Computationally-guided thermostabilization of the primary endoglucanase from Hypocrea jecorina for cellulosic biofuel production

The creation of thermostable enzymes has wide-ranging applications in industrial, scientific, and pharmaceutical settings. As various stabilization techniques exist, it is often unclear how to best proceed. To this end, we have redesigned Cel5A (HjCel5A) from Hypocrea jecorina (anamorph Trichoderma reesei) to comparatively evaluate several significantly divergent stabilization methods: 1) consensus design, 2) core repacking, 3) helix dipole stabilization, 4) FoldX ΔΔG approximations, 5) Triad ΔΔG approximations, and 6) entropy reduction through backbone stabilization. As several of these techniques require structural data, we initially solved the first crystal structure of HjCel5A to 2.05 Å. Results from the stabilization experiments demonstrate that consensus design works best at accurately predicting highly stabilizing and active mutations. FoldX and helix dipole stabilization, however, also performed well. Both methods rely on structural data and can reveal non-conserved, structure-dependent mutations with high fidelity. HjCel5A is a prime target for stabilization. Capable of cleaving cellulose strands from agricultural waste into fermentable sugars, this protein functions as the primary endoglucanase in an organism commonly used in the sustainable biofuels industry. Creating a long-lived, highly active thermostable HjCel5A would allow cellulose hydrolysis to proceed more efficiently, lowering production expenses. We employed information gleaned during the survey of stabilization techniques to generate HjCel5A variants demonstrating a 12-15 °C increase in the temperature at which 50% of the total activity persists, an 11-14 °C increase in optimal operating temperature, and a 60% increase over the maximal amount of hydrolysis achievable using the wild type enzyme. We anticipate that our comparative analysis of stabilization methods will prove useful in future thermostabilization experiments.

Relatório de estágio

Este relatório destina-se a descrever as acções desenvolvidas no estágio, do 1º Curso de Mestrado em Enfermagem Comunitária, realizado na Escola Superior de Saúde de Portalegre. A intervenção comunitária, realizou-se na comunidade escolar de Portalegre, tendo tido duas vertentes: Educação sexual na adolescência; Promoção de comportamentos saudáveis nos estilos de vida dos adolescentes. As ações realizadas abordaram a educação sexual na adolescência, com base no diagnóstico de situação e, através da elaboração de um projecto de intervenção, aplicado em metodologia de planeamento em saúde. A intervenção comunitária teve como base a promoção de comportamentos saudáveis nos estilos de vida dos adolescentes, a alunos das escolas de Portalegre do 9º e 12º anos, na área de ciências e tecnologias. Todo este trabalho foi desenvolvido na fase teórica e suportou a nossa intervenção em estágio. Assim, após a conclusão deste diagnóstico, seguiu-se a fase de estabelecimento de prioridades, fixação dos objectivos, selecção de estratégias, preparação da execução e avaliação. Na adolescência, a educação sexual deverá ser orientada para a maturação psicossexual, para os afetos, para a contraceção e planeamento familiar e para a prevenção de doenças. É da responsabilidade dos profissionais de saúde, da escola, mas sobretudo da família, proporcionar um ambiente favorável à aprendizagem e ao diálogo. O realce da educação sexual, deverá ser colocado na igualdade de papéis, na tolerância face ao pluralismo de orientação e de condutas sexuais. Por outro lado, é também na adolescência, que se adquirem hábitos e comportamentos saudáveis. Assim, os profissionais de saúde, conjuntamente com a escola e a família, poderão ter um papel preponderante, capacitando os jovens de competências que lhes permitam optar por comportamentos saudáveis, a nível de alimentação, álcool, uso de drogas, prevenção de infecções sexualmente transmissíveis e cidadania

As forças armadas e a segurança interna

A Constituição da República Portuguesa desde 1982, e após sete revisões constitucionais, faz a distinção vincada entre o que é Segurança Interna e Defesa Nacional. Assim, reserva para a primeira a intervenção das Forças da Segurança e para a segunda a as Forças Armadas, permitindo apenas a intervenção destas na Segurança Interna em estados de excepção, como o estado de emergência e o estado de sítio. Dada a delicadeza do tema optou-se pela construção de um questionário, para que as pessoas possam pronunciar-se de forma isenta sobre o tema, sem constrangimentos institucionais. Optou-se por uma abordagem qualitativa. Com base num guião, entrevistaram-se quatro peritos no assunto de forma a obter respostas sobre a viabilidade da intervenção das Forças Armadas na Segurança Interna, num estado de normalidade democrática. As respostas depois de transcritas constituíram o corpus que foi submetido a análise de conteúdo. Com base nesta análise de conteúdo, a partir dos excertos de discursos codificados nas categorias e subcategorias, procedeu-se à construção dos itens que figurarão numa primeira versão do questionário. A primeira versão do questionário apresenta-se em torno de quatro eixos (categorias), que se dividem em subcategorias: intervenção das Forças Armadas na Segurança Interna; legislação; mudança do paradigma de segurança; e, definição conceptual.

Structural and ultrastructural characteristics of interrenal gland and chromaffin cell of matrinxa, Brycon cephalus Gunther 1869 (Teleostei-Characidae)

This work presents the structure and ultrastructure of the interrenal gland and chromaffin cells, as well as the morphology of the head kidney of Brycon cephalus, the head kidney is composed of fused bilateral lobes located anterior to the swim bladder and ventrolateral to the spinal column, the parenchyma revealed lympho-haematopoietic tissue, melano-macrophage centres, interrenal gland and chromaffin cells. The interrenal gland consisted of cords or strands of cells grouped around the posterior cardinal vein and their branches. Chromaffin cells are found in small groups, closely associated with the interrenal gland and/or under the endothelium of the posterior cardinal vein. So far, the ultrastructural analysis has revealed only one interrenal cell type which contained abundant smooth endoplasmic reticulum and numerous mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Two types of chromaffin cells were observed. The first type was characterized by the presence of vesicles with round, strongly electron-dense granules, which were eccentrically located, Such cells were interpreted as noradrenaline cells, Meanwhile, cells which contained smaller vesicles and electron-lucent granules, with a small halo separating the granule from the vesicular limiting membrane, were identified as adrenaline cells.

Use of Fiber Reinforced Polymer Composite Cable for Post-tensioning Application

Corrosion of steel tendons is a major problem for post-tensioned concrete, especially because corrosion of the steel strands is often hard to detect inside grouted ducts. Non-metallic tendons can serve as an alternative material to steel for post-tensioning applications. Carbon fiber reinforced polymer (CFRP), given its higher strength and elastic modulus, as well as excellent durability and fatigue strength, is the most practical option for post-tensioning applications. The primary objective of this research project was to assess the feasibility of the use of innovative carbon fiber reinforced polymer (CFRP) tendons and to develop guidelines for CFRP in post-tensioned bridge applications, including segmental bridges and pier caps. An experimental investigation and a numerical simulation were conducted to compare the performance of a scaled segmental bridge model, post-tensioned with two types of carbon fiber strands and steel strands. The model was tested at different prestress levels and at different loading configurations. While the study confirms feasibility of both types of carbon fiber strands for segmental bridge applications, and their similar serviceability behavior, strands with higher elastic modulus could improve structural performance and minimize displacements beyond service loads. As the second component of the project, a side-by-side comparison of two types of carbon fiber strands against steel strands was conducted in a scaled pier cap model. Two different strand arrangements were used for post-tensioning, with eight and six strands, respectively representing an over-design and a slight under-design relative to the factored demand. The model was tested under service and factored loads. The investigation confirmed the feasibility of using carbon fiber strands in unbonded post-tensioning of pier caps. Considering both serviceability and overload conditions, the general performance of the pier cap model was deemed acceptable using either type of carbon fiber strands and quite comparable to that of steel strands. In another component of this research, creep stress tests were conducted with carbon fiber composite cable (CFCC). The anchorages for all the specimens were prepared using a commercially available expansive grout. Specimens withstood 95% of the guaranteed capacity provided by the manufacturer for a period of five months, without any sign of rupture.

Adaptive cultural transmission biases in children and nonhuman primates

Comparative and evolutionary developmental analyses seek to discover the similarities and differences between humans and non-human species that illuminate both the evolutionary foundations of our nature that we share with other animals, and the distinctive characteristics that make human development unique. As our closest animal relatives, with whom we last shared common ancestry, non-human primates have beenparticularly important in this endeavour. Such studies that have focused on social learning, traditions, and culture have discovered much about the ‘how’ of social learning, concerned with key underlying processes such as imitation and emulation. One of the core discoveries is that the adaptive adjustment of social learning options to different contexts is not unique to human infants, therefore multiple new strands of research have begun to focus on more subtle questions about when, from whom, and why such learning occurs. Here we review illustrative studies on both human infants and young children and on non-human primates to identify the similarities shared more broadly across the primate order, and the apparent specialisms that distinguish human development. Adaptive biases in social learning discussed include those modulated by task comprehension, experience, conformity to majorities, and the age, skill, proficiency and familiarity of potential alternative cultural models.

