceptors. They contain approximately 70 amino acids, three heme groups with bis-histidinyl axial coordination, and share between 57 and 77% sequence identity. The triheme cytochrome PpcA is highly abundant in Gs and is most likely the reservoir of electrons destined for outer surface. In addition to its role in electron transfer pathways this protein can perform e-/H+ energy transduction, a process that is disrupted when the strictly conserved aromatic residue phenylalanine 15 is replaced by a leucine (PpcAF15L). This Thesis focuses on the expression, purification and characterization of these proteins using Nuclear Magnetic Resonance and ultraviolet-visible spectroscopy. The orientations of the heme axial histidine ring planes and the orientation of the heme magnetic axis were determined for each Gs triheme cytochrome. The comparison of the orientations obtained in solution with the crystal structures available showed significant differences. The results obtained provide the paramagnetic constraints to include in the future refinement of the solution structure in the oxidized state. In this work was also determined the solution structure and the pH-dependent conformational changes of the PpcAF15L allowing infer the structural origin for e-/H+ energy transduction mechanism as shown by PpcA. Finally, the backbone and side chain NH signals of PpcA were used to map interactions between this protein and the putative redox partner 9,10-anthraquinone-2,6-disulfonate (AQDS). In this work a molecular interaction was identified for the first time between PpcA and AQDS, constituting the first step toward the rationalization of the Gs respiratory chain.

The making of saudade. National identity and ethnic psychology in Portugal

Roots and rituals.The construction of ethnic identities, Ton Dekker, John Helsloot Carla Wijers editors, p. 267-268; Selected papers of the 6TH SIEF conference on 'Roots & rituals', Amsterdam 20-25 April 1998.

Lithostratigraphy, sequence stratigraphy and depositional setting of the Pliensbachian and Toarcian series in the Lusitanian Basin, Portugal

In the Lusitanian Basin (Central Portugal), the Middle-Upper Liassic series are characterized by an expressive marly limestone accumulation, sediments that were deposited on a homoclinal carbonate ramp. These series belong to the Vale das Fontes, Lemede, S. Gião (and the lateral equivalents Prado and Cabo Carvoeiro Formations) and, partially, to the Póvoa da Lomba Formations. These units, in great part controlled by an accurate ammonite biostratigraphic scale, are organized into two secondorder transgressive-regressive sequences. The first one (SP) is dated of early Pliensbachian/lowermost early Toarcian age; the second (ST) is dated of early Toarcian to early Aalenian.

Multiple sequence alignment correction using constraints

Trabalho apresentado no âmbito do European Master in Computational Logics, como requisito parcial para obtenção do grau de Mestre em Computational Logics

THE MAKING OF THE IMMIGRANT IDENTITY IN THE PRESS: Constructing the Immigrant Imagined Community

A dissertation submitted in partial fulfilment of the requirements for the degree of Master in Arts

Constructing the National Canon in Ireland and Macedonia: The Function of Folklore in the Plays The Countess Cathleen and The Land of Heart’s Desire by William Butler Yeats, and Proud Flesh and The Black Hole by Goran Stefanovski and Their Role in Strengthening National Identity

Erasmus Mundus Masters “Crossways in European Humanities” June 2011

FROM NON-PLACE TO PLACE: A STUDY OF EUROPEAN PUBLIC SPACE AS A SPACE OF IDENTITY

Master Erasmus Mundus Crossways in European Humanities

Ambivalent Islam: the identity construction of Muslims under Portuguese colonial rule

This article wishes to contribute to the study of the historical processes that have been spotting Muslim populations as favourite targets for political analysis and governance. Focusing on the Portuguese archives, civil as well as military, the article tries to uncover the most conspicuous identity representations (mainly negative or ambivalent) that members of Portuguese colonial apparatus built around Muslim communities living in African colonies, particularly in Guinea-Bissau and Mozambique. The paper shows how these culturally and politically constructed images were related to the more general strategies by which Portuguese imagined their own national identity, both as ‘European’ and as ‘coloniser’ or ‘imperial people’. The basic assumption of this article is that policies enforced in a context of inter-ethnic and religious competition are better understood when linked to the identity strategies inherent to them. These are conceived as strategic constructions aimed at the preservation, the protection and the imaginary expansion of the subject, who looks for groups to be included in and out-groups to reject, exclude, aggress or eliminate. We think that most of the inter-ethnic relationships and conflicts, as well as the very experience of ethnicity, are born from this identity matrix.

