Development of stable cell lines for production or regulated expression using matrix attachment regions.

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.

Regulation of antibiotic production in root-colonizing Peudomonas spp. and relevance for biological control of plant disease.

Certain strains of fluorescent pseudomonads are important biological components of agricultural soils that are suppressive to diseases caused by pathogenic fungi on crop plants. The biocontrol abilities of such strains depend essentially on aggressive root colonization, induction of systemic resistance in the plant, and the production of diffusible or volatile antifungal antibiotics. Evidence that these compounds are produced in situ is based on their chemical extraction from the rhizosphere and on the expression of antibiotic biosynthetic genes in the producer strains colonizing plant roots. Well-characterized antibiotics with biocontrol properties include phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, lipopeptides, and hydrogen cyanide. In vitro, optimal production of these compounds occurs at high cell densities and during conditions of restricted growth, involving (i) a number of transcriptional regulators, which are mostly pathway-specific, and (ii) the GacS/GacA two-component system, which globally exerts a positive effect on the production of extracellular metabolites at a posttranscriptional level. Small untranslated RNAs have important roles in the GacS/GacA signal transduction pathway. One challenge in future biocontrol research involves development of new strategies to overcome the broad toxicity and lack of antifungal specificity displayed by most biocontrol antibiotics studied so far.

