Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element.

Notch is a transmembrane receptor that plays a critical role in cell fate determination. In Drosophila, Notch binds to and signals through Suppressor of Hairless. A mammalian homologue of Suppressor of Hairless, named CBF1 (or RBPJk), is a ubiquitous transcription factor whose function in mammalian Notch signaling is unknown. To determine whether mammalian Notch can stimulate transcription through a CBF1-responsive element (RE), we cotransfected a CBF1-RE-containing chloramphenicol acetyltransferase reporter and N1(deltaEC), a constitutively active form of human Notch1 lacking the extracellular domain, into DG75, COS-1, HeLa, and 293T cells, which all contain endogenous CBF1. N1(deltaEC) dramatically increased chloramphenicol acetyltransferase activity in these cells, indicating functional coupling of Notch1 and CBF1. The activity was comparable to that produced by the Epstein-Barr virus protein EBNA2, a well-characterized, potent transactivator of CBF1. To test whether CBF1 and Notch1 interact physically, we tagged CBF1 with an epitope from the influenza virus hemagglutinin or with the N-terminal domain of gal4, and transfected the tagged CBF1 plus N1(deltaEC) into COS-1 cells. Cell lysates were immunoprecipitated and immunoblotted with several anti-Notch1 antibodies [to detect N1(deltaEC)] or with antibodies to hemagglutinin or gal4 (to detect CBF1). Each immunoprecipitate contained a complex of N1(deltaEC) and CBF1. In summary, we find that the truncated, active form of human Notch1, N1(deltaEC), binds CBF1 and activates transcription through a CBF1-RE-containing promoter. We conclude that CBF1 is a critical downstream protein in the human Notch1 signaling pathway.

A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation.

Examination of the interactions involving transcription factor E2F activity during cell growth and terminal differentiation suggests distinct roles for Rb family members in the regulation of E2F accumulation. The major species of E2F in quiescent cells is a complex containing the E2F4 product in association with the Rb-related p130 protein. As cells enter the cell cycle, this complex disappears, and there is a concomitant accumulation of free E2F activity of which E2F4 is a major component. E2F4 then associates with the Rb-related p107 protein as cells enter S phase. Rb can be found in interactions with each E2F species, including E2F4, during G1, but there appears to be a limited amount of Rb with respect to E2F, likely due to the maintenance of most Rb protein in an inactive state by phosphorylation. A contrasting circumstance can be found during the induction of HL60 cell differentiation. As these cells exit the cell cycle, active Rb protein appears to exceed E2F, as there is a marked accumulation of E2F-Rb interactions, involving all E2F species, including E2F4, which is paralleled by the conversion of Rb from a hyperphosphorylated state to a hypophosphorylated state. These results suggest that the specific ability of Rb protein to interact with each E2F species, dependent on concentration of active Rb relative to accumulation of E2F, may be critical in cell-growth decisions.

Como estamos indo? Estudo do deslocamento ativo no Brasil

Introdução: O deslocamento ativo tem estreita relação com problemas de saúde pública da atualidade e sua promoção pode contribuir para melhorias quanto à mobilidade urbana, estado de saúde e proteção do meio ambiente. Entretanto, a maior parte das pesquisas sobre o tema tem sido desenvolvida em países de renda alta. A presente tese busca ampliar a investigação sobre o deslocamento ativo no Brasil. Objetivos: i) Descrever a frequência, a distribuição e a variação temporal de indicadores do deslocamento ativo em populações brasileiras; ii) Avaliar o impacto de mudanças no padrão de transporte da população sobre o deslocamento ativo, o tempo sedentário e desfechos de saúde em populações brasileiras. Métodos: Tese composta por sete manuscritos. O primeiro apresenta revisão sistemática de estudos com informações sobre a prática de deslocamento ativo na América Latina e Caribe; o segundo descreve estimativas representativas da população brasileira sobre a prática de deslocamento ativo para o trabalho; o terceiro e o quarto descrevem a frequência e tendência temporal do deslocamento ativo na Região Metropolitana de São Paulo (ciclistas e escolares); o quinto discute a questão da mobilidade urbana e do direito à cidade em São Paulo; o sexto e o sétimo avaliam o impacto de mudanças no padrão de mobilidade da metrópole paulistana sobre a prática de deslocamento ativo, tempo não-ativo de deslocamento e tempo total de deslocamento, bem como sobre a poluição do ar e saúde da população. Resultados: A prevalência mediana de deslocamento ativo encontrada em diferentes locais do Brasil foi de 12 por cento , variando entre 5,1 por cento em Palmas (Tocantins) a 58,9 por cento em Rio Claro (São Paulo) (Manuscrito 1). Um terço dos homens e das mulheres desloca-se a pé ou de bicicleta de casa para o trabalho no país. Em ambos os sexos, esta proporção diminui com o aumento da renda e da escolaridade e é maior entre os mais jovens, entre os que residem em áreas rurais, e na região Nordeste. Em todas as regiões metropolitanas estudadas, o quinto das pessoas de menor renda apresenta uma maior frequência de deslocamento ativo (Manuscrito 2). Entre os anos de 2007 e 2012, observamos redução no número de ciclistas em São Paulo e diferenças expressivas na proporção de ciclistas entre homens e mulheres (9,7 por mil habitantes versus 1,4 por mil habitantes em 2012) (Manuscrito 3). Também verificamos uma queda na proporção de crianças que se deslocam ativamente para a escola entre os anos de 1997 e 2012 (Manuscrito 4). O cenário epidemiológico do deslocamento ativo no país é resultante da disputa pelo direito à cidade, com repercussões na transição de mobilidade humana e na saúde e qualidade de vida da população, como podemos observar no caso de São Paulo (Manuscrito 5). A construção de uma São Paulo mais inclusiva, com menores distâncias para os deslocamentos cotidianos e maior frequência de caminhada e bicicleta, levaria à substancial redução do tempo total e do tempo sedentário despendidos nos deslocamentos, sem diminuir a duração do deslocamento ativo (Manuscrito 6). Traria também ganhos à saúde da população, sobretudo pelo aumento da prática de atividade física e da redução da poluição do ar (Manuscrito 7). Conclusões: A prática de deslocamento ativo no Brasil apresenta marcadas diferenças segundo região e características sociodemográficas. De um modo geral, esta prática vem diminuindo no país, o que deve contribuir negativamente para a saúde da população. A promoção de cidades mais inclusivas e compactas, com o favorecimento a modos ativos de deslocamento, pode contribuir para reverter esta preocupante tendência.

