Hepatitis C virus proteins: from structure to function.

Great progress has been made over the past years in elucidating the structure and function of the hepatitis C virus (HCV) proteins, most of which are now actively being pursued as antiviral targets. The structural proteins, which form the viral particle, include the core protein and the envelope glycoproteins E1 and E2. The nonstructural proteins include the p7 viroporin, the NS2 protease, the NS3-4A complex harboring protease and NTPase/RNA helicase activities, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. NS4B is a master organizer of replication complex formation while NS5A is a zinc-containing phosphoprotein involved in the regulation of HCV RNA replication versus particle production. Core to NS2 make up the assembly module while NS3 to NS5B represent the replication module (replicase). However, HCV proteins exert multiple functions during the viral life cycle, and these may be governed by different structural conformations and/or interactions with viral and/or cellular partners. Remarkably, each viral protein is anchored to intracellular membranes via specific determinants that are essential to protein function in the cell. This review summarizes current knowledge of the structure and function of the HCV proteins and highlights recent advances in the field.

OSIRISv1.2: a named entity recognition system for sequence variants of genes in biomedical literature

Background: Single Nucleotide Polymorphisms, among other type of sequence variants, constitute key elements in genetic epidemiology and pharmacogenomics. While sequence data about genetic variation is found at databases such as dbSNP, clues about the functional and phenotypic consequences of the variations are generally found in biomedical literature. The identification of the relevant documents and the extraction of the information from them are hampered by the large size of literature databases and the lack of widely accepted standard notation for biomedical entities. Thus, automatic systems for the identification of citations of allelic variants of genes in biomedical texts are required. Results: Our group has previously reported the development of OSIRIS, a system aimed at the retrieval of literature about allelic variants of genes http://ibi.imim.es/osirisform.html. Here we describe the development of a new version of OSIRIS (OSIRISv1.2, http://ibi.imim.es/OSIRISv1.2.html webcite) which incorporates a new entity recognition module and is built on top of a local mirror of the MEDLINE collection and HgenetInfoDB: a database that collects data on human gene sequence variations. The new entity recognition module is based on a pattern-based search algorithm for the identification of variation terms in the texts and their mapping to dbSNP identifiers. The performance of OSIRISv1.2 was evaluated on a manually annotated corpus, resulting in 99% precision, 82% recall, and an F-score of 0.89. As an example, the application of the system for collecting literature citations for the allelic variants of genes related to the diseases intracranial aneurysm and breast cancer is presented. Conclusion: OSIRISv1.2 can be used to link literature references to dbSNP database entries with high accuracy, and therefore is suitable for collecting current knowledge on gene sequence variations and supporting the functional annotation of variation databases. The application of OSIRISv1.2 in combination with controlled vocabularies like MeSH provides a way to identify associations of biomedical interest, such as those that relate SNPs with diseases.

Asymmetries in the opportunity structure. Intergenerational mobility trends in Europe

It remains unclear whether social mobility is increasing in the advancednations. The answer may depend on mobility patterns within very recentbirth cohorts. We use the inter-generational module in the 2005 EUSILCwhich allows us to include more recent cohorts. Comparingacross two Nordic and three Continental European countries, weestimate inter-generational mobility trends for sons both indirectly, viasocial origin effects on educational attainment, and directly in terms ofadult income attainment. In line with other studies we find substantiallymore mobility in Scandinavia, but also that traditionally less mobilesocieties, like Spain, are moving towards greater equality. We focusparticularly on non-linear relations. Most interestingly, we revealevident asymmetries in the process of equalizing life chances, inDenmark. The disadvantages associated with low social class originshave largely disappeared, but the advantages related to privilegedorigins persist.