The role of the c-myc gene in pigment cell development and homeostasis

Résumé La dérégulation de c-Myc est un événement fréquent de la transformation cellulaire. Une régulation positive de cette oncoprotéine a été démontrée dans divers mélanomes cutanés primaires et métastatiques et est associée à un pronostic défavorable (Grover et al., 1996; Zhuang et al., 2008). c-Myc est considéré comme une molécule centrale impliquée dans plusieurs processus de l'homéostasie cellulaire. En raison de sa contribution importante dans la progression tumorale, la fonction de c-Myc a été étudiée intensément. Cependant nous connaissons peu le rôle de ce facteur de transcription dans l'embryogenèse et dans la spécification tissulaire. Un déficit total de c-Myc pendant l'embryogenèse conduit à la mort embryonnaire avant 10.5 jours de gestation. Cette mort est causée par de multiples imperfections du développement touchant la taille de l'embryon, le coeur, le péricarde, le tube neural et les cellules sanguines (Davis et al., 1993; Trumpp et al., 2001). Récemment, il a été montré que la plupart de ces anomalies sont secondaires et résultent d'une insuffisance du placenta dans les embryons c-myc-/- (Dubois et al., 2008). Sachant que c-Myc est important dans la maintenance des lignées de la crête neurale (Wei et al., 2007), nous nous sommes intéressés au rôle de c-Myc dans le développement des cellules pigmentaires et à leur homéostasie après la naissance. Un allèle floxé de c-myc (Trumpp et al., 2001) a été utilisé pour supprimer ce gène spécifiquement dans la lignée mélanocytaire à l'aide d'une souris transgénique Tyr::Cre (Delmas et al., 2003). L'ablation des deux allèles de c-myc dans les mélanocytes des souris c-myccKO conduit au phénotype de grisonnement des poils, observé directement après la naissance et associé à une diminution du nombre de mélanocytes dans le bulbe des follicules pileux. Les cellules pigmentaires restantes expriment les marqueurs mélanogéniques (Tyr, TRP-1, Dct and MITF) et semblent être fonctionnelles puisqu'elles peuvent produire et transférer la mélanine. De plus, la capacité de prolifération des mélanocytes déficients en c-Myc dans le bulbe des follicules pileux ne semble pas être affectée chez les nouveaux-nés. Les cellules souches mélanocytaires sont présentes, mais en nombre réduit, dans le bulge des follicules pileux à la fin de la morphogenèse chez les souris c-myccKO âgées de huit jours. Ces cellules sont maintenues sans changement durant le premier cycle pileux (vérifié à l'âge de trente jours), ce qui sous-entend que la fonction de c-Myc n'est pas nécessaire pour ce processus. Ceci explique pourquoi, en supposant que des cellules souches mélanocytaires fonctionnelles sont présentes dans la peau, nous n'observons pas de dilution de couleur de la robe liée à l'âge. Cependant, la présence de ces cellules souches mélanocytaires dans la peau c-myccKO ne suffit pas à assurer une quantité normale de mélanocytes différenciés dans le bulbe des follicules pileux. Cette population de cellules pigmentaires matures est sévèrement affectée par la suppression de c-Myc, ce qui contribue amplement au phénotype de grisonnement des poils. De plus, c-Myc paraît être important pour le développement des mélanocytes. Ainsi, le nombre de mélanoblastes diminue dans les embryons c-myccKO à partir du douzième jour de gestation. A treize jours de gestation, au stade où les mélanoblastes pénètrent dans l'épiderme et prolifèrent, les mélanoblastes déficients en c-Myc ne s'adaptent pas aux signaux de prolifération et se retrouvent en nombre réduit dans l'épiderme. Finalement, nous nous sommes intéressés, au rôle de N-Myc, un homologue proche de c-Myc, dans la lignée mélanocytaire. Nos expériences ont montré que. N-Myc était superflu pour le développement et l'homéostasie des mélanocytes, une seule copie du gène c-myc étant suffisante pour maintenir une pigmentation normale de la robe des souris c-mycc-myccKO/+~N_ myccKO/KO. Cependant, le rôle essentiel de N-Myc dans la maintenance des cellules mélanocytaires précurseurs apparaît lorsque c-Myc est absent, puisque la suppression simultanée des deux Myc résulte en une perte complète de la coloration de la robe. Ceci implique la présence d'un mécanisme compensatoire entre c- et N-Myc dans la lignée mélanocytaire, avec un rôle prédominant de c-Myc. Summary Deregulation of c-Myc is known to be a common event in cellular transformation. Upregulation of this oncoprotein was shown in a variety of primary and metastatic cutaneous melanomas and has been associated with a poor prognosis (Grover et al., 1996; Zhuang et al., 2008). c-myc is seen as a central molecule involved in many aspects of cellular homeostasis. c-Myc function has been intensively studied mostly because of its significant contribution to tumour progression. However little is known on the role of this transcription factor in embryogenesis and tissue specification. Complete loss of c-Myc during embryogenesis results in embryonic death before E10.5 due to multiple developmental defects including embryonic size, heart, pericardium, neural tube and blood cells (Davis et al., 1993; Trumpp et al., 2001). Recently it was discovered that most of these abnormalities are secondary and results of placental insufficiency in c-Myc-/- embryos (Dubois et al., 2008). Here, we focused on the role of c-Myc in pigment cell development and homeostasis after birth, knowing that c-Myc is important in the maintenance of neural crest lineages (Wei et al., 2007). A floxed allele of c-Myc (Trumpp et al., 2001) was used to specifically delete this gene in the melanocyte lineage using Tyr::Cre transgenic mice (Delmas et al., 2003). Removal of both c-Myc alleles in melanocytes of c-MyccKO mouse led to the grey hair phenotype which is seen directly after birth and was associated with a decrease in the melanocyte number in the bulb of the hair follicle. The remaining population of pigment cells express melanogenic markers (Tyr, TRP-1, Dct and MITF) and seem functionally normal since they can produce and transfer melanin. Furthermore proliferation capacity of c-Myc deficient melanocytes in the bulb of hair follicle seems not to be affected in newborn animals. Melanocyte stem cells (MSCs) are present but reduced in numbers in the bulge of the hair follicle at the end of morphogenesis in 8 days old c-MyccKO mice. These cells are maintained through the first hair cycle (as verified at P30) without any further changes, suggesting that c-Myc function is not required for this process. This explains why we did not detect any agerelated coat color dilution, assuming a presence of functional MSCs in the skin. Importantly, presence of MSCs in c-MyccKO skin was not sufficient for assuring a normal number of differentiated melanocytes in the bulb of the hair follicle. This population of mature pigmented cells is severely affected upon c-myc deletion thus largely contributing to the grey hair phenotype. Moreover, c-Myc appears to be important for melanocyte development. Thus, melanoblast number is affected in c-MyccKO embryos day 12 of gestation onwards. At E13.5, when melanoblasts enter the epidermis and proliferate, c-myc deficient melanoblasts failed to adapt to proliferation signals and are therefore reduced in number in the epidermis. Finally, we addressed the role of N-Myc, a closest homologue of c-Myc, in the melanocyte lineage. In these experiments, N-Myc was dispensable for melanocyte development and homeostasis, and even one copy of the c-myc gene was sufficient to maintain normal coat color pigmentation in c-mycc-mycCKO/+ ,N-myccKO/KO mice. However the crucial role of N-Myc in maintenance of melanocyte precursor cells became apparent when c-myc is eliminated since simultaneous deletion of both Myc results in complete loss of coat color pigmentation. This suggests compensatory mechanisms between c- and N-Myc with a predominant role of c-Myc in melanocyte lineage.

