A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft. The proteins belong to a large family of secondary transporters, which includes bacterial glutamate transporters. The C-terminal half of the glutamate transporters is well conserved and thought to contain the translocation path and the binding sites for substrate and coupling ions. A serine-rich sequence motif in this part of the proteins is located in a putative intracellular loop. Cysteine-scanning mutagenesis was applied to this loop in the glutamate transporter GltT of Bacillus stearothermophilus. The loop was found to be largely intracellular, but three consecutive positions in the conserved serine-rich motif (S269, S270, and E271) are accessible from both sides of the membrane. Single-cysteine mutants in the serine-rich motif were still capable of glutamate transport, but modification with N-ethylmaleimide blocked the transport activity in six mutants (T267C, A268C, S269C, S270C, E271C, and T272C). Two milimolars l-glutamate effectively protected against the modification of the cysteines at position 269–271 from the periplasmic side of the membrane but was unable to protect cysteine modification from the cytoplasmic side of the membrane. The results indicate that the conserved serine-rich motif in the glutamate transporter forms a reentrant loop, a structure that is found in several ion channels but is unusual for transporter proteins. The reentrant loop is of crucial importance for the function of the glutamate transporter.

Identification of a novel p53 functional domain that is necessary for efficient growth suppression

Activation of the p53 tumor suppressor protein has been demonstrated to block cell growth by inducing either a transient cell cycle arrest or programmed cell death (apoptosis). Although evidence exists linking p53’s function as an activator of transcription to its ability to effect cell cycle arrest, the role of this activity in the induction of apoptosis remains unclear. To gain insight into the molecular mechanisms underlying p53-mediated antiproliferative pathways, a study was initiated to explore the functions of a putative p53 signaling domain. This region of the human p53 protein is localized between amino acids 61 and 94 (out of 393) and is noteworthy in that it contains five repeats of the sequence PXXP (where P represents proline and X any amino acid). This motif has been shown to play a role in signal transduction via its SH3 domain binding activity. A p53 cDNA deletion mutant (ΔproAE), which lacks this entire proline-rich domain (deleted for amino acids 62–91), was created and characterized for a variety of p53 functions. The entire domain has been shown to be completely dispensable for transcriptional activation. On the other hand, this deletion of the p53 proline-rich domain impairs p53’s ability to suppress tumor cell growth in culture. Amino acid substitution mutations at residues 22 and 23 of p53 (eliminates transcriptional activity) also impair p53-mediated inhibition of cell growth in culture. Unlike wild-type p53, the ΔproAE mutant cDNA can be stably expressed in tumor derived cell lines with few immediate detrimental effects. These cells express physiologic levels of p53 protein that are induced normally in response to DNA damage, indicating that removal of the proline-rich domain does not disrupt p53’s upstream regulation by DNA damage. These data indicate that, in addition to the transcriptional activation domain, the p53 proline-rich domain plays a critical role in the transmission of antiproliferative signals downstream of the p53 protein and may link p53 to a direct signal transduction pathway.

Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase

The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 Å resolution; it has a parallel α-β topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related helicases. Using data to 2.8 Å resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a “dumbbell” structure consisting of two compact domains connected by an extended linker. By using the structures of other helicases as a template, compact structures can be modeled for eIF4A that suggest (i) helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA helicase family but is absent from many other helicases, also binds RNA; and (iii) motifs V and VI “link” the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.

The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing.

Circuit-specific alterations of N-methyl-D-aspartate receptor subunit 1 in the dentate gyrus of aged monkeys.

Age-associated memory impairment occurs frequently in primates. Based on the established importance of both the perforant path and N-methyl-D-aspartate (NMDA) receptors in memory formation, we investigated the glutamate receptor distribution and immunofluorescence intensity within the dentate gyrus of juvenile, adult, and aged macaque monkeys with the combined use of subunit-specific antibodies and quantitative confocal laser scanning microscopy. Here we demonstrate that aged monkeys, compared to adult monkeys, exhibit a 30.6% decrease in the ratio of NMDA receptor subunit 1 (NMDAR1) immunofluorescence intensity within the distal dendrites of the dentate gyrus granule cells, which receive the perforant path input from the entorhinal cortex, relative to the proximal dendrites, which receive an intrinsic excitatory input from the dentate hilus. The intradendritic alteration in NMDAR1 immunofluorescence occurs without a similar alteration of non-NMDA receptor subunits. Further analyses using synaptophysin as a reflection of total synaptic density and microtubule-associated protein 2 as a dendritic structural marker demonstrated no significant difference in staining intensity or area across the molecular layer in aged animals compared to the younger animals. These findings suggest that, in aged monkeys, a circuit-specific alteration in the intradendritic concentration of NMDAR1 occurs without concomitant gross structural changes in dendritic morphology or a significant change in the total synaptic density across the molecular layer. This alteration in the NMDA receptor-mediated input to the hippocampus from the entorhinal cortex may represent a molecular/cellular substrate for age-associated memory impairments.

Interazione di nanoparticelle metalliche con menbrane biologiche: risultati di studi in vitro su cute, mucosa del cavo orale e meningi ed implicazioni per la salute

L’utilizzo di nanomateriali, ovvero una nuova classe di sostanze composte da particelle ultrafini con dimensioni comprese fra 1 e 100 nm (American Society for Testing Materials - ASTM), è in costante aumento a livello globale. La particolarità di tali sostanze è rappresentata da un alto rapporto tra la superficie e il volume delle particelle, che determina caratteristiche chimico-fisiche completamente differenti rispetto alle omologhe macrosostanze di riferimento. Tali caratteristiche sono tali da imporre una loro classificazione come nuovi agenti chimici (Royal Society & Royal Academy of Engineering report 2004). Gli impieghi attuali dei nanomateriali risultano in continua evoluzione, spaziando in diversi ambiti, dall’industria farmaceutica e cosmetica, all’industria tessile, elettronica, aerospaziale ed informatica. Diversi sono anche gli impieghi in campo biomedico; tra questi la diagnostica e la farmacoterapia. È quindi prevedibile che in futuro una quota sempre maggiore di lavoratori e consumatori risulteranno esposti a tali sostanze. Allo stato attuale non vi è una completa conoscenza degli effetti tossicologici ed ambientali di queste sostanze, pertanto, al fine di un loro utilizzo in totale sicurezza, risulta necessario capirne meglio l’impatto sulla salute, le vie di penetrazione nel corpo umano e il rischio per i lavoratori conseguente al loro utilizzo o lavorazione. La cute rappresenta la prima barriera nei confronti delle sostanze tossiche che possono entrare in contatto con l’organismo umano. Successivamente agli anni ‘60, quando si riteneva che la cute rappresentasse una barriera totalmente impermeabile, è stato dimostrato come essa presenti differenti gradi di permeabilità nei confronti di alcuni xenobiotici, dipendente dalle caratteristiche delle sostanze in esame, dal sito anatomico di penetrazione, dal grado di integrità della barriera stessa e dall’eventuale presenza di patologie della cute. La mucosa del cavo orale funge da primo filtro nei confronti delle sostanze che entrano in contatto con il tratto digestivo e può venir coinvolta in contaminazioni di superficie determinate da esposizioni occupazionali e/o ambientali. È noto che, rispetto alla cute, presenti una permeabilità all’acqua quattro volte maggiore, e, per tale motivo, è stata studiata come via di somministrazione di farmaci, ma, ad oggi, pochi sono gli studi che ne hanno valutato le caratteristiche di permeazione nei confronti delle nanoparticelle (NPs). Una terza importante barriera biologica è quella che ricopre il sistema nervoso centrale, essa è rappresentata da tre foglietti di tessuto connettivo, che assieme costituiscono le meningi. Questi tre foglietti rivestono completamente l’encefalo permettendone un isolamento, tradizionalmente ritenuto completo, nei confronti degli xenobiotici. L’unica via di assorbimento diretto, in questo contesto, è rappresentata dalla via intranasale. Essa permette un passaggio diretto di sostanze dall’epitelio olfattivo all’encefalo, eludendo la selettiva barriera emato-encefalica. Negli ultimi anni la letteratura scientifica si è arricchita di studi che hanno indagato le caratteristiche di assorbimento di farmaci attraverso questa via, ma pochissimi sono gli studi che hanno indagato la possibile penetrazione di nanoparticelle attraverso questa via, e nessuno, in particolar modo, ha indagato le caratteristiche di permeazione delle meningi. L’attività di ricerca svolta nell’ambito del presente dottorato ha avuto per finalità l’indagine delle caratteristiche di permeabilità e di assorbimento della cute, della mucosa del cavo orale e delle meningi nei confronti di alcune nanoparticelle, scelte fra quelle più rappresentative in relazione alla diffusione d’utilizzo a livello globale. I risultati degli esperimenti condotti hanno dimostrato, in vitro, che l’esposizione cutanea a Pt, Rh, Co3O4 e Ni NPs determinano permeazione in tracce dei medesimi metalli attraverso la cute, mentre per le TiO2 NPs tale permeazione non è stata dimostrata. È stato riscontrato, inoltre, che la mucosa del cavo orale e le meningi sono permeabili nei confronti dell’Ag in forma nanoparticellare.

Stop, Collaborate & Listen: How the Librarian/Publisher Relationship Can Facilitate the Development of the Information Literacy Curriculum

A librarian from the Florida Gulf Coast University (FGCU) and the Library Communications Manager at Taylor & Francis Group partnered to launch a collaborative information literacy pilot program focusing on assisting FGCU students and faculty navigate and understand the scholarly publishing process. This article describes how the idea was created, as well as steps involved in developing the publishing toolkit to help FGCU patrons. An overview of the pilot program was presented during the 2015 Charleston Conference as a poster session.

Factores determinantes de los resultados derivados de las actividades de patrocinio y mecenazgo

El objetivo de este trabajo consiste en analizar los resultados empresariales derivados de las acciones de patrocinio y mecenazgo, así como sus factores determinantes. La metodología aplicada utiliza el event study para estimar los excesos de rentabilidad de las acciones generadas en Bolsa por una muestra de 21 noticias de patrocinio y 37 noticias de mecenazgo, correspondientes a 26 empresas españolas. Adicionalmente se realiza un análisis de regresión para determinar la influencia de ciertas dimensiones (el origen del evento, así como el vínculo entre el evento y la actividad de la empresa) sobre dichas rentabilidades anormales. La aplicación empírica efectuada ha permitido detectar que sólo las acciones de patrocinio generan rentabilidad, siendo sus principales determinantes el tamaño y el vínculo entre el evento financiado y la actividad de la empresa.

La identidad como producto del aprendizaje en la práctica de enseñar matemáticas en profesores de primaria

Esta investigación estudia la identidad como producto del aprendizaje en la práctica situada de enseñar matemáticas de cuatro profesores de primaria en México. Las características de la identidad de “ser profesor de matemáticas” se infieren desde la trayectoria de formación y la práctica de los profesores. Los resultados indican que los profesores construyen y reconstruyen su identidad a partir de su práctica cotidiana lo que supone la negociación de maneras de realizar una práctica en un contexto a partir de tres aspectos: (i) El significado y sentido que los profesores atribuyen a las experiencias que acumulan en su trayectoria de formación, (ii) a la acción mediada con las tareas y los instrumentos que utilizan, y (iii) a la interacción entre el significado y sentido y la acción mediada al gestionar la clase de matemáticas.

Visual perception for the 3D recognition of geometric pieces in robotic manipulation

During grasping and intelligent robotic manipulation tasks, the camera position relative to the scene changes dramatically because the robot is moving to adapt its path and correctly grasp objects. This is because the camera is mounted at the robot effector. For this reason, in this type of environment, a visual recognition system must be implemented to recognize and “automatically and autonomously” obtain the positions of objects in the scene. Furthermore, in industrial environments, all objects that are manipulated by robots are made of the same material and cannot be differentiated by features such as texture or color. In this work, first, a study and analysis of 3D recognition descriptors has been completed for application in these environments. Second, a visual recognition system designed from specific distributed client-server architecture has been proposed to be applied in the recognition process of industrial objects without these appearance features. Our system has been implemented to overcome problems of recognition when the objects can only be recognized by geometric shape and the simplicity of shapes could create ambiguity. Finally, some real tests are performed and illustrated to verify the satisfactory performance of the proposed system.

Y-box binding protein 1 (YB-1) relevance in estrogen receptor-positive (ER+) breast cancer

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016

Referenciação de doentes oncológicos para os cuidados paliativos

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

The Single Supervisory Mechanism: A Sound First Step in Europe’s Banking Union? Egmont European Affairs Paper, March 2013

The financial and economic crisis has hit Europe in its core. While the crisis may not have originated in the European Union, it has laid bare structural weaknesses in the EU’s policy framework. Both public finances and the banking sector have been heavily affected. For a long time, the EU failed to take into account sufficiently the perverse link that existed between the two. Negative evolutions in one field of the crisis often dragged along the other in its downward spiral. In June 2012, in the early hours of a yet another EU Summit, the leaders of the eurozone finally decided to address the link between the banking and sovereign debt crises. Faced with soaring public borrowing costs in Spain and Italy, they decided to allow for the direct European recapitalisation of banks when the Member State itself would no longer be in a position to do so. In exchange, supervision of the banking sector would be lifted to the European level by means of a Single Supervisory Mechanism. The Single Supervisory Mechanism, or SSM in the EU jargon, is a first step in the broader revision of policies towards banks in Europe. The eventual goal is the creation of a Banking Union, which is to carry out effective surveillance and – if needed – crisis management of the banking sector. The SSM is to rely on national supervisors and the ECB, with the ECB having final authority on the matter. The involvement of the latter made it clear that the SSM would be centred on the eurozone – while it is to remain open to other Member States willing to join. Due to the ongoing problems and the link between the creation of the SSM and the recapitalisation of banks, the SSM became one of the key legislative priorities of the EU. In December 2012, Member States reached an agreement on the design of the SSM. After discussions with the European Parliament (which were still ongoing at the time of writing), the process towards making the SSM operational can be initiated. The goal is to have the SSM fully up and running in the first half of 2014. The decisions that were taken in June 2012 are likely to have had a bigger impact than the eurozone’s Heads of State and Government could have realised at the time for two important reasons. On the one hand, creating the SSM necessitates a full Banking Union and therefore shared risk. On the other hand, the decisions improved the ECB’s perception of the willingness of governments to take far-reaching measures. This undoubtedly played a significant role in the creation of the Outright Monetary Transactions programme by the ECB, which has led to a substantial easing of the crisis in the short-term. 1 These short-term gains should now be matched with a stable long-term framework for bank supervision and crisis management. The agreement on the SSM should be the first step in the direction of this goal. This paper provides an analysis of the SSM and its role in the creation of a Banking Union. The paper starts with a reminder of why the EU decided to put in place the SSM (§1) and the state of play of the ongoing negotiations on the SSM (§2). Subsequently, the supervisory responsibilities of the SSM are detailed, including its scope and the division of labour between the national supervisors and the ECB (§3). The internal functioning of the SSM (§4) and its relation to the other supervisors are discussed afterwards (§5). As mentioned earlier, the SSM is part of a wider move towards a Banking Union. Therefore, this paper sheds light on the other building blocks of this ambitious project (§6). The transition towards the Banking Union is important and will prove to be a bumpy ride. Before formulating a number of conclusions, this Working Paper therefore provides an overview of the planned road ahead (§7).

Linking Cohesion Policy to European Economic Governance: an Idea Up for Improvement. European Policy Brief No. 3, September 2011

The idea of linking cohesion policy to EU economic governance has received the support of several EU institutions. The nature of such link is still to be agreed upon and is likely to lead to intense discussions. This Policy Brief argues that while the Commission’s envisaged proposal has its merits, it would nevertheless result in a partial and inconsistent link between the cohesion policy and EU economic governance. A more flexible and coherent approach is proposed.

Co-authorship Networks in Swiss Political Research

Co-authorship is an important indicator of scientific collaboration. Co-authorship networks are composed of sub-communities, and researchers can gain visibility by connecting these insulated subgroups. This article presents a comprehensive co-authorship network analysis of Swiss political science. Three levels are addressed: disciplinary cohesion and structure at large, communities, and the integrative capacity of individual researchers. The results suggest that collaboration exists across geographical and language borders even though different regions focus on complementary publication strategies. The subfield of public policy and administration has the highest integrative capacity. Co-authorship is a function of several factors, most importantly being in the same subfield. At the individual level, the analysis identifies researchers who belong to the “inner circle” of Swiss political science and who link different communities. In contrast to previous research, the analysis is based on the full set of publications of all political researchers employed in Switzerland in 2013, including past publications.