Ambientes tecno-pedagógicos para o desenvolvimento de competências transversais para a inovação em engenharia.

Muito se fala na crescente velocidade de inovações tecnológicas que se apresenta ao mundo e que vai se acelerando. Antes, gerações se passavam para termos uma evolução tecnológica. Hoje, uma mesma geração presencia vários saltos tecnológicos. Neste contexto de inovações frequentes, surge a preocupação sobre como formar um profissional responsável, ético e que consiga acompanhar e ser protagonista de tais mudanças. O trabalho desenvolvido nesta tese busca colaborar na discussão sobre a formação do engenheiro, com foco na engenharia elétrica e de computação, revisitando as definições de conceitos como educação em engenharia, aprendizagem ativa, inovação, Design Thinking e competências transversais, e definindo, como contribuição de pesquisa, os conceitos de tecno-pedagogia e ambientes tecno-pedagógicos, como pressuposto de convergência de estruturas tecnológicas, estratégias pedagógicas e métodos de avaliação em aprendizagem ativa para a inovação. Apresenta um método para identificar e quantificar o grau de ênfase das competências transversais para a inovação a partir da demanda de mercado para engenheiros eletricistas e da computação; e um método de observação, coleta e análise de dados sobre o desenvolvimento de competências transversais na participação em duas experiências de aula: na disciplina global ME310 da Universidade de Stanford; e na disciplina 030-3410 da Escola Politécnica da Universidade de São Paulo. Com isso, foi possível elaborar um método para auxiliar no planejamento de disciplinas e cursos com foco em inovação, identificando as competências transversais que devem ser incentivadas e relacionando tais competências com estratégias de ensino e aprendizagem e sugerindo a estrutura tecnológica e método de avaliação a serem adotados.

The curricular value of mathematics in non-mathematics degree

Higher education should provide the acquisition of skills and abilities that allow the student to play a full and active role in society. The educational experience should offer a series of conceptual, procedural and attitudinal contents that encourage “learning to know, learning to do, learning to be and learning to live together”. It is important to consider the curricular value of mathematics in the education of university undergraduates who do not intend to study mathematics but for whom the discipline will serve as an instrumental. This work discusses factors that form part of the debate on the curricular value of mathematics in non-mathematics degrees.

Music and movement: A comparative study between the BAPNE and Suzuki Methods

The aim of this article is to compare the Suzuki and BAPNE methods based on bibliography published for both approaches. In the field of musical and instrumental education and especially for the childhood stage, the correct use of the body and voice are of fundamental importance. These two methods differ from one another; one principally musical and instrumental, which is the Suzuki method, and one non-musical, the BAPNE method, which aims at stimulating attention, concentration, memory and the executing function of the pupil through music and body percussion. Comparing different approaches may provide teachers with a useful insight for facing different issues related to their discipline.