Numerical modelling of braided fibres for reinforced concrete

Fire has been always a major concern for designers of steel and concrete structures. Designing fire-resistant structural elements is not an easy task due to several limitations such as the lack of fire-resistant construction materials. Concrete reinforcement cover and external insulation are the most commonly adopted systems to protect concrete and steel from overheating, while spalling of concrete is minimised by using HPFRC instead of standard concrete. Although these methodologies work very well for low rise concrete structures, this is not the case for high-rise and inaccessible buildings where fire loading is much longer. Fire can permanently damage structures that cost a lot of money. This is unsafe and can lead to loss of life. In this research, the author proposes a new type of main reinforcement for concrete structures which can provide better fire-resistance than steel or FRP re-bars. This consists of continuous braided fibre rope, generally made from fire-resistant materials such as carbon or glass fibre. These fibres have excellent tensile strengths, sometimes in excess of ten times greater than steel. In addition to fire-resistance, these ropes can produce lighter and corrosive resistant structures. Avoiding the use of expensive resin binders, fibres are easily bound together using braiding techniques, ensuring that tensile stress is evenly distributed throughout the reinforcement. In order to consider braided ropes as a form of reinforcement it is first necessary to establish the mechanical performance at room temperature and investigate the pull-out resistance for both unribbed and ribbed ropes. Ribbing of ropes was achieved by braiding the rope over a series of glass beads. Adhesion between the rope and concrete was drastically improved due to ribbing, and further improved by pre-stressing ropes and reducing the slacked fibres. Two types of material have been considered for the ropes: carbon and aramid. An implicit finite element approach is proposed to model braided fibres using Total Lagrangian formulation, based on the theory of small strains and large rotations. Modelling tows and strands as elastic transversely isotropic materials was a good assumption when stiff and brittle fibres such as carbon and glass fibres are considered. The rope-to-concrete and strand-to-strand bond interaction/adhesion was numerically simulated using newly proposed hierarchical higher order interface elements. Elastic and linear damage cohesive models were used effectively to simulate non-penetrative 'free' sliding interaction between strands, and the adhesion between ropes and concrete respectively. Numerical simulation showed similar de-bonding features when compared with experimental pull-out results of braided ribbed rope reinforced concrete.

Occurrence of plastic debris in the stomach of the invasive crab Eriocheir sinensis

The Chinese mitten crab is known as a pest causing damage to fishing gears and fish. On the other hand, this highly invasive species is considered a delicacy by Asian migrants and therefore commercially fished and sold in many countries. The ingestion of plastic by the Chinese mitten crab Eriocheir sinensis from the Baltic coastal waters (Poland) and the Tagus Estuary (Portugal) was studied based on stomach content analysis. As many as 13% of the 302 analysed males and females (38.07–89.07 mm carapace width) from both regions, contained microplastic in the form of strands and balls. Most of them were transparent. Ingested plastic particles were identified as fragments of fishing gears. Contamination with plastic may have a negative impact on this species as well as on higher trophic levels feeding on crabs.

Dall'uomo esposto al soggetto esposto: il concetto di interfaccia in alcuni filoni di riflessione sulla tecnica dal Settecento a Marcel Mauss

La nozione di ambiente non è una nozione recente. Al giorno d’oggi il riferimento all’ambiente compare in maniera sempre più diffusa nei dibattiti, nelle opere scientifiche e artistiche, negli spazi di divulgazione e sempre più entra all’interno degli spazi affettivi individuali e all’interno delle teorie e delle prassi politiche individuali e collettive. L’ambiente tanto entra nel campo assiologico, quanto vi entra come elemento fragile, indissociabile dall’azione antropica individuale e collettiva, attraverso ciò che noi riassumiamo con la nozione di crisi ambientale. La crisi ambientale permette di riaprire degli ambiti di valore e di interesse nel presente, allo stesso tempo provoca una reinterpretazione e un riorientamento delle teorie e delle prassi del passato. Dal nostro presente di crisi ambientale, è possibile rintracciare delle figure che, nel passato, hanno rappresentato l’inerenza degli enti con il loro ambiente e, tra questi, dell’uomo con il suo ambiente di vita. Sono due le principali figure attraverso le quali si intende riprendere un rapporto con il nostro passato letto attraverso la crisi ambientale: la figura dell’interfaccia come ciò che demarca un rapporto di inerenza e di separazione tra un interno e un esterno che hanno un rapporto tra loro necessario e tuttavia contingente, e la figura della relazione di “esposizione”, tramite la quale si vuole pensare a forme di soggettività vulnerabili, esposte alle relazioni e ai concatenamenti nei quali sono inserite e che tuttavia non possono che esporre a loro volta le altre soggettività e l’ambiente di vita comune a continue modificazioni. Tramite i concetti di interfaccia e di esposizione abbiamo potuto seguire il prodursi di forme di sapere relative all’inerenza tra forme e modi dell’interiorità rispetto all’ambiente che si sono prodotte nella storia e che sono emerse tramite la provocazione di un passato indotto dalla trasformazione che è il nostro presente di crisi ambientale.

Simulating the aggregation of DNA oligonucleotides

In this work we have studied, by means of Molecular Dynamics simulations, the process of denaturation and self-assembly of short oligonucleotides. Supramolecular ordering of DNA short strands is a promising field which is constantly enriched with new findings. Examples are provided by micellar and fibrils formations and due to the selectivity of DNA bindings, "intelligent" devices have been developed to perform simple logic operations. It is worth to notice that computer simulations of these DNA nanosystems would complement experiments with detailed insight into processes involved in self-assembly. In order to obtain an accurate description of the interactions involved in the complex structure of DNA we used oxDNA, a coarse-grained model developed by Ouldridge. We simulated the melting transition of 4, 6, and 8 base pair sequences. Sequence and length dependence were analyzed, specifically we compared thermodynamic parameters DeltaH, DeltaS and the melting temperature with literature results. Moreover, we have attempted to reproduce liquid crystal ordering of the ultrashort sequence GCCG at relatively high saline concentration, until now only experimentally observed in Bellini's works. We found that our simple model successfully reproduces the experimental phase sequence (isotropic, nematic, columnar) at T= 5 °C as a function of oligonucleotide concentration, and we fully characterized the microscopic structure of the three phases.

Different views on Digital Scholarship: separate worlds or cohesive research field?

This article presents a systematic review of the literature on Digital Scholarship, aimed at better understanding the collocation of this research area at the crossroad of several disciplines and strands of research. The authors analysed 45 articlesin order to draw a picture of research in this area. In the first phase, the articles were classified, and relevant quantitative and qualitative data were analysed. Results showed that three clear strands of research do exist: Digital Libraries, Networked Scholarship and Digital Humanities. Moreover, researchers involved in this research area tackle the problems related to technological uptake in the scholar's profession from different points of view, and define the field in different – often complementary – ways, thus generating the perception of a research area still in need of a unifying vision. In the second phase, authors searched for evidence of the disciplinary contributions and interdisciplinary cohesion of research carried out in this area through the use of bibliometric maps. Results suggest that the area of Digital Scholarship, still in its infancy, is advancing in a rather fragmented way, shaping itself around the above-mentioned strands, each with its own research agenda. However, results from the cross-citation analysis suggest that the Networked Scholarship strand is more cohesive than the others in terms of cross-citations.

Rebooting the constitutional debate: deliberative constitutionalism in the European Union

The aim of my thesis is to investigate the possibility and necessity to rethink a constitutional framework and debate in a transnational polity such as the European Union. My effort focuses on a promising theory called deliberative constitutionalism, which carries on new insights on how democracy and constitutions relate each other. The EU is a unique political entity which poses unanswered questions about its political legitimacy and constitutional foundation, if a Constitution will ever be possible. Going beyond the classical conception of the national and sovereign ‘people’, we keep wondering how citizens may deliberate and discuss about their rights and political communities across borders, in what could be defined as a transnational civic society. The development of the latter brings with it necessary constitutional changes, if not an evolution of constitutionalism itself. Chapter 1 deals with defining the theoretical framework, which develops the distinctiveness of the deliberative constitutional paradigm not only with respect to other more 'classical' models of democracy, but also with respect to other deliberative models that have marked the constructivist debate. Chapter 2 presents a conceptual history of constituent power, mainly studying the evolution of the constitution-sovereignty-constituent power dialectic, up to contemporary theories that explain the negation, separation, union or plurality of a transnational constituent with respect to its national counterparts. Chapter 3 develops the discourse of constitutional pluralism, through its main claims and strands that especially pertain to Neil Walker's (2002, 2016) institutional and epistemic claims. Chapter 4 applies a deliberative constitutionalist framework to the case of the European Union. Through the exposition of DC normative tenets, a form of self-learning process is proposed that can reconcile the heterarchical arrangement of constitutional claims and the new demand for legitimacy, as well as the relationship between European peoples and European citizens.