Characterization of N-cadherin association to lipid rafts in melanoma

RESUME La peau est un organe complex composé de deux parties distinctes: l'épiderme et le derme, séparé par une membrane basale. Dans la couche basale de l'épiderme, les melanocytes synthétisent la mélanine dans des mélanosomes. Les mélanosomes sont ensuite transportés des mélanocytes vers les kératinocytes, protégeant ainsi la peau des dégâts dus aux radiations U.V. La E-cadhérine assure l'adhésion entre les mélanocytes et les kératinocytes. Au cours de la transformation du mélanocyte en cellule malignes, les mélanocytes perdent l'expression de la E-cadhérine et, simultanément, se mettent à exprimer la N-cadhérine, ce phénomène est nommé « cadherin switch ». La perte de l'expression de la E-cadhérine permet au mélanocytes d'échapper au contrôle des kératinocytes, tandis que l'expression de la N-cadhérine promeut l'invasion métastasique des cellules de mélanome. Préalablement, nous avons trouvé qu'une fraction de la N-cadhérine était localisée les microdomaines membranaires spécialisés, enrichi en cholestérol et en glycosphingolipides, appelés « lipid rafts ». Une des particularité des « lipid rafts » est qu'ils sont riches en molécules permettant la transmission de signaux d'activation. De plus, des travaux récents rapportent qu'un sous-type de « lipid rafts » appelé caveolae pourrai contribuer à la progression tumorale. S'appuyant sur le rôle prépondérant de la N-cadhérine dans la progression du mélanome ainsi que sur sa présence dans les « lipid rafts », nous avons émis l'hypothèse que l'association de la N-cadhérine avec les « lipid rafts » pourrai contribuer à la progression du mélanome. Le but de ce projet à été de caractériser l'association de la Ncadhérine avec les « lipid rafts » au cours de la progression du mélanome. Au moyen de lignées cellulaires humaines, dérivées de mélanomes à différents stades de progression, nous avons trouvé que (1) la N-cadhérine est partiellement associée aux «lipid rafts » dans six lignées dérivées de mélanome en phase avancée de progression et dans des tumeurs expérimentales, mais pas dans deux lignées dérivées de mélanome à un stade plus précoce ; (2) l'association de la N-cadhérine dans les « lipid rafts » ne dépent pas de son niveau d'expression ; (3) la E-cadhérine n'est pas présente dans les « lipid rafts »d'une lignée de cellule de mélanome ayant conservé l'expression de la E-cadhérine ; (4) la localisation de la N-cadhérine dans les « lipid rafts »n'est pas modulée par les facteurs de croissance bFGF, IGF-I, et HRG1-β1, ni par des voies de signalisation impliquant MEK, PKA, les kinases de la famille Src, et PI3K ; (5) l'association de la N-cadhérine avec les « lipid rafts » n'est pas requise pour la stabilisation des jonctions adhérentes et n'est pas perturbée par la destruction de ces dernières ; (6) la N-cadhérine dans les « lipid rafts » forme un complexe avec β-caténine, p 120ctn et α-caténine. En conclusion, cette étude originale montre pour la première fois que dans des cellules de mélanome agressifs, une fraction de la N-cadhérine est localisée dans les « lipid rafts » en association avec β-caténine, p 120ctn et α-caténine. Comme la présence de la N-cadhérine dans les « lipid rafts » ne contribue pas à la formation de jonction adhérentes, cette étude suggère une nouvelle fonction pour la N-cadhérine dans les « lipid rafts ». SUMMARY Human skin is a complex organ composed of two layers separated by a basement membrane: the epidermis and the dermis. In the basal layer of the epidermis, the melanin-producing cells of the skin, the melanocytes deliver melanin-containing melanosomes to keratinocytes, thereby protecting the epidermis and the dermis from the deleterious effects of ultraviolet light. Melanocytes physically interact with keratinocytes through E-cadherin-mediated adhesion. During malignant transformation into melanoma cells, melanocytes lose E-cadherin expression and concomitantly gain expression of N-cadherin, a phenomenon referred to as "cadherin switch". Loss of E-cadherin allows melanocytes to escape the regulatory effects of neighbouring keratinocytes, while gain of N-cadherin expression promotes migration, invasion and metastatic abilities of melanoma cells. In preliminary experiments, we found that a fraction of N-cadherin localized to specialized membrane microdomains enriched in cholesterol- and glycosphingolipid, called lipid rafts. One particular feature of lipid rafts is that they are rich in signalling molecules and they possibly modulate transmembrane signalling events. Moreover, recent reports suggested that a specialized type of rafts called caveolae might contribute to tumor progression. Based on the documented role of N-cadherin in melanoma progression and its presence in lipid rafts of melanoma cells, we raised the hypothesis that the association of N-cadherin with lipid rafts might be relevant to melanoma progression. The aim of this project was to characterize N-cadherin associated to lipid rafts during melanoma progression. Using human melanoma cell lines derived from melanoma at different stages of progression, we found that (1) N-cadherin is partly associated to lipid rafts in six cell lines derived from melanomas at late stages of progression and in experimental tumors, but not in two melanoma cell lines derived from early stages; (2) N-cadherin targeting to lipid rafts does not depend on its expression level; (3) E-cadherin is not localized in lipid rafts of a melanoma cell line that retained E-cadherin expression; (4) N-cadherin localization to lipid rafts is not modulated by the growth factors bFGF, IGF-I, and HRG1-β1, nor by MEK-, PKA-, Src family kinases-, and PI3K-mediated signalling events; (5) the association of N-cadherin with lipid rafts is not required for adherens junctions stability nor it is perturbed by adherens junctions disruption; (6) N-cadherin in lipid rafts is in complex with β-catenin, p 120ctm and α-catenin. In conclusion, this study provides original evidence that in aggressive melanoma cells a pool of N-cadherin is localized in lipid rafts in association with β-catenin, p 120 and α-catenin. The presence of N-cadherin in lipid rafts independently of its involvement in adherens junctions formation, suggests a possible new role for N-cadherin recruited to lipid rafts. Further studies investigating the biological meaning of this localization promise to uncover new properties of this molecule.

Introduction d'un questionnaire de dépistage de l'Etat de stress post traumatique (ESPT) : quel impact sur le diagnostic dans une unité d'urgence psychiatrique ambulatoire ?

Contexte: Impression clinique que l'Etat de Stress Post-traumatique (ESPT) est sous-diagnostiqué dans la prise en charge des patients qui sont évalués dans le cadre d'une urgence psychiatrique. Objectifs: (i) identifier la prévalence de l'ESPT dans une unité d'urgence psychiatrique au moyen d'un instrument diagnostic et la comparer avec le diagnostic clinique retenu dans un échantillon historique (ii) évaluer la perception des cliniciens quant à l'utilisation systématique d'un instrument diagnostic Méthodes: la prévalence de l'ESPT a été évaluée chez des patients consécutifs (N = 403) qui ont bénéficié d'une consultation par des psychiatres de l'Unité urgence-crise du Service de Psychiatrie de Liaison (PLI) du CHUV, en utilisant le module J du Mini Mental Neuropsychologic Interview (MINI 5.0.0, version CIM-10). Ce résultat a été comparé avec la prévalence de l'ESPT mentionné comme diagnostic dans les dossiers (N = 350) d'un échantillon historique. La perception des médecins-assistants de psychiatrie quant au dépistage systématique de l'ESPT avec un instrument a été étudiée en se basant sur la conduite d'un focus group d'assistants travaillant dans l'Unité urgence-crise du PLI. Résultats: Parmi les patients (N = 316) évalués à l'aide de l'instrument diagnostic, 20,3% (n = 64) réunissaient les critères de l'ESPT. Cela constitue un taux de prévalence significativement plus élevé que la prévalence d'ESPT documentée dans les dossiers de l'échantillon historique (0,57%). Par ailleurs, la prévalence de l'ESPT est significativement plus élevée parmi les groupes socio- économiques précarisés, tels que réfugiés et sans papiers (50%), patients venant d'un pays à histoire de guerre récente (47,1%), patients avec quatre (44,4%) ou trois comorbidités psychiatriques (35,3%), migrants (29,8%) et patients sans revenus professionnels (25%). Le focus groupe composé de 8 médecins-assistants a révélé que l'utilisation systématique d'un outil- diagnostic ne convenait pas dans le setting d'urgence psychiatrique, notamment parce que l'instrument a été considéré comme non adapté à une première consultation ou jugé avoir un impact négatif sur l'entretien clinique. Toutefois, après la fin de l'étude, les médecins-assistants estimaient qu'il était important de rechercher activement l'ESPT et continuaient à intégrer les éléments principaux du questionnaire dans leur travail clinique. Conclusion et perspectives: cette étude confirme que l'ESPT est largement sous-diagnostiqué dans le contexte des urgences psychiatriques, mais que l'usage systématique d'un outil diagnostic dans ce cadre ne satisfait pas les praticiens concernés. Pour améliorer la situation et au vu du fait qu'un instrument diagnostic est considéré comme non-adapté dans ce setting, il serait peut-être bénéfique d'envisager un dépistage ciblé et/ou de mettre en place une stratégie de formation institutionnelle.

ACS800-kaappimoduulien pakkaamon ulkoistaminen

Tässä insinöörityössä selvitetään ABB Oy System AC -liiketoimintayksikön pakkaamon ulkoistamiseen liittyviä ongelmia sekä mahdollisia ratkaisuja näihin ongelmiin. Koko ongelman käsittely on liian laaja projektin yhdelle insinöörityölle. Tässä työssä keskitytään ratkaisemaan ko. ulkoistamiseen liittyvät keskeisimmät ACS800-kaappimoduulien kuljetuslogistiset ongelmat. Näitä ongelmia ovat kaappimoduulien kiinnitystarve kuljetusalustaansa sekä kuljetuspaketoinnin puuttuessa kaappimoduulien suojaaminen maantiekuljetuksen ajaksi. Työssä on otettu huomioon viranomaisten maantiekuljetuksia koskevat vaatimukset. Työssä on suunniteltu uusia kiinnitysvälineitä sekä muutoksia olemassa oleviin kuljetusalustoihin, joita käytetään tehtaan sisäisissä kaappimoduulien siirroissa. Kiinnitysvälineistä on tehty lujuuslaskelmia ja määritelty kiristysmomentteja. Tuotettavuuden ja kustannustehokkuuden kannalta on selvitetty kiinnitysvälineisiin soveltuvia materiaalivaihtoehtoja. Kuljetusalustojen muutoksista on myös tehty ohjeistusta. Tämän työn tulos on uusien kiinnitysvälineiden ja kuljetusalustojen mekaanisten muutosten suunnittelu. Samalla on todistettu, että kaappimoduulien siirto tehtaan ulkopuoliseen pakkaamoon ilman kuljetuspaketointia on teoriassa mahdollista.

VIKI : a semiotic-based system for multilingual knowledge retrieval

Abstract Since its creation, the Internet has permeated our daily life. The web is omnipresent for communication, research and organization. This exploitation has resulted in the rapid development of the Internet. Nowadays, the Internet is the biggest container of resources. Information databases such as Wikipedia, Dmoz and the open data available on the net are a great informational potentiality for mankind. The easy and free web access is one of the major feature characterizing the Internet culture. Ten years earlier, the web was completely dominated by English. Today, the web community is no longer only English speaking but it is becoming a genuinely multilingual community. The availability of content is intertwined with the availability of logical organizations (ontologies) for which multilinguality plays a fundamental role. In this work we introduce a very high-level logical organization fully based on semiotic assumptions. We thus present the theoretical foundations as well as the ontology itself, named Linguistic Meta-Model. The most important feature of Linguistic Meta-Model is its ability to support the representation of different knowledge sources developed according to different underlying semiotic theories. This is possible because mast knowledge representation schemata, either formal or informal, can be put into the context of the so-called semiotic triangle. In order to show the main characteristics of Linguistic Meta-Model from a practical paint of view, we developed VIKI (Virtual Intelligence for Knowledge Induction). VIKI is a work-in-progress system aiming at exploiting the Linguistic Meta-Model structure for knowledge expansion. It is a modular system in which each module accomplishes a natural language processing task, from terminology extraction to knowledge retrieval. VIKI is a supporting system to Linguistic Meta-Model and its main task is to give some empirical evidence regarding the use of Linguistic Meta-Model without claiming to be thorough.

