Structure-function relationships in a glycosyltransferase, a phosphatase and an oxidoreductase

Dissertation presented to obtain the Ph.D degree in BiochemistryEnzyme evolution is often constrained by aspects of catalysis. Mechanistically diverse enzymes evolved from a common ancestor still preserve those structural signatures essential to the core chemistry retained by all members of the superfamily. Indeed, these shared features allow superfamilies to be accurately classified, while derived features allow nested families and subfamilies to be identified in a hierarchical fashion. Accurate classification has helped elucidate mechanisms promoting functional diversification, for example catalytic promiscuity, and protein engineering by rational design. Nowadays, a holistic view of enzymes` regulatory mechanisms and catalytic proficiency is provided by the identification of conserved features of molecular architecture in combination with aspects of reaction dynamics. My work focused on the structural elucidation and analysis of three enzymes: a glycosyltransferase; a phosphatase and an oxidorreductase. “Snapshots” along the reaction coordinate of each enzyme were obtained by combining X-ray diffraction with “cryo-trapping” ligand-binding methods. These were used to characterize the molecular mechanisms involved in substrate recognition and binding. They were also used to distinguish between models proposed for the catalytic mechanisms of each enzyme, and provide insights into enzyme dynamics essential for catalysis and the stereo and regio-selective strategies at work.(...)Apoio financeiro da FCT e do POPH/FSE no âmbito do Quadro Comunitário de Apoio, Bolsa Nº SFRH/BD/23222/2005

Via régia

Via Régia é um trabalho sobre a violência da linguagem na obra de Peter Handke. "R.S.I." ou "O Discurso da Histérica", a partir de Self-Accusation de Peter Handke é uma performance solo com a duração de 27 minutos; ABSTRACT: Via Régia is a work about verbal violence in Peter Handke's drama. "R.S.I." or "O Discurso da Histérica", is a 27-minute long solo performance based on Self-Accusation by Peter Handke

Synthesis of Reactive Nanoparticles for Coatings with Enhanced Chemical Resistance

Trabalho apresentado em BIT's 5th Annual World Congress of Advanced Materials, 6-8 junho de 2016, Chongqing, ChinaN/

Social Perceptions of Nonhumans in Tombali (Guinea-Bissau, West Africa): a contribution to chimpanzee (Pan troglodytes verus) conservation

Rainforest biodiversity is particularly vulnerable to loss, since the distribution of forests is limited and the vertebrate species that live within these forests have a limited potential to re-colonize deforested areas, especially when their abundance declines to critical levels. Guinea-Bissau (West Africa) is experiencing significant loss of habitats and species diversity; as such, the establishment of an effective conservation programme is urgent in its remaining forested areas. Despite six legislated protected areas, Guinean forests and their wildlife are not safe in reality. This lack of on-the-ground protection is the case for Cantanhez National Park (Tombali region), where this research took place. The park was established in 2007 to protect remnant forests containing unique and endemic Guinean biodiversity, such as the endangered West African chimpanzees (Pan troglodytes verus). Local inhabitant’s attitudes towards protected areas and associated externallydriven conservation programmes are seldom examined in depth in relation to understanding the drivers (livelihood, socio-cultural, and local) of perceptions, which makes conservation problematic. Understanding attitudes to animals, habitats and livelihood risks were the focus of this project, specifically in order to assess perceptions of chimpanzees. Chimpanzees are currently suffering catastrophic declines due to human actions across Africa. Thus a focus on understanding, managing and enhancing people’s perceptions and attitudes towards this species could be vital to its long-term survival. The theoretical approach is based upon (i) examining the construct of sociozoologic scales in this specific socio-cultural context, (ii) elucidating issues in humanwildlife interaction (e.g. conflict such as crop-raiding and positive such as ecotourism potential), (iii) local economies (i.e. level of dependency on forest resources), and (iv) understanding people’s expectations about the future of the National Park as a potential constraint or opportunity for their welfare and livelihoods. Quantitative and qualitative methods were combined to approach these questions. 17 The Guinean sociozoologic scale of Cantanhez clearly divides vertebrate species into (i) “tame”, considered good (e.g. gazelles) and (ii) “hazardous”, considered bad (e.g. hyaenas). Chimpanzees lay exactly in the midpoint. They are considered humans’ close relatives; however, they “misbehave” as astute crop thieves sufficiently to be perceived as a competitor for resources. Since chimpanzees are also seen as very similar to humans, their meat consumption is taboo, which adds the potential for protection. Gender and religion both influence the way locals perceive of and relate to chimpanzees. Women and Muslims tend to be more negative towards this species and the protected area than are men and non-Muslims. Women never exhibited positive attitudes in relation to the protected area, while men appeared to be more engaged with “capitalized” principles, with some awareness about the importance chimpanzees might have in catalyzing the National Park and local economy. This study highlights the need for a management plan to mitigate crop-raiding and the development of sustainable strategies that provide livelihood benefits for both men and women, addressing their distinct needs, outside the protected area