Eficacia e ineficacia de la comunicación horizontal en los movimientos sociales. Caso análisis: Oposiciones justas en Cataluña 2011

Las redes sociales están generando un amplio entramado de comunicaciones, a distintos niveles y con distintos objetivos. En este artículo analizamos un caso concreto, fruto de la observación y el seguimiento de un grupo creado para la defensa de unas oposiciones justas para el acceso a la función pública de docentes para educación secundaria. Se trata de un grupo creado como reacción a una serie de posibles irregularidades. Interesa observar tanto la creación del grupo, como las distintas fases que sigue la evolución, muy ligada a la efectividad de los procesos de comunicación, y la vinculación de estos últimos a las posibilidades de acción y resolución del conflicto que existe en la base del grupo

Volume-targeted and whole-heart coronary magnetic resonance angiography using an intravascular contrast agent.

PURPOSE: To compare volume-targeted and whole-heart coronary magnetic resonance angiography (MRA) after the administration of an intravascular contrast agent. MATERIALS AND METHODS: Six healthy adult subjects underwent a navigator-gated and -corrected (NAV) free breathing volume-targeted cardiac-triggered inversion recovery (IR) 3D steady-state free precession (SSFP) coronary MRA sequence (t-CMRA) (spatial resolution = 1 x 1 x 3 mm(3)) and high spatial resolution IR 3D SSFP whole-heart coronary MRA (WH-CMRA) (spatial resolution = 1 x 1 x 2 mm(3)) after the administration of an intravascular contrast agent B-22956. Subjective and objective image quality parameters including maximal visible vessel length, vessel sharpness, and visibility of coronary side branches were evaluated for both t-CMRA and WH-CMRA. RESULTS: No significant differences (P = NS) in image quality were observed between contrast-enhanced t-CMRA and WH-CMRA. However, using an intravascular contrast agent, significantly longer vessel segments were measured on WH-CMRA vs. t-CMRA (right coronary artery [RCA] 13.5 +/- 0.7 cm vs. 12.5 +/- 0.2 cm; P < 0.05; and left circumflex coronary artery [LCX] 11.9 +/- 2.2 cm vs. 6.9 +/- 2.4 cm; P < 0.05). Significantly more side branches (13.3 +/- 1.2 vs. 8.7 +/- 1.2; P < 0.05) were visible for the left anterior descending coronary artery (LAD) on WH-CMRA vs. t-CMRA. Scanning time and navigator efficiency were similar for both techniques (t-CMRA: 6.05 min; 49% vs. WH-CMRA: 5.51 min; 54%, both P = NS). CONCLUSION: Both WH-CMRA and t-CMRA using SSFP are useful techniques for coronary MRA after the injection of an intravascular blood-pool agent. However, the vessel conspicuity for high spatial resolution WH-CMRA is not inferior to t-CMRA, while visible vessel length and the number of visible smaller-diameter vessels and side-branches are improved.

Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants.