The hematology laboratory in blood doping (bd): 2014 update on the athlete biological passport (APB)

Introduction: Blood doping (BD) is the use of Erythropoietic Stimulating Agents (ESAs) and/or transfusion to increase aerobic performance in athletes. Direct toxicologic techniques are insufficient to unmask sophisticated doping protocols. The Hematological module of the ABP (World Anti-Doping Agency), associates decision support technology and expert assessment to indirectly detect BD hematological effects. Methods: The ABP module is based on blood parameters, under strict pre-analytical and analytical rules for collection, storage and transport at 2-12°C, internal and external QC. Accuracy, reproducibility and interlaboratory harmonization fulfill forensic standard. Blood samples are collected in competition and out-ofcompetition. Primary parameters for longitudinal monitoring are: - hemoglobin (HGB); - reticulocyte percentage (RET); - OFF score, indicator of suppressed erythropoiesis, calculated as [HGB(g/L) * 60-√RET%]. Statistical calculation predicts individual expected limits by probabilistic inference. Secondary parameters are RBC, HCT, MCHC-MCH-MCV-RDW-IFR. ABP profiles flagged as atypical are review by experts in hematology, pharmacology, sports medicine or physiology, and classified as: - normal - suspect (to target) - likely due to BD - likely due to pathology. Results: Thousands of athletes worldwide are currently monitored. Since 2010, at least 35 athletes have been sanctioned and others are prosecuted on the sole basis of abnormal ABP, with a 240% increase of positivity to direct tests for ESA, thanks to improved targeting of suspicious athletes (WADA data). Specific doping scenarios have been identified by the Experts (Table and Figure). Figure. Typical HGB and RET profiles in two highly suspicious athletes. A. Sample 2: simultaneous increases in HGB and RET (likely ESA stimulation) in a male. B. Samples 3, 6 and 7: "OFF" picture, with high HGB and low RET in a female. Sample 10: normal HGB and increased RET (ESA or blood withdrawal). Conclusions: ABP is a powerful tool for indirect doping detection, based on the recognition of specific, unphysiological changes triggered by blood doping. The effect of factors of heterogeneity, such as sex and altitude, must also be considered. Schumacher YO, et al. Drug Test Anal 2012, 4:846-853. Sottas PE, et al. Clin Chem 2011, 57:969-976.

Neuronal responses in mouse barrel cortex:Comparison of two signal discriminators

Barrels are discrete cytoarchitectonic neurons cluster located in the layer IV of the somatosensory¦cortex in mice brain. Each barrel is related to a specific whisker located on the mouse snout. The¦whisker-to-barrel pathway is a part of the somatosensory system that is intensively used to explore¦sensory activation induced plasticity in the cerebral cortex.¦Different recording methods exist to explore the cortical response induced by whisker deflection in¦the cortex of anesthetized mice. In this work, we used a method called the Single-Unit Analysis by¦which we recorded the extracellular electric signals of a single barrel neuron using a microelectrode.¦After recording the signal was processed by discriminators to isolate specific neuronal shape (action¦potentials).¦The objective of this thesis was to familiarize with the barrel cortex recording during whisker¦deflection and its theoretical background and to compare two different ways of discriminating and¦sorting cortical signal, the Waveform Window Discriminator (WWD) or the Spike Shape Discriminator (SSD).¦WWD is an electric module allowing the selection of specific electric signal shape. A trigger and a¦window potential level are set manually. During measurements, every time the electric signal passes¦through the two levels a dot is generated on time line. It was the method used in previous¦extracellular recording study in the Département de Biologie Cellulaire et de Morphologie (DBCM) in¦Lausanne.¦SSD is a function provided by the signal analysis software Spike2 (Cambridge Electronic Design). The¦neuronal signal is discriminated by a complex algorithm allowing the creation of specific templates.¦Each of these templates is supposed to correspond to a cell response profile. The templates are saved¦as a number of points (62 in this study) and are set for each new cortical location. During¦measurements, every time the cortical recorded signal corresponds to a defined number of templates¦points (60% in this study) a dot is generated on time line. The advantage of the SSD is that multiple¦templates can be used during a single stimulation, allowing a simultaneous recording of multiple¦signals.¦It exists different ways to represent data after discrimination and sorting. The most commonly used¦in the Single-Unit Analysis of the barrel cortex are the representation of the time between stimulation¦and the first cell response (the latency), the representation of the Response Magnitude (RM) after¦whisker deflection corrected for spontaneous activity and the representation of the time distribution¦of neuronal spikes on time axis after whisker stimulation (Peri-Stimulus Time Histogram, PSTH).¦The results show that the RMs and the latencies in layer IV were significantly different between the¦WWD and the SSD discriminated signal. The temporal distribution of the latencies shows that the¦different values were included between 6 and 60ms with no peak value for SSD while the WWD¦data were all gathered around a peak of 11ms (corresponding to previous studies). The scattered¦distribution of the latencies recorded with the SSD did not correspond to a cell response.¦The SSD appears to be a powerful tool for signal sorting but we do not succeed to use it for the¦Single-Unit Analysis extracellular recordings. Further recordings with different SSD templates settings¦and larger sample size may help to show the utility of this tool in Single-Unit Analysis studies.

Coaching como factor de Diferenciação nas Organizações

O mundo empresarial tem sido alvo de mudanças impulsionadas pela globalização. Neste sentido, as empresas que pretendem sobreviver e atingir um bom nível de competitividade devem focalizar-se na capacitação, no desenvolvimento e na motivação constante dos seus colaboradores. Devido à importância dos recursos humanos como diferencial competitivo, o processo de coaching como modelo de gestão da mudança, tem vindo a merecer muita importância no contexto organizacional. O processo de coaching em consonância com programas de desenvolvimento da liderança no contexto organizacional/empresarial visa ajudar os colaboradores a desenvolverem novas atitudes e competências ao alcance das metas organizacionais. O intuito deste trabalho é apresentar esclarecimentos acerca do termo, abordando os aspectos mais relevantes e o seu respectivo impacto no contexto organizacional. Realizou-se um Estudo de Caso baseado numa empresa de produção e prestação de serviços em São Vicente, com a finalidade de avaliar a motivação dos seus colaboradores, através da aplicação de um questionário aos mesmos. Na sequência da análise dos resultados sugeriu-se um modelo que mais se adapta à realidade da empresa. The business world has been challenged by several changes driven by globalization. Considering the present situation, companies that intend to survive and achieve a somewhat good level of competiveness should focus on their employee’s constant capacity building, development and motivation. Considering the Human Resources importance as a competitive differential, the coaching process as a change management module has been a very importance on the organizational context. The coaching process together with the leadership development programmes on the organizational/entrepreneurial context enables the employees to develop new attitudes and skills aiming for organizational goals. This paper purpose is to enlighten the reader on the subject, addressing the most relevant issues and its impact on the organizational context. We’ve conducted a Case Study, of a production and service company on S. Vicente Island, aiming to assess the employee’s motivation, through the application of a questionnaire. Following the results analysis, a model was proposed to better fit the company’s reality.

GENETIC AND BIOCHEMICAL ANALYSIS OF THE TESTIS-SPECIFIC ZINC FINGER PROTEIN CTCFL/BORIS

Abstract en FrançaisCTCFL a d'abord été identifié comme un paralogue de la protéine ubiquitaire CTCF en raison de sa forte homologie entre leurs onze « zinc fingers », un domaine de liaison à l'ADN. Parmi ses nombreux rôles, la liaison des zinc fingers de CTCF à la région de contrôle de l'empreinte (ICR) maternelle non-méthylée Igf2/H19, contrôle l'expression empreinte (monoallélique) de H19 et IGF2 dans les cellules somatiques. La méthylation de l'ICR Igf2/H19 paternelle est nécessaire à l'expression empreinte de ces deux gènes. Bien que le mécanisme par lequel l'ICR est méthylé soit mal compris, il est connu que l'établissement de la méthylation se produit pendant le développement des cellules germinales mâles et que les ADN méthyltransférases de novo DNMT3A et DNMT3L sont essentiels. Par conséquent, CTCFL fournit un bon candidat pour un rôle dans la méthylation de l'ICR paternelle Igf2/H19 en raison de son expression restreinte à certains types de cellules où la méthylation de l'ICR a lieu (spermatogonies et spermatocytes) ainsi qu'en raison sa capacité à lier les ICR lgf2/HÎ9 dans ces cellules. Les premiers travaux expérimentaux de cette thèse portent sur le rôle possible des mutations de CTCFL chez les patients atteints du syndrome de Silver-Russell (SRS), où une diminution de la méthylation de l'ICR IGF2/H19 a été observée chez 60% d'entre eux. Admettant que CTCFL pourrait être muté chez ces patients, j'ai examiné les mutations possibles de CTCFL chez 35 d'entre eux par séquençage de l'ADN et analyse du nombre de copies d'exons. N'ayant trouvé aucune mutation chez ces patients, cela suggère que les mutations de CTCFL ne sont pas associées au SRS. Les travaux expérimentaux suivants ont porté sur les modifications post-traductionnelles de CTCFL par la protéine SU MO « small ubiquitin-like modifier » (SUMO). La modification de protéines par SU MO change les interactions avec d'autres molécules (ADN ou protéines). Comme CTCFL régule sans doute l'expression d'un certain nombre de gènes dans le cancer et que plusieurs facteurs de transcription sont régulés par SUMO, j'ai mené des expériences pour déterminer si CTCFL est sumoylé. En effet, j'ai observé que CTCFL est sumoylated in vitro et in vivo et j'ai déterminé les deux résidus d'attachement de SUMO aux lysines 181 et 645. Utilisant les mutants de CTCFL K181R et K645R ne pouvant pas être sumoylated, j'ai évalué les conséquences fonctionnelles de la modification par SUMO. Je n'ai trouvé aucun changement significatif dans la localisation subcellulaire, la demi-vie ou la liaison à l'ADN, mais ai constaté que la sumoylation module à la fois {'activation CTCFL-dépendante et la répression de l'expression génique. Il s'agit de la première modification post-traductionnelle décrite pour CTCFL et les conséquences possibles de cette modification sont discutées pour le cancer et les testicules normaux. Avec cette thèse, j'espère avoir ajouté des résultats importants à l'étude de CTCFL et donné quelques idées pour de futures recherches.AbstractJeremiah Bernier-Latmani, Institute of Pathology, University of Lausanne, CHUVCTCFL was first identified as a paralog of the ubiquitous protein CTCF because of high homology between their respective eleven zinc fingers, a DNA binding domain. Among its many roles, CTCF zinc finger-mediated binding to the unmethylated maternal Igf2/H19 imprinting control region (ICR), controls the imprinted (monoallelic) expression of Igf2 and H19 in somatic cells. Methylation of the paternal Igf2/H19 ICR is necessary for the imprinted expression of the two genes. Although the mechanism by which the ICR is methylated is incompletely understood, it is known that establishment of methylation occurs during male germ cell development and the de novo DNA methyltransferases DNMT3A and DNMT3L are essential. Therefore, CTCFL provided a good candidate to play a role in methylation of the paternal Igf2/H19 ICR because of its restricted expression to cell types where ICR methylation takes place (spermatogonia and spermatocytes) and its ability to bind the Igf2/H19 ICR in these cells. The first experimental work of this thesis investigated the possible role of CTCFL mutations in Silver-Russell syndrome (SRS) patients, where it has been observed that 60% of the patients have reduced methylation of the IGF2/HÎ9 ICR. Reasoning that CTCFL could be mutated in these patients, I screened 35 patients for mutations in CTCFL by DNA sequencing and exon copy number analysis, I did not find any mutations in these patients suggesting that mutations of CTCFL are not associated with SRS. The next experimental work of my thesis focused on posttranslational modification of CTCFL by small ubiquitin-like modifier (SUMO) protein. SUMO modification of proteins changes the interactions with other molecules (DNA or protein). As CTCFL arguably regulates the expression of a number of genes in cancer and many transcription factors are regulated by SUMO, I conducted experiments to assess whether CTCFL is sumoylated. I found that CTCFL is sumoylated in vitro and in vivo and determined the two residues of SUMO attachment to be lysines 181 and 645. Using K181R, K645R mutated CTCFL- which cannot be detected to be sumoylated-1 assessed the functional consequences of SUMO modification. I found no significant changes in subcellular localization, half-life or DNA binding, but found that sumoylation modulates both CTCFL-dependent activation and repression of gene expression. This is the first posttranslational modification described for CTCFL and possible consequences of this modification are discussed in both cancer and normal testis. With this thesis, I hope I have added important findings to the study of CTCFL and provide some ideas for future research.

The use of textbooks in Capeverdean secondary schools: The Relationship between Use, Evaluation and Adaptation of Textbooks

This study examines the relationship between teacher’s use of English textbooks and the way teachers evaluate and adapt them, looking at a particular context, the Capeverdean secondary schools, specifically in Praia. The referred relationship was analyzed through teachers’ responses about how they use, evaluate and adapt their textbooks. The results of the study revealed that, on the one hand, the way teachers use their textbooks influences the way they evaluate the same textbooks; on the other hand, the use of textbooks doesn’t necessarily influence the way teachers adapt them. Moreover, the findings revealed that, in general, due to some particular constraints the Capeverdean English teachers are using their textbooks as resources, in which several textbooks are used in combination with one another. Additionally, although teachers assume that they are doing their best, they still need more confidence concerning the way they use, evaluate and adapt available textbooks. Teachers’ confidence in the way they are using their textbooks can be reinforced by establishing an intensive teacher training module on materials evaluation and adaptation, taking into account that a textbook is one of the most important tools in the process of teaching and learning. I hope that the elements presented may lead to further studies on this matter, specifically regarding textbook evaluation and adaptation.

Molecular and cellular analysis of genodermatoses

Summary Skin is the essential interface between our body and its environment; not only does it prevent water loss and protect us from external insults it also plays an essential role in the central nervous system acting as a major sense organ primarily for touch and pain. The main cell type present in skin, keratinocyte, undergoes a differentiation process leading to the formation of this protecting barrier. This work is intended to contribute to the understanding of how keratinocyte differentiates and skin functions. To do this, we studied two genetic skin diseases: Erythrokeratodermia variabilis and Mal de Meleda. Our approach was to examine the expression and localization of proteins implicated in these two pathologies in normal and diseased tissues and to determine the influence of mutant proteins at the molecular and cellular levels. Connexins are major components of gap junctions, channels allowing direct communication between cells. Our laboratory has identified mutations in both connexin 30.3 (Cx30.3) and 31 (Cx31) to be causally involved in erythrokeratodermia variabilis (EKV), an autosomal dominant disorder of keratinization. In the first chapter, we show a new mutation of Cx31, L209P-Cx31, in 3 EKV patients, extending the field of EKV-causing mutations although the mechanism by which connexin mutations lead to the disease is unclear. In the second chapter, we studied the effect of F137L-Cx30.3 on expression, trafficking and localization of cotransfected Cx31 and Cx30.3 in connexin-deficient HeLa cells. The F137 amino acid, highly conserved in connexin family, is oriented towards the channel pore and F137L mutation in either Cx30.3 or Cx31 lead to EKV. As two genes can lead to EKV when mutated, our hypothesis was that Cx31 and Cx30.3 might cooperate at a molecular level. We were able to demonstrate a physical interaction between Cx31 and Cx30.3. The presence of F137L-Cx30.3 disturbed the trafficking of both connexins, less connexins were integrated into gap junctions and thus, the coupling between cell was diminished. Connexins formed in the presence of F137L-Cx30.3 are degraded at their exit from the endoplasmic reticulum. In conclusion, our results indicate that the genetic heterogeneity of EKV is due to mutations in two interacting proteins. F137L-Cx30.3 has a dominant negative effect and affects Cx31, disturbing cellular communication in epidermal cells. Mal de Meleda is an autosomal recessive inflammatory and a keratotic palmoplantar skin disorder due to mutations in SLURP1 (secreted LY6/PLAUR-related protein 1). SLURP1 belongs to the LY6/PLAUR family of proteins and has the particularity of being secreted instead of being GPI-anchored. The high degree of structural similarity between SLURP1 and the three fingers motif of snake neurotoxins and LYNX 1-C suggests that this protein could interact with the neuronal acetylcholine receptors. In the third chapter, we show that SLURP1 potentiates responses of the a7 nicotinic acetylcholine receptor (nAchR) to acetylcholine. These results identify SLURP1 as a secreted epidermal neuromodulator that is likely to be essential for palmoplantar skin. In the fourth chapter, we show that SLURP1 is expressed in the granular layer of the epidermis but is absent from skin biopsies of Mal de Meleda patients. SLURP1 is also present in secretions such as sweat, tears or saliva. An in vitro analysis on two mutant of SLURP-I demonstrates that W15R-SLURP1 is absent in cells while G86R-SLURP1 is expressed and secreted, suggesting that SLURP1 can lead to the disease by either an absent or an abnormal protein. Finally, in the fifth chapter, we analyse the expression and biological properties of other LY6/PLAUR members, clustered around SLURP] on chromosome 8. Their GPI-anchored or secreted status were analysed in vitro. SLURP1, LYNX1-A and -B are secreted while LYPDC2 and LYNX 1-C are GPI anchored. Three of these proteins are expressed in the epidermis and in cultured keratinocytes. These results suggest that these LY6/PLAUR members may have an important role in skin homeostasis. Résumé Résumé La peau est la barrière essentielle entre notre corps et l'environnement, nous protégeant des agressions extérieures, de la déshydratation et assurant aussi un rôle dans le système nerveux central en tant qu'organe du toucher et de la douleur. Le principal type de cellules présent dans la peau est le kératinocyte qui suit un processus de différenciation aboutissant à la formation de cette barrière protectrice. Ce travail est destiné à comprendre la différenciation des kératinocytes et le fonctionnement de la peau. Pour cela, nous avons étudié deux maladies génodermatoses : l'Erthrokeratodermia Variabilis (EKV) et le Mal de Meleda. Nous avons examiné l'expression et la localisation des protéines impliquées dans ces deux pathologies dans des tissus normaux et malades puis déterminé l'influence des protéines mutantes aux niveaux moléculaires et cellulaires. Les connexines (Cx) sont les composants majeurs des jonctions communicantes, canaux permettant la communication directe entre les cellules. Notre laboratoire a identifié des mutations dans les Cx30.3 et Cx31 comme responsables de l'EKV, génodermatose de transmission autosomique dominante. Dans le ler chapitre, nous décrivons une nouvelle mutation de Cx31, L209-Cx31, et contribuons à l'établissement du catalogue des mutations de Cx31 entraînant cette maladie. Cependant, le mécanisme par lequel les mutations de Cx31 et C3x0.3 provoquent l'EKV est inconnu. Dans le 2ème chapitre, nous étudions les effets de la mutation F137L-Cx30.3 sur l'expression, le trafic et la localisation des Cx31 et Cx30.3 transfectées dans des cellules HeLa, déficientes en connexines. Comme deux gènes peuvent causer une EKV quand ils sont mutés, notre hypothèse était que Cx31 et Cx30.3 pourraient coopérer au niveau moléculaire. Nous avons montré l'existence d'une interaction physique entre ces deux connexines. La présence de la mutation F137L-Cx30.3 perturbe le trafic des deux connexines, moins de connexines sont intégrées dans les jonctions communicantes et donc le couplage entre les cellules est diminué. Les connexons formés en présence de cette mutation sont dégradés à leur sortie du réticulum endoplasmique. En conclusion, nos résultats indiquent que l'hétérogénéité génétique de EKV est due à des mutations dans deux protéines qui interagissent. F137L-Cx30.3 a un effet dominant négatif et affecte Cx31, perturbant la communication entre les cellules épidermiques. Le Mal de Meleda est une maladie récessive de la peau palmoplantaire due à des mutations dans SLURP1. SLURP1 appartient à la famille des protéines contenant un domaine LY6/PLAUR et a la particularité d'être sécrétée. La grande homologie de structure existant entre SLURP1, les neurotoxines de serpent et LYNX1-C suggère que la protéine pourrait interagir avec des récepteurs à acétylcholine (Ach). Dans le 3ème chapitre, nous montrons que SLURP1 module la réponse à l'Ach du récepteur nicotinique α7. Ces résultats identifient SLURP1 comme un neuromodulateur épidermique sécrété, probablement essentiel pour la peau palmoplantaire. Dans le 4ème chapitre, nous montrons que SLURP1 est exprimé dans la couche granuleuse de l'épiderme et qu'il est absent des biopsies des patients. SLURP1 a aussi été détecté dans des sécrétions telles que la sueur, les lamies et la salive. Une analyse in vitro de deux mutants de SLURP1 a montré que W15R-SLURP1 est absent des cellules tandis que G86R-SLURP1 est exprimé et sécrété, suggérant qu'une absence ou une anomalie de SLURP1 peuvent causer la maladie. Finalement, dans le 5ème chapitre, nous analysons l'expression et les propriétés biologiques d'autres membres de la famille LY6/PLAUR localisés autour de SLURP1 sur le chromosome 8. Leur statut de protéines sécrétées ou liées à la membrane par une ancre GPI est analysé in vitro. SLURP1, LYNXI-A et -B sont sécrétées alors que LYPDC2 et LYNX1-C sont liés à la membrane. Trois de ces protéines sont exprimées dans l'épiderme et dans des kératinocytes cultivés. Ces résultats suggèrent que la famille LY6/PLAUR pourrait avoir un rôle important dans l'homéostasie de la peau.

Gut symbionts from distinct hosts exhibit genotoxic activity via divergent colibactin biosynthetic pathways.

Secondary metabolites produced by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways are chemical mediators of microbial interactions in diverse environments. However, little is known about their distribution, evolution, and functional roles in bacterial symbionts associated with animals. A prominent example is "colibactin", a largely unknown family of secondary metabolites produced by Escherichia coli via a hybrid NRPS-PKS biosynthetic pathway, inflicting DNA damage upon eukaryotic cells and contributing to colorectal cancer and tumor formation in the mammalian gut. Thus far, homologs of this pathway have only been found in closely related Enterobacteriaceae, while a divergent variant of this gene cluster was recently discovered in a marine alphaproteobacterial Pseudovibrio strain. Herein, we sequenced the genome of Frischella perrara PEB0191, a bacterial gut symbiont of honey bees, and identified a homologous colibactin biosynthetic pathway related to those found in Enterobacteriaceae. We show that the colibactin genomic island (GI) has conserved gene synteny and biosynthetic module architecture across F. perrara, Enterobacteriaceae and the Pseudovibrio strain. Comparative metabolomics analyses of F. perrara and E. coli further reveal that these two bacteria produce related colibactin pathway-dependent metabolites. Finally, we demonstrate that F. perrara, like E. coli, causes DNA damage in eukaryotic cells in vitro in a colibactin pathway-dependent manner. Together, these results support that divergent variants of the colibactin biosynthetic pathway are widely distributed among bacterial symbionts, producing related secondary metabolites and likely endowing its producer with functional capabilities important for diverse symbiotic associations.

Efeitos do sexo, do vigor e do tamanho da planta hospedeira sobre a distribuição de insetos indutores de galhas em Baccharis pseudomyriocephala Teodoro (Asteraceae)

Several hypotheses have been proposed to explain the patterns of host plant utilization by herbivorous insects in natural communities. We tested four hypotheses aiming to understand the pattern of attack by gall-inducing insects on the dioecious shrub, Baccharis pseudomyriocephala (Asteraceae). The shrub occurs in the Parque Estadual do Itacolomi, Southeastern Brazil, and supports ten species of galling insects. The following hypotheses were tested: i) male plants are more attacked by galling insects than female plants; ii) larger plant modules are preferentially attacked by galling insects; iii) galling insects perform better on larger modules than on smaller modules; iv) galling insects increase in abundance with meristematic availability. To address these questions, 240 plants (120 of each sex) were sampled in both reproductive and vegetative periods. We recorded the growth rate (4 cm), inflorescence and fruit production, attack rates of the galling insects, and their survivorship and mortality per shoot (module). Modules were separated into size classes (cm) and analyzed by regressions and ANCOVAs. Module size and reproductive effort were positively correlated with host plant size. We did not observe any effect of host plant gender on either variables. In the same way, host plant sex did not show any influence on the abundance and richness of galling insects. Although the abundance of galling insects showed a positive correlation with shoot size, the trend disappeared when the analyses were performed taking into consideration the number of galls per unit of growth (number of galls/cm of shoot) or biomass (number of galls/dry weight). Larval survivorship was not influenced by shoot size. Also, we observed that the abundance of one species of hemipteran galling insect showed a positive relation with leaf biomass. Therefore, we conclude that gender and vigor of this plant species do not influence the community structure of its galling herbivores.