Os níveis fundacionais da Idade do ferro de Mesas do Castelinho (Almodôvar): os contextos arqueológicos na (re) construção do povoado

Tese de mestrado, Arqueologia, Universidade de Lisboa, Faculdade de Letras, 2011Analisam-se e discutem-se os dados da cultura material (arquitectura e espólio) decorrentes de várias campanhas de escavação arqueológica no sítio da II Idade do Ferro de Mesas do Castelinho (Almodôvar, Baixo Alentejo), de acordo com critérios de ordem estratigráfica. Confirma-se e afina-se a cronologia sidérica deste povoado, entre os finais do séc. V. a.C. e o séc. II a.C., ao longo de três fases de ocupação. Caracteriza-se o sítio do ponto de vista do seu estabelecimento - numa fronteira geográfica, entre os contrafortes da serra algarvia e a peneplanície alentejana, mas em altimetria pouco marcada e num quadro de localização junto a uma via natural de comunicação, com recurso à edificação de um perímetro amuralhado que faz desenvolver as actividades do povoado, de cariz essencialmente rural, no seu interior. Ao mesmo tempo, contrapõe-se esta informação com os conhecimentos acerca de outros sítios desta área geográfica, apresentando os pontos de convergência e de divergência entre uns e outros, procurando demonstrar as muitas possibilidades de modelos de povoamento e evidenciar os sinais da continuidade, patentes no acervo artefactual e na diversidade das localizações e na dinâmica arquitectónica.Resumen Se analizan y discuten los datos de la cultura material (arquitectura y espolio) decurrentes de varias campañas de excavación arqueológica en el sitio de la II Edad del Hierro de Mesas do Castelinho (Almodôvar, Bajo Alentejo, Portugal), de acuerdo con criterios de orden estratigráfico. Se confirma y apura la cronología sidérica de este poblado, entre los finales del s. V. a.C. y lo s. II a.C., en tres fases de ocupación. Se caracteriza el sitio desde el punto de vista de su asentamiento – en una frontera geográfica, entre los contrafuertes de la Serra del Algarve y la peneplanície alentejana, pero en altimetria poco marcada y en un cuadro de localización junto a un vía natural de comunicación, con el recurso a la edificación de un perímetro amurallado que hace desarrollar las actividades del poblado, de fondo esencialmente rural, en su interior. A la vez, se compara esta información con los conocimientos de otros sitios de este área geográfico, presentando los puntos de convergencia y de divergencia entre unos y otros, procurando demostrar las muchas posibilidades de modelos de poblamiento y evidenciar los señales de la continuidad, presentes en el conjunto artefactual y en la diversidad de los emplazamientos y en la dinámica arquitectónica.Abstract Material culture data (architecture and artefactual assemblages) from several archaeological excavation campaigns in the II Iron Age site of Mesas do Castelinho (Almodôvar, Baixo Alentejo, Portugal) are analyzed and discussed, according to statigraphic criteria. Its chronology is confirmed and redefined, between the late Fifth and Second centuries B. C., all along three occupation phases. The site is characterized in terms of its establishment – in a geographic frontier, between the Serra do Algarve hillforts and the Alentejo Plains, but in a modest shaped altimetry and in a location near a natural communication route, with a fortified wall perimeter structure, which organize the inner activities of a rural community. At the same time, this information is counter parted with data from other sites of this geographic area, presenting the convergence and divergence points between ones and an others, in the pursuit to demonstrate the many possibilities of settlement models and to put in light the permanence signs, present in their artefactual assemblages, in the location variability and in the architectural dynamics