In this study, we enlarged our previous investigation focusing on the biodiversity of chlamydiae and amoebae in a drinking water treatment plant, by the inclusion of two additional plants and by searching also for the presence of legionellae and mycobacteria. Autochthonous amoebae were recovered onto non-nutritive agar, identified by 18S rRNA gene sequencing, and screened for the presence of bacterial endosymbionts. Bacteria were also searched for by Acanthamoeba co-culture. From a total of 125 samples, we recovered 38 amoebae, among which six harboured endosymbionts (three chlamydiae and three legionellae). In addition, we recovered by amoebal co-culture 11 chlamydiae, 36 legionellae (no L. pneumophila), and 24 mycobacteria (all rapid-growers). Two plants presented a similar percentage of samples positive for chlamydiae (11%), mycobacteria (20%) and amoebae (27%), whereas in the third plant the number of recovered bacteria was almost twice higher. Each plant exhibited a relatively high specific microbiota. Amoebae were mainly represented by various Naegleria species, Acanthamoeba species and Hartmannella vermiformis. Parachlamydiaceae were the most abundant chlamydiae (8 strains in total), and in this study we recovered a new genus-level strain, along with new chlamydiae previously reported. Similarly, about 66% of the recovered legionellae and 47% of the isolated mycobacteria could represent new species. Our work highlighted a high species diversity among legionellae and mycobacteria, dominated by putative new species, and it confirmed the presence of chlamydiae in these artificial water systems.

Continuous monitoring and quantification of multiple parameters of daily physical activity in ambulatory Duchenne muscular dystrophy patients.

Multiple motor function and strength assessment tools exist for the evaluation of neuromuscular diseases, but most do not directly assess functional ability in the patients' daily physical activity in their home environment. In this study our aim was to assess: 1) the feasibility and accuracy of physical activity monitoring during two days in a home environment of five DMD patients using a non-commercialized monitor containing a 3D accelerometer and a gyroscope, 2) if a difference in the physical activity parameters could be measured before and one month after starting prednisolone. We reliably quantified the time spend sitting, standing, lying, walking, the number of steps taken, the cadence, the number of walking episodes and their duration as well as how these were distributed over the day. Parameters possibly reflecting endurance, such as the duration of the walking episodes or the succession of two or three walking episodes lasting more than 30 s were the most improved after prednisolone treatment. This degree of detailed determination of physical activity in a home environment has not been previously reported in neuromuscular disorders to our knowledge and some of the reported parameters are potential new outcome measures in clinical trials.

Statistical properties of population differentiation estimators under stepwise mutation in a finite island model.

Microsatellite loci mutate at an extremely high rate and are generally thought to evolve through a stepwise mutation model. Several differentiation statistics taking into account the particular mutation scheme of the microsatellite have been proposed. The most commonly used is R(ST) which is independent of the mutation rate under a generalized stepwise mutation model. F(ST) and R(ST) are commonly reported in the literature, but often differ widely. Here we compare their statistical performances using individual-based simulations of a finite island model. The simulations were run under different levels of gene flow, mutation rates, population number and sizes. In addition to the per locus statistical properties, we compare two ways of combining R(ST) over loci. Our simulations show that even under a strict stepwise mutation model, no statistic is best overall. All estimators suffer to different extents from large bias and variance. While R(ST) better reflects population differentiation in populations characterized by very low gene-exchange, F(ST) gives better estimates in cases of high levels of gene flow. The number of loci sampled (12, 24, or 96) has only a minor effect on the relative performance of the estimators under study. For all estimators there is a striking effect of the number of samples, with the differentiation estimates showing very odd distributions for two samples.

Executive Order Number Eighty-Six September, 2014

In this order the governor declares that The University of Northern Iowa's Center for Violence Prevention shall establish the Governor's Office for Bullying Prevention. The Office's mission shall be to empower schools to provide and every student with a safe and respectful learning environment.

Computational and statistical analysis of copy number variants in normal and cancer genomes

AbstractAlthough the genomes from any two human individuals are more than 99.99% identical at the sequence level, some structural variation can be observed. Differences between genomes include single nucleotide polymorphism (SNP), inversion and copy number changes (gain or loss of DNA). The latter can range from submicroscopic events (CNVs, at least 1kb in size) to complete chromosomal aneuploidies. Small copy number variations have often no (lethal) consequences to the cell, but a few were associated to disease susceptibility and phenotypic variations. Larger re-arrangements (i.e. complete chromosome gain) are frequently associated with more severe consequences on health such as genomic disorders and cancer. High-throughput technologies like DNA microarrays enable the detection of CNVs in a genome-wide fashion. Since the initial catalogue of CNVs in the human genome in 2006, there has been tremendous interest in CNVs both in the context of population and medical genetics. Understanding CNV patterns within and between human populations is essential to elucidate their possible contribution to disease. But genome analysis is a challenging task; the technology evolves rapidly creating needs for novel, efficient and robust analytical tools which need to be compared with existing ones. Also, while the link between CNV and disease has been established, the relative CNV contribution is not fully understood and the predisposition to disease from CNVs of the general population has not been yet investigated.During my PhD thesis, I worked on several aspects related to CNVs. As l will report in chapter 3, ! was interested in computational methods to detect CNVs from the general population. I had access to the CoLaus dataset, a population-based study with more than 6,000 participants from the Lausanne area. All these individuals were analysed on SNP arrays and extensive clinical information were available. My work explored existing CNV detection methods and I developed a variety of metrics to compare their performance. Since these methods were not producing entirely satisfactory results, I implemented my own method which outperformed two existing methods. I also devised strategies to combine CNVs from different individuals into CNV regions.I was also interested in the clinical impact of CNVs in common disease (chapter 4). Through an international collaboration led by the Centre Hospitalier Universitaire Vaudois (CHUV) and the Imperial College London I was involved as a main data analyst in the investigation of a rare deletion at chromosome 16p11 detected in obese patients. Specifically, we compared 8,456 obese patients and 11,856 individuals from the general population and we found that the deletion was accounting for 0.7% of the morbid obesity cases and was absent in healthy non- obese controls. This highlights the importance of rare variants with strong impact and provides new insights in the design of clinical studies to identify the missing heritability in common disease.Furthermore, I was interested in the detection of somatic copy number alterations (SCNA) and their consequences in cancer (chapter 5). This project was a collaboration initiated by the Ludwig Institute for Cancer Research and involved other groups from the Swiss Institute of Bioinformatics, the CHUV and Universities of Lausanne and Geneva. The focus of my work was to identify genes with altered expression levels within somatic copy number alterations (SCNA) in seven metastatic melanoma ceil lines, using CGH and SNP arrays, RNA-seq, and karyotyping. Very few SCNA genes were shared by even two melanoma samples making it difficult to draw any conclusions at the individual gene level. To overcome this limitation, I used a network-guided analysis to determine whether any pathways, defined by amplified or deleted genes, were common among the samples. Six of the melanoma samples were potentially altered in four pathways and five samples harboured copy-number and expression changes in components of six pathways. In total, this approach identified 28 pathways. Validation with two external, large melanoma datasets confirmed all but three of the detected pathways and demonstrated the utility of network-guided approaches for both large and small datasets analysis.RésuméBien que le génome de deux individus soit similaire à plus de 99.99%, des différences de structure peuvent être observées. Ces différences incluent les polymorphismes simples de nucléotides, les inversions et les changements en nombre de copies (gain ou perte d'ADN). Ces derniers varient de petits événements dits sous-microscopiques (moins de 1kb en taille), appelés CNVs (copy number variants) jusqu'à des événements plus large pouvant affecter des chromosomes entiers. Les petites variations sont généralement sans conséquence pour la cellule, toutefois certaines ont été impliquées dans la prédisposition à certaines maladies, et à des variations phénotypiques dans la population générale. Les réarrangements plus grands (par exemple, une copie additionnelle d'un chromosome appelée communément trisomie) ont des répercutions plus grave pour la santé, comme par exemple dans certains syndromes génomiques et dans le cancer. Les technologies à haut-débit telle les puces à ADN permettent la détection de CNVs à l'échelle du génome humain. La cartographie en 2006 des CNV du génome humain, a suscité un fort intérêt en génétique des populations et en génétique médicale. La détection de différences au sein et entre plusieurs populations est un élément clef pour élucider la contribution possible des CNVs dans les maladies. Toutefois l'analyse du génome reste une tâche difficile, la technologie évolue très rapidement créant de nouveaux besoins pour le développement d'outils, l'amélioration des précédents, et la comparaison des différentes méthodes. De plus, si le lien entre CNV et maladie a été établit, leur contribution précise n'est pas encore comprise. De même que les études sur la prédisposition aux maladies par des CNVs détectés dans la population générale n'ont pas encore été réalisées.Pendant mon doctorat, je me suis concentré sur trois axes principaux ayant attrait aux CNV. Dans le chapitre 3, je détaille mes travaux sur les méthodes d'analyses des puces à ADN. J'ai eu accès aux données du projet CoLaus, une étude de la population de Lausanne. Dans cette étude, le génome de plus de 6000 individus a été analysé avec des puces SNP et de nombreuses informations cliniques ont été récoltées. Pendant mes travaux, j'ai utilisé et comparé plusieurs méthodes de détection des CNVs. Les résultats n'étant pas complètement satisfaisant, j'ai implémenté ma propre méthode qui donne de meilleures performances que deux des trois autres méthodes utilisées. Je me suis aussi intéressé aux stratégies pour combiner les CNVs de différents individus en régions.Je me suis aussi intéressé à l'impact clinique des CNVs dans le cas des maladies génétiques communes (chapitre 4). Ce projet fut possible grâce à une étroite collaboration avec le Centre Hospitalier Universitaire Vaudois (CHUV) et l'Impérial College à Londres. Dans ce projet, j'ai été l'un des analystes principaux et j'ai travaillé sur l'impact clinique d'une délétion rare du chromosome 16p11 présente chez des patients atteints d'obésité. Dans cette collaboration multidisciplinaire, nous avons comparés 8'456 patients atteint d'obésité et 11 '856 individus de la population générale. Nous avons trouvés que la délétion était impliquée dans 0.7% des cas d'obésité morbide et était absente chez les contrôles sains (non-atteint d'obésité). Notre étude illustre l'importance des CNVs rares qui peuvent avoir un impact clinique très important. De plus, ceci permet d'envisager une alternative aux études d'associations pour améliorer notre compréhension de l'étiologie des maladies génétiques communes.Egalement, j'ai travaillé sur la détection d'altérations somatiques en nombres de copies (SCNA) et de leurs conséquences pour le cancer (chapitre 5). Ce projet fut une collaboration initiée par l'Institut Ludwig de Recherche contre le Cancer et impliquant l'Institut Suisse de Bioinformatique, le CHUV et les Universités de Lausanne et Genève. Je me suis concentré sur l'identification de gènes affectés par des SCNAs et avec une sur- ou sous-expression dans des lignées cellulaires dérivées de mélanomes métastatiques. Les données utilisées ont été générées par des puces ADN (CGH et SNP) et du séquençage à haut débit du transcriptome. Mes recherches ont montrées que peu de gènes sont récurrents entre les mélanomes, ce qui rend difficile l'interprétation des résultats. Pour contourner ces limitations, j'ai utilisé une analyse de réseaux pour définir si des réseaux de signalisations enrichis en gènes amplifiés ou perdus, étaient communs aux différents échantillons. En fait, parmi les 28 réseaux détectés, quatre réseaux sont potentiellement dérégulés chez six mélanomes, et six réseaux supplémentaires sont affectés chez cinq mélanomes. La validation de ces résultats avec deux larges jeux de données publiques, a confirmée tous ces réseaux sauf trois. Ceci démontre l'utilité de cette approche pour l'analyse de petits et de larges jeux de données.Résumé grand publicL'avènement de la biologie moléculaire, en particulier ces dix dernières années, a révolutionné la recherche en génétique médicale. Grâce à la disponibilité du génome humain de référence dès 2001, de nouvelles technologies telles que les puces à ADN sont apparues et ont permis d'étudier le génome dans son ensemble avec une résolution dite sous-microscopique jusque-là impossible par les techniques traditionnelles de cytogénétique. Un des exemples les plus importants est l'étude des variations structurales du génome, en particulier l'étude du nombre de copies des gènes. Il était établi dès 1959 avec l'identification de la trisomie 21 par le professeur Jérôme Lejeune que le gain d'un chromosome supplémentaire était à l'origine de syndrome génétique avec des répercussions graves pour la santé du patient. Ces observations ont également été réalisées en oncologie sur les cellules cancéreuses qui accumulent fréquemment des aberrations en nombre de copies (telles que la perte ou le gain d'un ou plusieurs chromosomes). Dès 2004, plusieurs groupes de recherches ont répertorié des changements en nombre de copies dans des individus provenant de la population générale (c'est-à-dire sans symptômes cliniques visibles). En 2006, le Dr. Richard Redon a établi la première carte de variation en nombre de copies dans la population générale. Ces découvertes ont démontrées que les variations dans le génome était fréquentes et que la plupart d'entre elles étaient bénignes, c'est-à-dire sans conséquence clinique pour la santé de l'individu. Ceci a suscité un très grand intérêt pour comprendre les variations naturelles entre individus mais aussi pour mieux appréhender la prédisposition génétique à certaines maladies.Lors de ma thèse, j'ai développé de nouveaux outils informatiques pour l'analyse de puces à ADN dans le but de cartographier ces variations à l'échelle génomique. J'ai utilisé ces outils pour établir les variations dans la population suisse et je me suis consacré par la suite à l'étude de facteurs pouvant expliquer la prédisposition aux maladies telles que l'obésité. Cette étude en collaboration avec le Centre Hospitalier Universitaire Vaudois a permis l'identification d'une délétion sur le chromosome 16 expliquant 0.7% des cas d'obésité morbide. Cette étude a plusieurs répercussions. Tout d'abord elle permet d'effectuer le diagnostique chez les enfants à naître afin de déterminer leur prédisposition à l'obésité. Ensuite ce locus implique une vingtaine de gènes. Ceci permet de formuler de nouvelles hypothèses de travail et d'orienter la recherche afin d'améliorer notre compréhension de la maladie et l'espoir de découvrir un nouveau traitement Enfin notre étude fournit une alternative aux études d'association génétique qui n'ont eu jusqu'à présent qu'un succès mitigé.Dans la dernière partie de ma thèse, je me suis intéressé à l'analyse des aberrations en nombre de copies dans le cancer. Mon choix s'est porté sur l'étude de mélanomes, impliqués dans le cancer de la peau. Le mélanome est une tumeur très agressive, elle est responsable de 80% des décès des cancers de la peau et est souvent résistante aux traitements utilisés en oncologie (chimiothérapie, radiothérapie). Dans le cadre d'une collaboration entre l'Institut Ludwig de Recherche contre le Cancer, l'Institut Suisse de Bioinformatique, le CHUV et les universités de Lausanne et Genève, nous avons séquencés l'exome (les gènes) et le transcriptome (l'expression des gènes) de sept mélanomes métastatiques, effectués des analyses du nombre de copies par des puces à ADN et des caryotypes. Mes travaux ont permis le développement de nouvelles méthodes d'analyses adaptées au cancer, d'établir la liste des réseaux de signalisation cellulaire affectés de façon récurrente chez le mélanome et d'identifier deux cibles thérapeutiques potentielles jusqu'alors ignorées dans les cancers de la peau.