Changing Banking Supervision in the Eurozone: the ECB as a Policy Entrepreneur. Bruges Political Research Paper No. 38, December 2014

In 2012, the European Union adopted a transformational change to its banking policy for the Eurozone. It dropped the model of decentralized supervision and regulatory competition between countries, and replaced it with a single supervisor and harmonization. Transferring banking supervision to the ECB also alters the existing constitutional order. The policy process leading to this transformational change was rapid and highly political, which was different compared to earlier incremental changes to banking policy. Kingdon's model, whereby policy entrepreneurs seize opportunities at times when the independent streams of solutions, problems and politics converge, partly explains this transformation. The study of EU banking policy suggests, however, that the multiple streams framework should pay more attention to the way in which entrepreneurs engineer fluctuations within the streams and thereby contribute to creating opportunities for change. This paper identifies the ECB as an effective entrepreneur which also played an active role in political bargaining.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

Artisans, génies et vedettes : le statut des compositeurs dans la presse musicale française

Ce mémoire propose d’explorer quel(s) statut(s) sont accordés aux compositeurs contemporains dans le contexte musical parisien des premières années du XXe siècle à travers une analyse de la presse musicale spécialisée de l’époque. Le corpus de notre recherche est constitué de trois revues, chacune ancrée dans des sphères de sociabilités bien distinctes : La Revue musicale (histoire et critique), une « revue savante » proche du milieu de la Schola cantorum ; Le Mercure musical, une « petite revue » d’avant-garde militant en faveur de la musique de Maurice Ravel ; et Musica, une « grande revue » destinée à un lectorat issu de la petite bourgeoisie en plein essor, essentiellement féminin et pratiquant la musique en amateur. Cette étude révèle que la traditionnelle opposition entre la conception du compositeur-artisan et celle du compositeur-génie issue de la fin du XVIIIe siècle et du XIXe siècle voit apparaître un troisième joueur à l’aube du XXe siècle : le compositeur-vedette, un statut qui était jusqu’alors généralement réservé aux interprètes. Ces trois statuts coexistent dans le panorama de la presse musicale du tournant du XXe siècle, et leur promotion par certains organes de presse spécifiques répond à une logique tantôt esthétique, tantôt économique. Il se dégage de cette étude que la presse musicale constitue non seulement un indice des variations qu’a subies la figure du compositeur dans le spectre de la grandeur en culture, mais qu’elle a également joué un rôle actif dans ces transformations.

Digital Classics Outside the Echo-Chamber : Teaching, Knowledge Exchange and Public Engagement /

The international perspectives on these issues are especially valuable in an increasingly connected, but still institutionally and administratively diverse world. The research addressed in several chapters in this volume includes issues around technical standards bodies like EpiDoc and the TEI, engaging with ways these standards are implemented, documented, taught, used in the process of transcribing and annotating texts, and used to generate publications and as the basis for advanced textual or corpus research. Other chapters focus on various aspects of philological research and content creation, including collaborative or community driven efforts, and the issues surrounding editorial oversight, curation, maintenance and sustainability of these resources. Research into the ancient languages and linguistics, in particular Greek, and the language teaching that is a staple of our discipline, are also discussed in several chapters, in particular for ways in which advanced research methods can lead into language technologies and vice versa and ways in which the skills around teaching can be used for public engagement, and vice versa. A common thread through much of the volume is the importance of open access publication or open source development and distribution of texts, materials, tools and standards, both because of the public good provided by such models (circulating materials often already paid for out of the public purse), and the ability to reach non-standard audiences, those who cannot access rich university libraries or afford expensive print volumes. Linked Open Data is another technology that results in wide and free distribution of structured information both within and outside academic circles, and several chapters present academic work that includes ontologies and RDF, either as a direct research output or as essential part of the communication and knowledge representation. Several chapters focus not on the literary and philological side of classics, but on the study of cultural heritage, archaeology, and the material supports on which original textual and artistic material are engraved or otherwise inscribed, addressing both the capture and analysis of artefacts in both 2D and 3D, the representation of data through archaeological standards, and the importance of sharing information and expertise between the several domains both within and without academia that study, record and conserve ancient objects. Almost without exception, the authors reflect on the issues of interdisciplinarity and collaboration, the relationship between their research practice and teaching and/or communication with a wider public, and the importance of the role of the academic researcher in contemporary society and in the context of cutting edge technologies. How research is communicated in a world of instant- access blogging and 140-character micromessaging, and how our expectations of the media affect not only how we publish but how we conduct our research, are questions about which all scholars need to be aware and self-critical.

Comparisons of physical separation methods of Kunjin virus-induced membranes

The two sets of connected membranes induced in Kunjin virus-infected cells are characterized by the presence of NS3 helicase/protease in both, and by RNA-dependent RNA polymerase (RdRp) activity plus the associated double-stranded RNA (dsRNA) template in vesicle packets (VP), or by the absence of both the VP-specific markers in the convoluted membranes/paracrystalline arrays (CM/PC). Attempts were made to separate flavivirus-induced membranes by sedimentation or flotation analyses in density gradients of sucrose or iodixanol, respectively, after treatment of cell lysates by sonication, osmotic shock, or tryptic digestion. Only osmotic shock treatment provided suggestive evidence of separation. This was explored by flow cytometry analysis (FCA) of RdRp active membrane fractions from a sucrose gradient, using dual fluorescent labelling via antibodies to NS3 and dsRNA. FCA revealed the presence of a dual labelled membrane population indicative of VP, and in a faster sedimenting fraction a membrane population able to be labelled only in NS3, representative of CM/PC and associated (R)ER. It was postulated that osmotic shock ruptured the bounding membrane of the VP, releasing the enclosed small vesicles associated with the Kunjin virus replication complex characterized previously. Notably, the presence of the full spectrum of nonstructural proteins in some membrane fractions was not a reliable marker for RdRp activity. These experiments may provide the opportunity for isolation of relatively pure flavivirus replication complexes in their native membrane-associated state by fluorescence-activated cell sorting. (C) 2004 Elsevier B.V. All rights reserved.

A distributed equivalent magnetic current based FDTD method for the calculation of E-fields induced by gradient coils

This paper evaluates a new, low-frequency finite-difference time-domain method applied to the problem of induced E-fields/eddy currents in the human body resulting from the pulsed magnetic field gradients in MRI. In this algorithm, a distributed equivalent magnetic current is proposed as the electromagnetic source and is obtained by quasistatic calculation of the empty coil's vector potential or measurements therein. This technique circumvents the discretization of complicated gradient coil geometries into a mesh of Yee cells, and thereby enables any type of gradient coil modelling or other complex low frequency sources. The proposed method has been verified against an example with an analytical solution. Results are presented showing the spatial distribution of gradient-induced electric fields in a multi-layered spherical phantom model and a complete body model. (C) 2004 Elsevier Inc. All rights reserved.

Better prediction of protein contact number using a support vector regression analysis of amino acid sequence

Background: Protein tertiary structure can be partly characterized via each amino acid's contact number measuring how residues are spatially arranged. The contact number of a residue in a folded protein is a measure of its exposure to the local environment, and is defined as the number of C-beta atoms in other residues within a sphere around the C-beta atom of the residue of interest. Contact number is partly conserved between protein folds and thus is useful for protein fold and structure prediction. In turn, each residue's contact number can be partially predicted from primary amino acid sequence, assisting tertiary fold analysis from sequence data. In this study, we provide a more accurate contact number prediction method from protein primary sequence. Results: We predict contact number from protein sequence using a novel support vector regression algorithm. Using protein local sequences with multiple sequence alignments (PSI-BLAST profiles), we demonstrate a correlation coefficient between predicted and observed contact numbers of 0.70, which outperforms previously achieved accuracies. Including additional information about sequence weight and amino acid composition further improves prediction accuracies significantly with the correlation coefficient reaching 0.73. If residues are classified as being either contacted or non-contacted, the prediction accuracies are all greater than 77%, regardless of the choice of classification thresholds. Conclusion: The successful application of support vector regression to the prediction of protein contact number reported here, together with previous applications of this approach to the prediction of protein accessible surface area and B-factor profile, suggests that a support vector regression approach may be very useful for determining the structure-function relation between primary sequence and higher order consecutive protein structural and functional properties.

Simultaneous liquid chromatographic determination of doxorubicin and its major metabolite doxorubicinol in parrot plasma

A new microscale method is reported for the determination of doxorubicin and its active metabolite, doxorubicinol, in parrot plasma. Sample workup involved acetonitrile protein precipitation, ethyl acetate extraction, followed by back extraction into HCl. Separations were achieved on a phenyl-hexyl column at 30 degrees C using acetonitrile (17%, v/v) in 0.01 M orthophosphoric acid (83%, v/v) delivered via a linear flow program. Fluorometric detection wavelengths were 235 nm (excitation) and 550 nm (emission). Calibration plots were linear (1 2 > 0.999), and recoveries were 71-87% from 20 to 400 ng/mL. Assay imprecision was

Enhancing the ratio of molecular ions to non-covalent compounds in the electrospray interface of LC-MS in quantitative analysis

A common problem encountered during the development of MS methods for the quantitation of small organic molecules by LGMS is the formation of non-covalently bound species or adducts in the electrospray interface. Often the population of the molecular ion is insignificant compared to those of all other forms of the analyte produced in the electrospray, making it difficult to obtain the sensitivity required for accurate quantitation. We have investigated the effects of the following variables: orifice potential, nebulizer gas flow, temperature, solvent composition and the sample pH on the relative distributions of ions of the types MH+, MNa+, MNH+, and 2MNa(+), where M represents a 4 small organic molecule: BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile). Orifice potential, solvent composition and the sample pH had the greatest influence on the relative distributions of these ions, making these parameters the most useful for optimizing methods for the quantitation of small molecules.