exploring a local setting in Portugal

In Portugal, the majority of immigrants come from Portuguese-speaking countries. Among children, overweight and obesity are serious public health concerns, but a few studies include children with immigrant background. This study aimed to estimate the prevalence of overweight and obesity and risk of abdominal obesity in school-age children and to explore potential determinants, considering the origin of the children’s parents (both mother and father are non-Portuguese, only one Portuguese, and both are Portuguese). A cross-sectional study included a random sample of 789 children (5–11 years old, 49.2% of males and 50.8% female) attending public primary schools in Barreiro, district of Setubal, Portugal. Fieldwork occurred from 20th April to 3rd July 2009. Data concerning socioeconomic, eating habits, and sports outside school were obtained through a questionnaire applied to the children’s person in charge. Anthropometric measures were collected by certified technicians. One-third of the children presented overweight and obesity (33.7%, 95% CI [30.0; 37.7]) and risk of abdominal obesity (16.4%, 95% CI [13.6; 19.7]) without differences according to parents’ origin. Children with immigrant background presented higher consumption of low-priced, high-sugar, and high-fat foods, with a worse situation for girls. Females from both non-Portuguese parents also practiced less sports outside school than those where one or two parents are Portuguese. Thus, promoting and monitoring a healthy diet and physical activity in this group should be prioritized in this local setting.publishersversionpublishe