Compositional and functional analysis of polycomb target chromatin

Dissertation presented to obtain the Ph.D degree in Molecular Biology

Ambivalent Islam: the identity construction of Muslims under Portuguese colonial rule

This article wishes to contribute to the study of the historical processes that have been spotting Muslim populations as favourite targets for political analysis and governance. Focusing on the Portuguese archives, civil as well as military, the article tries to uncover the most conspicuous identity representations (mainly negative or ambivalent) that members of Portuguese colonial apparatus built around Muslim communities living in African colonies, particularly in Guinea- Bissau and Mozambique. The paper shows how these culturally and politically constructed images were related to the more general strategies by which Portuguese imagined their own national identity, both as ‘European’ and as ‘coloniser’ or ‘imperial people’. The basic assumption of this article is that policies enforced in a context of interethnic and religious competition are better understood when linked to the identity strategies inherent to them. These are conceived as strategic constructions aimed at the preservation, protection and imaginary expansion of the subject, who looks for groups to be included in and out-groups to reject, exclude, aggress or eliminate. The author argues that most of the inter-ethnic relationships and conflicts, as well as the very experience of ethnicity, are born from this identity matrix.

"Joãozinho", Hospital de S. João do Porto: A proposal for a brand identity for the new pediatric hospital

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Identity dynamics in non-profit organizations: Ideological and methodological identity orientations

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Nestum vs private labels: A renewed identity for Nestum

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Expressão e purificação da proteína E3 do vírus chikungunya (CHIKV)

RESUMO: O vírus chikungunya (CHIKV) é um vírus de RNA, com invólucro, da família Togaviridae, transmitido por mosquitos Aedes spp. Distribuído por largas regiões de África e Ásia, causa grandes epidemias de artrite grave. A semelhança de sintomas com outras doenças como a dengue e a malária e a persistência de IgM específicas, dificultam o diagnóstico da infeção por CHIKV. A deteção no sangue de E3, uma glicoproteína viral secretada, a incluir num ensaio imunoenzimático poderá melhorar o diagnóstico nos países onde as técnicas de biologia molecular são de difícil acesso. Para testar a utilidade de E3 num ensaio de diagnóstico, esta deverá ser expressa em quantidade, purificada e usada para produção de anticorpos específicos. Para expressar E3 numa forma solúvel, suscetível de ser purificada num único passo cromatográfico sem proteases, recorreu-se à estratégia da fusão com o domínio de ligação à quitina (CBD)-inteína (IMPACT™ System, NEB). A sequência codificadora de E3 foi amplificada a partir de RNA viral, clonada em pTYB21 e expressa em E. coli como uma proteína de fusão insolúvel de 64 kDa. A expressão a 12ºC induzida por IPTG 0,1 mM aumentou a solubilidade de CBD-inteína-E3. A aplicação de lisados celulares em colunas de quitina originou a retenção de CBD-inteína-E3 na matriz. Porém, a autoclivagem da inteína na coluna, induzida com reagentes tiol, foi pouco eficiente e mesmo a proteína E3 separada não eluiu da coluna. E3 foi ainda expressa em E. coli com uma cauda de seis histidinas (E3[His]6) por clonagem no vetor pET28b(+). Lisados celulares aplicados em colunas de níquel permitiram a eluição de uma proteína de 9 kDa, compatível com a massa molecular estimada para E3[His]6, ainda que com outros contaminantes proteicos. A identidade da proteína de 9 kDa será confirmada pela indução de anticorpos com esta preparação e reatividade daqueles com células infetadas com CHIKV.----------------ABSTRACT: Chikungunya virus (CHIKV) is an enveloped, positive strand RNA virus belonging to the family Togaviridae. Transmitted by Aedes spp mosquitoes, CHIKV causes large epidemics of severe arthritogenic disease in Africa and Asia and represents a serious threat in countries where vectors are present. Symptoms similarity with other diseases, e.g. dengue and malaria, along with CHIKV IgM persistence turns accurate CHIKV diagnosis a difficult task in low-income countries. Detection of E3, a small secreted viral glycoprotein, to be included in an immunoenzymatic test was envisaged as a possible improvement in CHIKV diagnosis. To test the diagnostic value of E3, recombinant E3 should be expressed and purified to generate antibodies. In order to express CHIKV E3 in a soluble form amenable to purification by a single step affinity chromatography, the chitin binding domain (CBD)-intein fusion strategy without proteases (IMPACT™ System, NEB) was employed. The E3 coding sequence was amplified from viral RNA, cloned in pTYB21 and expressed in E. coli ER2566 as an insoluble 64 kDa CBD-intein-E3 fusion protein. Solubility was partially achieved by lowering the expression temperature to 12ºC and the inducer (IPTG) concentration to 0.1 mM. Clarified cell lysate loaded onto a chitin column allowed ligation of the fusion protein but the intein-mediated cleavage efficiency was low and E3 failed to elute from the column as demonstrated by SDS-PAGE. E3 was further expressed with a six histidine tag, E3[His]6, employing the pET System (Novagen). E3[His]6 was expressed in E. coli Rosetta (30ºC, 0.4 mM IPTG) as a 9 kDa protein. Soluble cell extracts in 20-40 mM imidazole, applied onto a nickel column and eluted with 500 mM imidazole yielded a protein preparation enriched in the 9kDa protein. The 9 kDa will be used as antigen to generate antibodies that upon reaction with CHIKV infected cells will confirm its identity.

A brand Identity for the Parish of Nossa Senhora de Fátima (in Lisbon)

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics