Millora de la resposta electro-mecànica per a ressonadors d'alta freqüència basats en MEMS

Report for the scientific sojourn at the Department of Micro and Nanotechnology of the Technical University of Denmark from August until December 2006. The research was focused on designing and carrying out a technological process for fabricating high frequency resonators with dielectric solid transducer gaps.

Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

From creative ideas to new emerging ventures: the process of identification and exploitation among finnish design entrepreneurs

This study focuses on identification and exploitation processes among Finnish design entrepreneurs (i.e. selfemployed industrial designers). More specifically, this study strives to find out what design entrepreneurs do when they create new ventures, how venture ideas are identified and how entrepreneurial processes are organized to identify and exploit such venture ideas in the given industrial context. Indeed, what does educated and creative individuals do when they decide to create new ventures, where do the venture ideas originally come from, and moreover, how are venture ideas identified and developed into viable business concepts that are introduced on the markets? From an academic perspective: there is a need to increase our understanding of the interaction between the identification and exploitation of emerging ventures, in this and other empirical contexts. Rather than assuming that venture ideas are constant in time, this study examines how emerging ideas are adjusted to enable exploitation in dynamic market settings. It builds on the insights from previous entrepreneurship process research. The interpretations from the theoretical discussion build on the assumption that the subprocesses of identification and exploitation interact, and moreover, they are closely entwined with each other (e.g. McKelvie & Wiklund, 2004, Davidsson, 2005). This explanation challenges the common assumption that entrepreneurs would first identify venture ideas and then exploit them (e.g. Shane, 2003). The assumption is that exploitation influences identification, just as identification influences exploitation. Based on interviews with design entrepreneurs and external actors (e.g. potential customers, suppliers and collaborators), it appears as identification and exploitation of venture ideas are carried out in close interaction between a number of actors, rather than alone by entrepreneurs. Due to their available resources, design entrepreneurs have a desire to focus on identification related activities and to find external actors that take care of exploitation related activities. The involvement of external actors may have a direct impact on decisionmaking and various activities along the processes of identification and exploitation, which is something that previous research does not particularly emphasize. For instance, Bhave (1994) suggests both operative and strategic feedback from the market, but does not explain how external parties are actually involved in the decisionmaking, and in carrying out various activities along the entrepreneurial process.

Quality control and conduct of genome-wide association meta-analyses.

Rigorous organization and quality control (QC) are necessary to facilitate successful genome-wide association meta-analyses (GWAMAs) of statistics aggregated across multiple genome-wide association studies. This protocol provides guidelines for (i) organizational aspects of GWAMAs, and for (ii) QC at the study file level, the meta-level across studies and the meta-analysis output level. Real-world examples highlight issues experienced and solutions developed by the GIANT Consortium that has conducted meta-analyses including data from 125 studies comprising more than 330,000 individuals. We provide a general protocol for conducting GWAMAs and carrying out QC to minimize errors and to guarantee maximum use of the data. We also include details for the use of a powerful and flexible software package called EasyQC. Precise timings will be greatly influenced by consortium size. For consortia of comparable size to the GIANT Consortium, this protocol takes a minimum of about 10 months to complete.

Implementación de técnicas de estimación y sincronización para sistemas OFDM: aplicación directa sobre la implementación de la capa física del estándar DVB-T

El sistema de modulación ODFM es utilizado en diversas aplicaciones de banda ancha, tanto en comunicaciones por cable como en aplicaciones inalámbricas. Presenta numerosas ventajas frente a sistemas de banda ancha de portadora única ya que permite una alta eficiencia espectral, una fácil ecualización y una reducción del ISI. Por el contrario, presenta dificultades inherentes a su estructura, que son de vital importancia solventar, entre las cuales se encuentran los altos requisitos de sincronización. Este proyecto presenta métodos de sincronización de tiempo y frecuencia implementados y evaluados sobre una plataforma software basada en Matlab®, que recoge el sistema completo de transmisión basado fielmente en el estándar DVB-T. Tras una presentación de los principios de la modulación OFDM, en este documento se presenta un estudio detallado de este sistema de transmisión y su implementación, formando conjuntamente una plataforma de simulación para la evaluación de los estimadores implementados.

Ear acupuncture and fMRI: a pilot study for assessing the specificity of auricular points.

In recent years research explored different acupuncture stimulation techniques but interest has focused primarily on somatic acupuncture and on a limited number of acupoints. As regards ear Acupuncture (EA) there is still some criticism about the clinical specificity of auricular points/areas representing organs or structures of the body. The aim of this study was to verify through (Functional magnetic resonance imaging) fMRI the hypothesis of EA point specificity using two auricular points having different topographical locations and clinical significance. Six healthy volunteers underwent two experimental fMRI sessions: the first was dedicated to the stimulation of Thumb Auricular Acupoint (TAA) and the second to the stimulation of Brain Stem Auricular Acupoint (BSAA). The stimulation of the needle placed in the TAA of the left ear produced an increase in activation bilaterally in the parietal operculum, region of the secondary somatosensory area SII. Stimulation of the needle placed in the BSAA of the left ear showed a pattern that largely overlapped regions belonging to the pain matrix, as shown to be involved in previous somatic acupuncture studies but with local differences in the left amygdala, anterior cingulate cortex, and cerebellum. The differences in activation patterns between TAA and BSAA stimulation support the specificity of the two acupoints. Moreover, the peculiarity of the regions involved in BSAA stimulation compared to those involved in the pain matrix, is in accordance with the therapeutic indications of this acupoint that include head pain, dizziness and vertigo. Our results provide preliminary evidence on the specificity of two auricular acupoints; further research is warranted by means of fMRI both in healthy volunteers and in patients carrying neurological/psychiatric syndromes.

Immunochip SNP array identifies novel genetic variants conferring susceptibility to candidaemia.

Candidaemia is the fourth most common cause of bloodstream infection, with a high mortality rate of up to 40%. Identification of host genetic factors that confer susceptibility to candidaemia may aid in designing adjunctive immunotherapeutic strategies. Here we hypothesize that variation in immune genes may predispose to candidaemia. We analyse 118,989 single-nucleotide polymorphisms (SNPs) across 186 loci known to be associated with immune-mediated diseases in the largest candidaemia cohort to date of 217 patients of European ancestry and a group of 11,920 controls. We validate the significant associations by comparison with a disease-matched control group. We observe significant association between candidaemia and SNPs in the CD58 (P = 1.97 × 10(-11); odds ratio (OR) = 4.68), LCE4A-C1orf68 (P = 1.98 × 10(-10); OR = 4.25) and TAGAP (P = 1.84 × 10(-8); OR = 2.96) loci. Individuals carrying two or more risk alleles have an increased risk for candidaemia of 19.4-fold compared with individuals carrying no risk allele. We identify three novel genetic risk factors for candidaemia, which we subsequently validate for their role in antifungal host defence.

Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family.

NR2E3, a photoreceptor-specific nuclear receptor (PNR), represses cone-specific genes and activates several rod-specific genes. In humans, mutations in NR2E3 have been associated with the recessively-inherited enhanced short-wavelength sensitive S-cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G>A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined the dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) cosegregated with the adRP-linked p.G56R mutation, and the compound heterozygotes exhibited an ESCS-like phenotype, which in 1 of the 2 cases was strikingly "milder" than the patients carrying the p.G56R mutation alone. Impaired repression of cone-specific genes by the corepressors atrophin-1 (dentatorubral-pallidoluysian atrophy [DRPLA] gene product) and atrophin-2 (arginine-glutamic acid dipeptide repeat [RERE] protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q variant to the p.G56R mutation is discussed.

Negative rebound and disinvestment effects in response to an improvement in energy efficiency in the UK economy

This paper uses a computable general equilibrium (CGE) framework to investigate the conditions under which rebound effects may occur in response to increases in energy efficiency in the UK national economy. Previous work for the UK has suggested that rebound effects will occur even where key elasticities of substitution in production are set close to zero. The research reported in this paper involves carrying out a systematic sensitivity analysis, where relative price sensitivity is gradually introduced into the system, focusing specifically on elasticities of substitution in production and trade parameters, in order to determine conditions under which rebound effects become a likely outcome. The main result is that, while there is positive pressure for rebound effects even where (direct and indirect) demands for energy are very price inelastic, this may be partially or wholly offset by negative income, competitiveness and disinvestment effects, which also occur in response to falling energy prices. The occurrence of disinvestment effects is of particular interest. These occur where falling energy prices reduce profitability in domestic energy supply sectors, leading to a contraction in capital stock in these sectors, which may in turn lead to rebound effects that are smaller in the long run than in the short run, a result that runs contrary to the predictions of previous theoretical work in this area.

Mass spectrometry as a rapid and powerful alternative to antibodies for detecting LPXTG wall-associated proteins of Staphylococcus aureus

Functional characterization of transformed or natively present bacterial virulence proteins can be achieved employing various model systems. A prerequisite is to verify the correct expression of the transformed protein or the presence of the native protein in the microbe. Traditionally, antibodies are raised against the protein or a peptide thereof, followed by Western blot analysis or by fluorescence-activated cell sorting. Alternatively, the protein-coding gene can be fused with a downstream reporter gene, the expression of which reports the simultaneous expression of the upstream recombinant protein. Although being powerful, these methods are time consuming, especially when multiple proteins must be assessed. Here we describe a novel way to validate the expression of Gram-positive surface proteins covalently attached to the peptidoglycan. Eighteen out of the 21 known LPXTG-motif carrying cell wall-associated proteins of Staphylococcus aureus were cloned in Lactoccocus lactis either alone, in combinations or as truncated forms, and their correct expression was assessed by liquid chromatography coupled to mass spectrometry (LC-MS). The method is rapid, sensitive and precise. It can identify multiple proteins in transformed constructs without the time and cost needed for raising and testing multiple sets of antibodies.

Solid-phase synthesis and NMR studies of modified oligonucleotides forming triplex helix and of oligonucleopeptides mimicking the topoisomerase I-DNA covalent complex

Report for the scientific sojourn carried out at the Institut de Biologia Molecular de Barcelona of the CSIC –state agency – from april until september 2007. Topoisomerase I is an essential nuclear enzyme that modulates the topological status of DNA, facilitating DNA helix unwinding during replication and transcription. We have prepared the oligonucleotide-peptide conjugate Ac-NLeu-Asn-Tyr(p-3’TTCAGAAGC5’)-LeuC-CONH-(CH2)6-OH as model compound for NMR studies of the Topoisomerase I- DNA complex. Special attention was made on the synthetic aspects for the preparation of this challenging compound especially solid supports and protecting groups. The desired peptide was obtained although we did not achieve the amount of the conjugate needed for NMR studies. Most probably the low yield is due to the intrinsic sensitive to hydrolysis of the phosphate bond between oligonucleotide and tyrosine. We have started the synthesis and the structural characterization of oligonucleotides carrying intercalating compounds. At the present state we have obtained model duplex and quadruplex sequences modified with acridine and NMR studies are underway. In addition to this project we have successfully resolved the structure of a fusion peptide derived from hepatitis C virus envelope synthesized by the group of Dr. Haro and we have synthesized and started the characterization of a modified G-quadruplex.

Ecological-economic modelling of interactions between wild and commercial bees and pesticide use

The decline in extent of wild pollinators in recent years has been partly associated with changing farm practices and in particular with increase of pesticide use. In this paper we combine ecological modelling with economic analysis of a single farm output under the assumption that both pollination and pest control are essential inputs. We show that the drive to increase farm output can lead to a local decline in the wild bee population. Commercial bees are often considered an alternative to wild pollinators, but we show that their introduction can lead to further decline and finally local extinction of wild bees. The transitions between different outcomes are characterised by threshold behaviour and are potentially difficult to predict and detect in advance. Small changes in economic (input prices) and ecological (wild bees carrying capacity and effect of pesticides on bees) can move the economic-ecological system beyond the extinction threshold. We also show that increasing the pesticide price or decreasing the commercial bee price might lead to reestablishment of wild bees following their local extinction. Thus, we demonstrate the importance of combining ecological modelling with economics to study the provision of ecosystem services and to inform sustainable management of ecosystem service providers.

Chemical genetic analysis of cdc2p : new insights into the regulation of cytokinesis in "Schizosaccharomyces pombe"

RÉSUMÉ La protéine kinase cyciine-cdc2p (Cdk) joue un rôle fondamental dans la progression du cycle cellulaire dans la levure de fission Schizosaccharomyces pombe. Nous avons étudié le rôle de cdc2p dans la régulation de la cascade de septation ou SIN (septation initiation network) en mitose et en méiose. Le SIN contrôle l'initiation de la cytokinèse à la fin de la mitose, et est supposé être négativement régulé par cdc2p. Nous avons mutagénéisé le site actif de cdc2p afin qu'il puisse lier un analogue de l'ATP (PP1) qui agit comme inhibiteur. Cet analogue ne peut pas lier la kinase de type sauvage. Cette approche dite «chemical genetics» permet une meilleure résolution temporelle comparée à l'approche classique utilisant les mutants sensibles à une température élevée. Nous avons montré que ce mutant cdc2-as (analogue sensitive) est fonctionnel et que, in vitro, l'activité kinase est inhibée en présence de l'analogue. Les cellules portant cette mutation, contrairement aux cellules de type sauvage s'arrêtent de manière irréversible soit en G2 soit en G1 et G2, suivant la concentration de l'inhibiteur. L'inactivation de cdc2p-as dans des cellules arrêtées en métaphase conduit au recrutement asymétrique des protéines du SIN sur le pôle du fuseau mitotique et au recrutement des composants du SIN, ainsi que de la ß-(1,3)glucan synthase à l'anneau contractile. De plus, nos résultats montrent que l'orthologue de la phosphatase cdc14p dans S. pombe, fip1p/clp1p, joue un rôle dans la régulation de la localisation des protéines du SIN suite à l'inactivation de cdc2p. Finalement, l'activité de cdc2p est requise pour maintenir la polo-like kinase plo1p sur les pôles du fuseau mitotique dans les premiers stages de la mitose. C'est pourquoi nous concluons que l'inactivation de cdc2p est suffisante pour activer le SIN et promouvoir la cytokinèse. Dans une étude séparée, nous avons caractérisé des potentiellement nouveaux composants ou régulateurs du SIN qui ont été isolés dans deux criblages génétiques visant à isoler des mutants atténuants la signalisation du SIN. Summary : The cyclin dependent protein kinase (Cdk) cdc2p plays a central role in the cell cycle progression of fission yeast Schizosaccharomyces pombe. We have studied the role of cdc2p in regulating the septation initiation network (SIN) in mitosis and meiosis. The SIN regulates the initiation of cytokinesis at the end of mitosis and is thought to be inhibited by cdc2p. We have mutated the active site of cdc2p to permit binding of an inhibitory ATP analogue (PP1), which is unable to bind unmodified kinases. This "chemical genetic" approach provides a much higher temporal resolution than it can be achieved with classical temperature-sensitive mutants. We demonstrate that cdc2-as (analogue sensitive) is functional and that addition of PP1 inhibits cdc2p kinase activity in vitro. Cells carrying the cdc2-as allele, but not cdc2+, undergo reversible cell cycle arrest following addition of PP1 either in G2, or at both major commitment points in the cell cycle (G1 and G2), depending upon the concentration of PP1. Inactivation of cdc2p-as in cells arrested in early mitosis promotes both the asymmetric recruitment of SIN proteins to the spindle pole bodies (SPBs), and the recruitment of the most downstream SIN components and ß-(1,3)-glucan synthase to the contractile ring. Furthermore, our results indicate that the S. pombe orthologue of Cdc14p, flp1p/clp1p, plays a role in regulating the relocalisation of SIN proteins following inactivation of cdc2p, and that cdc2p activity is required to retain the polo like kinase plot p on the SPBs in early mitosis. Thus, we conclude that inactivation of cdc2p is sufficient to activate the SIN and to promote cytokinesis. In a separate study, we have initially characterised potential novel components or regulators of the SIN pathway identified by two genetic screens for mutants attenuating SIN signaling.

Pets as the main source of two zoonotic species of the Trichophyton mentagrophytes complex in Switzerland, Arthroderma vanbreuseghemii and Arthroderma benhamiae

In cases of highly inflammatory dermatophytosis in humans, it is important to identify the possible source of animal transmission in order to prevent recurrence, family outbreaks or rapidly progressing epidemics. A survey of dermatophytes in pets during a 14-month period in Switzerland revealed, in addition to Microsporum canis, two different species of the Trichophyton mentagrophytes complex, Arthroderma benhamiae and Arthroderma vanbreuseghemii, all causing inflammatory dermatophytoses. Arthroderma benhamiae was only and frequently isolated from guinea pigs. Arthroderma vanbreuseghemii was isolated mainly from European short hair cats, but also from dogs and in one case from a pure-bred cat. Ninety-three percent of the cats carrying A. vanbreuseghemii were hunters and all had skin lesions. In contrast, cats with skin lesions that were strictly indoors were found to be almost exclusively infected by M. canis. Therefore, it can be suspected that infection with A. vanbreuseghemii occurred during hunting and that the natural source of this dermatophyte is either soil or an animal other than the cat, most probably a rodent.

PSMA-Specific CAR-Engineered T Cells Eradicate Disseminated Prostate Cancer in Preclinical Models.

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting.