Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in E. coli.

Micropropagation in elite corck oak trees: a tool for suber production improvement?

Dada a extrema importância económica e ambiental que o montado de sobro tem em Portugal, e dado o declínio deste devido a várias razões (e.g. doença, idade das plantas) é premente desenvolver estratégias de preservação de sobreiros elite e optimizar técnicas para a propagação destes genótipos. No primeiro Capítulo expõe-se uma breve introdução sobre o montado actual e as técnicas actuais de regeneração/propagação do sobreiro. Descreve-se ainda as principais técnicas de preservação e avaliação de estabilidade genética referidas na literatura para sobreiro e outras lenhosas. No Capítulo II é apresentado um estudo de melhoramento das condições actuais de maturação de embriões somáticos de sobreiro com vista a aperfeiçoar o processo de conversão em plantas. Neste capítulo é apresentado um protocolo melhorado em relação ao actual que permite um desenvolvimento dos embriões somáticos dum modo semelhante aos embriões zigóticos em termos de substâncias de reserva. O Capítulo III mostra um estudo efectuado com o objectivo principal de avaliar estabilidade genética durante todo o processo de embriogénese somática. Neste capítulo são apresentados resultados duma análise feita por RAPD em fases distintas da embriogénese somática de sobreiro. Neste estudo mostra-se que não existem diferenças significativas entre plantas de campo, embriões somáticos e plantas regeneradas. No Capítulo VI, pretende-se complementar o estudo anterior. Neste Capítulo descreve-se a dinâmica do ciclo celular durante as primeiras fases de embriogénese somática na presença de reguladores de crescimento. Este trabalho permitiu concluir a importância dos reguladores de crescimento na indução e perceber o peso do factor genótipo durante o processo. Considerando os resultados anteriores, a necessidade de um processo eficiente de preservação de genótipos elite torna-se fundamental. No Capítulo V descreve-se um protocolo de criopreservação eficiente sem recursos a substâncias tóxicas. Nesta secção é ainda feita uma análise de variabilidade genética após criopreservação através de FCM, AFLP e SSR. Todos os resultados obtidos anteriormente são postos a prova no Capítulo VI onde se faz uma monitorização extensiva de 10 genótipos elite, tendo em conta a sua capacidade de produção de cortiça, através do processo de embriogénese somática. Durante esta secção são utilizados os protocolos desenvolvidos anteriormente e avaliados na sua eficiência. Neste capítulo é descrita a integração de vários segmentos deste estudo num só protocolo eficiente de regeneração e preservação de sobreiros através de embriogénese somática. Finalmente, no Capítulo VI são apresentadas as conclusões da presente Tese de Doutoramento, com especial incidência para linhas de investigação futuras a serem tomadas. Discute-se a importância deste novo protocolo na optimização da produção da cortiça e traçam-se possíveis aplicações alternativas.

U-TRACER® - the use of communication technology in higher education - an information visualization tool for the context of Portuguese public higher education

Information Visualization is gradually emerging to assist the representation and comprehension of large datasets about Higher Education Institutions, making the data more easily understood. The importance of gaining insights and knowledge regarding higher education institutions is little disputed. Within this knowledge, the emerging and urging area in need of a systematic understanding is the use of communication technologies, area that is having a transformative impact on educational practices worldwide. This study focused on the need to visually represent a dataset about how Portuguese Public Higher Education Institutions are using Communication Technologies as a support to teaching and learning processes. Project TRACER identified this need, regarding the Portuguese public higher education context, and carried out a national data collection. This study was developed within project TRACER, and worked with the dataset collected in order to conceptualize an information visualization tool U-TRACER®. The main goals of this study related to: conceptualization of the information visualization tool U-TRACER®, to represent the data collected by project TRACER; understand higher education decision makers perception of usefulness regarding the tool. The goals allowed us to contextualize the phenomenon of information visualization tools regarding higher education data, realizing the existing trends. The research undertaken was of qualitative nature, and followed the method of case study with four moments of data collection.The first moment regarded the conceptualization of the U-TRACER®, with two focus group sessions with Higher Education professionals, with the aim of defining the interaction features the U-TRACER® should offer. The second data collection moment involved the proposal of the graphical displays that would represent the dataset, which reading effectiveness was tested by end-users. The third moment involved the development of a usability test to the UTRACER ® performed by higher education professionals and which resulted in the proposal of improvements to the final prototype of the tool. The fourth moment of data collection involved conducting exploratory, semi-structured interviews, to the institutional decision makers regarding their perceived usefulness of the U-TRACER®. We consider that the results of this study contribute towards two moments of reflection. The challenges of involving end-users in the conceptualization of an information visualization tool; the relevance of effective visual displays for an effective communication of the data and information. The second relates to the reflection about how the higher education decision makers, stakeholders of the U-TRACER® tool, perceive usefulness of the tool, both for communicating their institutions data and for benchmarking exercises, as well as a support for decision processes. Also to reflect on the main concerns about opening up data about higher education institutions in a global market.

MUSA: A Scalable Multi-Touch and Multi-Perspective Variability Management Tool

Variability management is one of the main activities in the Software Product Line Engineering process. Common and varied features of related products are modelled along with the dependencies and relationships among them. With the increase in size and complexity of product lines and the more holistic systems approach to the design process, managing the ever- growing variability models has become a challenge. In this paper, we present MUSA, a tool for managing variability and features in large-scale models. MUSA adopts the Separation of Concerns design principle by providing multiple perspectives to the model, each conveying different set of information. The demonstration is conducted using a real-life model (comprising of 1000+ features) particularly showing the Structural View, which is displayed using a mind-mapping visualisation technique (hyperbolic trees), and the Dependency View, which is displayed graphically using logic gates.

Coupling anchovy eggs and hydrodynamic model as an assessement tool for estuarine ecohydrological management

Nitrate and urban waste water directives have raised the need for a better understanding of coastal systems in European Union. The incorrect application of these directives can lead to important ecological or social penalties. In the paper this problem is addressed to Ria Formosa Coastal Lagoon. Ria Formosa hosts a Natural Park, important ports of the southern Portuguese coast and significant bivalve aquaculture activity. Four major urban waste water treatment plants discharging in the lagoon are considered in this study. Its treatment level must be selected, based on detailed information from a monitoring program and on a good knowledge of the processes determining the fate of the material discharged in the lagoon. In this paper the results of a monitoring program and simulations using a coupled hydrodynamic and water quality / ecological model, MOHID, are used to characterise the system and to understand the processes in Ria Formosa. It is shown that the water residence time in most of the lagoon is quite low, of the order of days, but it can be larger in the upper parts of the channels where land generated water is discharged. The main supply of nutrients to the lagoon comes from the open sea rather than from the urban discharges. For this reason the characteristics and behaviour of the general lagoon contrasts with the behaviour of the upper reaches of the channels where the influence of the waste water treatment plants are high. In this system the bottom mineralization was found to be an important mechanism, and the inclusion of that process in the model was essential to obtain good results.

Transformers on-load exciting current Park`s vector approach as a tool for winding faults diagnostics

This paper presents the development of a new approach for diagnosing the occurrence of inter-turn short-circuits in the windings of three-phase transformers, which is based on the on-line monitoring of the on-load exciting current Park's Vector patterns. Experimental and simulated results demonstrate the effectiveness of the proposed technique for detecting winding inter-turn insulation faults in operating three-phase transformers.

Modelling underwater acoustic noise as a tool for coastal management

Dissertação de Mestrado, Gestão da Água e da Costa, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2009

O estudo das funções com as F-Tools: o conceito F-Tool

O presente relatório, orientado pela Doutora Ana Conceição, foi elaborado no âmbito da Prática de Ensino Supervisionada (PES), integrada no plano de estudos do Mestrado em Ensino de Matemática no 3.º Ciclo do Ensino Básico e no Ensino Secundário, da Universidade do Algarve (UAlg). Apresento uma breve caraterização das escolas, dos orientadores cooperantes e das turmas. Menciono as aulas assistidas e as aulas lecionadas com a supervisão da orientadora e relato os trabalhos desenvolvidos em cada escola. Dedico um capítulo à importância das tecnologias no processo de ensino e aprendizagem pois, na minha opinião, na formação atual de qualquer professor deve ser tido em conta o uso de tecnologias em sala de aula. Partilhando a minha orientadora da mesma opinião, colocou-me um desafio: a criação de um software educacional com recurso ao sistema de álgebra computacional Mathematica, tendo em vista a melhoria do processo de ensino e aprendizagem da Matemática (em particular, no estudo das funções reais de variável real). Aceitei de imediato e, juntamente com a Dra. Cristina Simão e o Dr. José Pereira, iniciamos um projeto inovador na área de ensino, posteriormente distinguido com o prémio Timberlake1. Neste relatório apresento o conceito F-Tool2, isto é, ferramentas de ensino visuais, dinâmicas e interativas que permitem explorar de uma forma inovadora alguns dos principais conceitos nas áreas de Pré-Cálculo e Cálculo Diferencial, nos níveis básico, secundário e universitário. O conceito F-Tool foi utilizado nas aulas que lecionei em regime de par pedagógico com a Dra. Cristina Simão. Devido à sua importância na minha PES, ficou claro que o tema do meu relatório teria de ter uma referência explícita ao estudo das funções com as F-Tool. Reflito ainda sobre os seminários que apresentei na UAlg, no âmbito da PES, e apresento uma reflexão final de toda a Prática de Ensino Supervisionada.

Development of an operational tool for oil spill forecast: application to oil exposed regions

Tese de doutoramento, Ciências do Mar, da Terra e do Ambiente (Modelação), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

The Beliefs about Medicines Questionnaire utility as a tool in medication review

Background:Identification of potentially non-adherent patients is a milestone in the way to the rational use of drugs. Detecting beliefs that influence intentional non-adherence behaviours may improve pharmacists[simplequote] counselling. The aim of this study was to assess the Beliefs about Medicines Questionnaire (BMQ) as a proxy for necessity..

Choosing the Right Promotional Tool in the Hotel Industry: The Case of the Chinese Consumer.

The rapid growth of the Chinese tourism has stimulated competition within tourism-related industries, such as the hospitality industry. The purpose of this study is to examine the Chinese consumer reaction to different promotional tools used by hotels in China and, thus, to provide a deeper understanding for marketers of how to use sales promotion effectively to generate appropriate consumer responses. An experimental survey was administered yielding a total sample of 319 Chinese customers, who were probed using different types of sales promotion tools. Data analysis indicates that bonus packs (e.g. a 3-night stay at a hotel for the price of 2) induced the highest consumer perceived value, brand switching, and purchase acceleration intention, whereas price discounts resulted in the highest intention to spend more. Although this study has its limitations given its reliance on a convenience sample, it offers insightful practical implications for hotel business owners in Asia regarding targeting the right customers with the right promotional tools, where it is proposed that bonus packs successfully attract new Chinese customers and price discounts support in generating more sales.

The Potential of Participedia as a Crowdsourcing Tool for Comparative Analysis of Democratic Innovations

For two reasons, our capacity for systematic comparison of innovative participatory democratic processes remains limited. First, the category of participatory democratic innovations remains relatively vague when compared to more traditional democratic institutions and practices. Second, until recently there existed no large-sample databases that captured relevant variables in the practice of democratic innovation. The lone exception to these patterns is the Participedia database, located online. Participedia is well placed to respond to the two obstacles to systematic comparative research on democratic innovation. First, its crowdsourced data collection strategy means that many of the cases on the platform are not well known and have not been the subject of sustained academic analysis. Second, the data captured in the articles provides the basis for systematic comparative analysis of democratic innovations both within type (e.g., participatory budgeting, mini-publics) and across types. The platform allows for systematic content analysis of text descriptions and/or statistical analysis of the datasets generated from the structured data fields. This article describes the data about innovative participatory democratic processes available from Participedia, and furnishes examples of the kinds of quantitative and qualitative insights about those processes that Participedia enables.

Laccase bio-grafting a versatile modification tool from green chemistry technologies

Today more than 99% of plastics are petroleum-based because of availability and cost of the raw material. The durability of these disposed plastics contributes to the environmental problems as waste and their persistence in the environment causes deleterious effects on the ecosystem. Environmental pollution awareness and the demand for green technology have drawn considerable attention of both academia and industry into biodegradable polymers. In this regard green chemistry technology has the potential to provide solution to this problematic issue. Laccase bio-grafting has recently been the focus of green chemistry technologies due to the growing environmental concerns, legal restrictions and increasing availability of scientific knowledge. In the last several years, research covering various applications of laccases has been increased rapidly particularly in the field of grafting. In principle, laccase-assisted graft co-polymerization may impart a variety of new functionalities to a polymer. The modified polymers through grafting have a bright future and their development is practically boundless. In present work, novel biodegradable graft copolymers combining the advantages of bacterial cellulose backbone and PHB side chains will be prepared by introducing enzymatic grafting technique. The present research will be a first step in the biopolymer modification. To date no report has been found in literature explaining the enzymatic grafting of PHAs. The technique would also provide an efficient modulation approach to improve the biodegradability and biocompatibility of the graft copolymer. The newly grafted copolymers will exhibit unique functionalities with wider range of potential applications mainly in tissue engineering, biosensors, pharmaceutical industry (drug delivery systems) and bio-plastics.