The role of IFNγ in higher brain function: in health and under chronic stress

Tese de Doutoramento em Ciências da SaúdeThe neuroimmunology field is at an exciting stage due to a set of revolutionary discoveries challenging the now old-fashioned dogma of the brain being “protected” from the peripheral immune system action. Immune components such as T lymphocytes and the cytokines they produce, once regarded as detrimental to the brain, are now considered integrant parts of the healthy nervous system since their regulated actions control immune surveillance but also modulate higher brain functions. The cytokine interferon-gamma (IFNγ), produced mainly by T lymphocytes, is a potent pro-inflammatory molecule, whose levels are altered in many neuropsychiatric and neurodegenerative diseases. Though studies assessing the effects of this cytokine, when administered into the brain, have shown that it affects different cellular and synaptic mechanisms underlying behavioural dimensions, it is still unclear whether this is a collateral damage of the inflammatory response or if IFNγ indeed plays a role in the modulation of non-pathological brain function. As so, we sought to explore the role of this cytokine in the modulation of brain function in physiological conditions and also after exposure to chronic stress – a paradigm known to trigger the development of psychiatric complications and also accelerate neurodegenerative processes. In the first part of the thesis (2nd Chapter) we demonstrate that, in a healthy brain, the absence of IFNγ enhances dorsal hippocampus plasticity and associated cognitive function. At the structural level, an enlargement of the dorsal hippocampus volume contrasted with the absence of alterations observed in the ventral part, highlighting that the effects of this cytokine are more selective for cognitive behaviours. Moreover, the absence of this cytokine amplifies neuroplastic phenomena in the dorsal hippocampus, namely neurogenesis, size of neuronal dendritic arborisations and presynaptic functioning, most likely contributing for the enhanced cognitive performance. On the 3rd chapter, we demonstrate that there are gender-differences on the behavioural phenotype of IFNγ KO mice, and discuss the possible association of estrogen and the IFNγ expression in the central nervous system. In the following chapter (4th Chapter) we describe the optimisation of a chronic unpredictable stress (CUS) paradigm for use in C57BL/6 mice, a strain with higher resistance to stress. This mice model of stress-related disorders exhibits, beyond the stress-related neuroendocrine and behavioural alterations, mild changes in thymic cellular populations and relevant splenic myeloid cellular alterations, with an increased number of neutrophils as the most striking change. At last (5th Chapter), we discuss the contributory role of IFNγ for the development of the immune maladaptive response to chronic stress. By submitting mice to the optimized CUS protocol, it was observed that mRNA levels of Ifnγ are elevated in the brain, specifically in the medial prefrontal and orbitofrontal cortices. Moreover, exposure to chronic stress leads to an increase of the adrenergic innervation of the spleen as to alterations on the percentage of neutrophils and monocytes/macrophages populations in the spleen. Importantly, the absence of this cytokine blunts the stress-related changes on these cell populations in the spleen. The recognition of the proinflammatory cytokine – IFNγ, as a negative regulator of hippocampal plasticity and associated cognitive function, together with its contributory role for the stress-related immune dysfunction, suggests that this cytokine may articulate the complex network that underlies the inflammatory component of neuropsychiatric disorders.A área da neuroimunologia está a atravessar uma fase excitante devido a um conjunto de descobertas revolucionárias que desafiam o seu agora antiquado dogma que visiona o cérebro como um órgão “protegido” da ação do sistema imunitário periférico. Componentes imunitários, tal como os linfócitos T e respetivas citocinas que estes produzem, outrora vistos apenas como prejudiciais para o cérebro, são agora considerados partes integrantes do sistema nervoso, uma vez que a sua ação regulada controla a vigilância imunitária mas também a modulação de funções cerebrais superiores. O interferão gama (IFNγ), uma citocina produzida principalmente por linfócitos T, é uma molécula proinflamatória cujos níveis estão alterados em diversas doenças neuropsiquiátricas e neurodegenerativas. Apesar de estudos demonstrarem que a administração desta citocina no cérebro afeta diferentes mecanismos celulares e sinápticos que estão na base de dimensões comportamentais, não é ainda claro se este efeito é um dano colateral da resposta inflamatória ou se o IFNγ tem de fato um papel na modulação da função do cérebro num contexto não patológico. Como tal, iremos aqui explorar o papel desta citocina na modulação de funções cerebrais em condições fisiológicas como também após exposição ao stress crónico – um paradigma que desencadeia o desenvolvimento de complicações psiquiátricas e acelera processos neurodegenerativos. Na primeira parte da presente tese (2º Capítulo) nós demonstramos que a ausência de IFNγ no cérebro saudável leva a uma melhoria da plasticidade do hipocampo dorsal e função cognitiva associada. Ao nível estrutural, um aumento do volume do hipocampo dorsal contrasta com a ausência de alterações volumétricas na parte ventral, sublinhando assim que esta citocina afeta seletivamente o comportamento cognitivo. A ausência desta citocina leva também a uma amplificação dos fenómenos neuroplásticos do hipocampo dorsal, nomeadamente da neurogénese, o tamanho da arborização dendrítica neuronal e o funcionamento pré-sináptico, contribuindo, muito provavelmente, para a melhoria da performance cognitiva. No 3º capítulo, nós demonstramos que existem diferenças entre géneros no fenótipo comportamental dos murganhos IFNγ KO, e discutimos a possível associação entre o estrogénio e a expressão de IFNγ no sistema nervoso central. No capítulo seguinte (4º Capítulo) descrevemos a otimização de um paradigma de stress crónico imprevisível (CUS) para murganhos C57BL/6, uma estirpe que apresenta uma maior resistência ao stress. Este modelo de murganho de disfunção associada ao stress apresenta para além das alterações neuroendócrinas e comportamentais relacionadas com o stress, alterações moderadas nas populações celulares do timo e importantes alterações celulares mielóides no baço, sendo o aumento de neutrófilos a alteração mais impressionante. Por último (5º Capítulo), discutimos o papel contributório desta citocina para o desenvolvimento da resposta imunitária maladaptativa ao stress crónico. Após expor murganhos ao protocolo otimizado de CUS foi observado um aumento nos níveis de mRNA de Ifnγ, mais especificamente nos córtices préfrontal medial e orbitofrontal. Para além disso, a exposição ao stress crónico leva a um aumento da inervação adrenérgica do baço assim como a alterações nas percentagens das populações de neutrófilos e monócitos/macrófagos no baço. A ausência de IFNγ preveniu as alterações induzidas pelo stress crónico nestas populações celulares do baço. O reconhecimento da citocina pró-inflamatória – IFNγ, como um regulador negativo da plasticidade hippocampal e função cognitiva associada, juntamente com o seu papel contributório para a disfunção imunitária associada com o stress sugere que esta citocina poderá articular a rede complexa que está na base da componente inflamatória das doenças neuropsiquiátricas.This work was funded by the European Commission (FP7): “SwitchBox” (Contract HEALTH-F2- 2010-259772) and co-financed by the Portuguese North Regional Operational Program (ON.2 – O Novo Norte) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER). Susana Isabel Gonçalves Monteiro was supported by a PhD fellowship from the Portuguese Science Foundation (FCT)/MEC with the reference SFRH/BD/69311/2010

Use of immunohistochemical techniques to investigate museum specimens of neurological disease

Mestrado em Métodos BiomolecularesOs espécimes de museu são um recurso valioso tanto para o ensino como para a investigação, mas geralmente têm sido utilizados para demonstrar a morfologia macroscópica. O objectivo da presente dissertação é determinar se modernas técnicas neuropatológicas, tal como a histologia e a imunohistoquímica, podem ser aplicadas com sucesso a espécimes de museu preservados por períodos superiores a 50 anos. Poderá ser esperado que a qualidade do material histológico recuperado de espécimes de museu seja afectada por vários factores. Estes incluem a idade do espécime, o tipo e a duração da fixação/preservação, condições do acondicionamento do espécime, informação relativa ao paciente e o período post-mortem. É importante compreender como é que os métodos de preservação mudaram com o passar dos tempos, consequentemente uma breve revisão desta história foi feita. É descrito o processo de selecção dos espécimes para este estudo e é apresentada uma investigação às características das diferentes soluções de montagem. Grande parte desta dissertação refere-se ao desenvolvimento de formas que permitam a aplicação de técnicas histológicas modernas a espécimes de museu. É demonstrado que uma pós-fixação em formaldeído 10% e um ajuste do pH da solução corante de eosina para um intervalo entre 4.8 e 5.0, são especialmente importantes em tecidos envelhecidos. Foi obtida uma coloração imunohistoquímica satisfactória utilizando o sistema EnVision+ (Dako), um polímero acoplado com HRP, com o anticorpo anti-neurofilamento (Dako). No entanto, apenas alcançou-se resultados satisfatórios com cortes de crióstato, os quais, aparentemente, produzem melhor coloração imunohistoquímica quando comparados com cortes de tecidos fixados em formaldeído e incluídos em parafina (FFPE), sugerindo assim, que o processamento em solventes orgânicos e as altas temperaturas da parafina derretida alteram a conformação do antigénio impedindo a detecção imunohistoquímica. Um estudo detalhado de três casos datados de 1953, 1954 e 1955 confirma que técnicas modernas (incluíndo imunohistoquímica) podem ser utilizadas em tecidos envelhecidos a um ponto de serem consideradas diagnosticamente válidas. Este estudo demonstra que com ajustes cuidados aos protocolos é possível obter-se notáveis resultados histológicos de alta qualidade em tecidos que foram preservados por muitos anos. Confirma também, que espécimes de museu representam um valioso recurso para ensino e para a investigação a um nível ultraestrutural. ABSTRACT: Museum Specimens are a valuable resource both for teaching and research but have generally been used to show the gross morphology. The aim of this dissertation is to determine if modern neuropathological techniques, such as histology and immunohistochemistry, can be applied successfully in museum specimens that have been preserved for periods in excess of 50 years. It may be expected that many factors could affect the quality of histological material retrieved from museum specimens. These include the age of the specimen, type and length of fixation/preservation, storage conditions, patient information and post-mortem period. It is important to understand how preservation methods had changed over time, and therefore, the complex history of “potting” is reviewed. The process of selecting specimens for this study is described and an investigation into the characteristics of mounting solutions is presented. The major part of the dissertation concerns the development of ways in which to apply modern histological techniques to museum specimens. It is shown that post-fixation in formaldehyde 10% and an adjustment of the eosin stain solution to a pH ranging between 4.8 and 5.0 are especially important in older tissues. Satisfactory immunohistochemistry staining was obtained using EnVision + (Dako) system, a HRP labeled polymer, with anti-neurofilament antibody (Dako). Nevertheless, successful results were only achieved with cryostat sections, as they appeared to produce better immunohistochemistry staining when compared to formalin-fixed paraffin-embedded (FFPE) sections, suggesting that processing in organic solvents and high temperatures of molten paraffin alters the conformation of the antigen hampering immunohistochemistry detection. A detailed study of three cases from 1953, 1954 and 1955 confirms that modern techniques (including immunohistochemistry) can be used in aged tissue to the point where they are useful diagnostically. This study shows that with careful adjustment to protocols it is possible to achieve remarkably high quality histological results in tissues that have been preserved for many years. It confirms that specimens in museums represent a valuable resource for teaching and research at an ultrastructural level

The association between personal belief in a just world, voice and burnout: A cross-sectional study in a sample of national guard police officers

Voice is crucial both for the organizations, which benefit from new insights, and for the workers, who are able to express their concerns and ideas, having a great impact on their mental health (Cox et al., 2006). Actually, individuals who are afraid to voice are likely to show high levels of burnout (Hammond et al., 2019). In addition, continued exposure to job stressors put workers at risk of developing burnout (Penalba et al., 2008), as the case of the Republican National Guard Officers. The present study examined two parallel mediation models. The first model was a replication of a study conducted by Cheng et al. (2020), namely the association between personal belief in a just world (BJW) and voice, mediated by perceived voice efficacy (PVE) and perceived voice risk (PVR). The second model aimed to test the association between personal BJW and burnout (exhaustion and disengagement), mediated by PVE and PVR. Self-reported data was collected from National Guard Police Officers (N = 475). As expected, the results showed a positive association between personal BJW and employee voice, and a negative association between personal BJW and both dimensions of burnout. Additionally, the mediating effect of PVE was significant in both models. However, the mediating effect of PVR was only significant to the second model. These findings add knowledge to the existing literature on the fields of social psychology of justice, organizational citizenship behavior and mental health, and highlight the importance of reinforce organizational justice, by promoting BJW and voice behaviors.O comportamento de voz é fundamental quer para as organizações, que beneficiam de novos insights, quer para os trabalhadores, tendo um grande impacto na sua saúde mental (Cox et al., 2006). De facto, indivíduos com receio de adotar comportamentos de voz têm maior probabilidade de mostrar maiores níveis de burnout (Hammond et al., 2019). Ainda, a exposição continuada a stressores do trabalho coloca os trabalhadores em risco de desenvolverem burnout (Penalba et al., 2008), como é o caso dos agentes da Guarda Nacional Republicana. O presente estudo examinou dois modelos de mediação paralela. O primeiro modelo foi uma replicação do estudo de Cheng et al. (2020), nomeadamente a associação entre a crença num mundo justo (CMJ) pessoal e a voz, mediada pela perceção de eficácia de voz (PEV) e de risco (PRV). O segundo modelo teve como propósito testar a associação entre a CMJ pessoal e o burnout (distanciamento e exaustão), mediada pela PEV e pela PRV. Foram analisadas medidas de autorrelato de agentes da Guarda Nacional Republicana (N=475). Conforme esperado, os resultados mostraram uma associação positiva entre a CMJ pessoal e a voz do trabalhador, e uma associação negativa entre a CMJ pessoal e as duas dimensões do burnout. Ainda, o efeito da mediadora PEV foi significativo nos dois modelos. No entanto, o efeito mediador da PRV apenas foi significativo no segundo modelo. Estes resultados acrescentam conhecimento à literatura existente e sublinham a importância de reforçar a justiça organizacional, através da promoção da CMJ e dos comportamentos de voz