Recent skin injuries in children with motor disabilities.

OBJECTIVE: To determine the frequency of recent skin injuries in children with neuromotor disabilities and its association with disability. DESIGN: Cross-sectional study of 168 children with neuromotor disabilities aged 2-16 years. SETTING: Two outpatient child rehabilitation centres. MAIN OUTCOME MEASURES: Children were classified as unrestricted walkers, restricted walkers or wheelchair dependent. Each participant's body surface was systematically examined for recent skin injuries with the exception of the anal-genital area. RESULTS: The mean age of our sample was 7.8 (SD 3.7) years with a 3:2 male/female ratio. Overall, 64% had cerebral palsy, 17% a neuromuscular disease and 19% other motor disabilities. Participants had on average 5.3 (SD 4.5) recent skin injuries (max 19), of which 2.5 were bruises (SD 3.3, max 16), 2.4 were abrasions, scratches or cuts (SD 3.0, max 16) and 0.4 were pressure lesions (SD 0.8, max 4). There was a significant decrease in the frequency of recent skin injuries and of bruises with increasing severity of motor disability. Most of this variation was accounted for by injuries to the lower limbs. There were no significant effects of gender, learning disabilities or other comorbidities. CONCLUSIONS: Children with neuromotor disabilities present a progressive reduction in the number of skin injuries with decreasing mobility. Therefore, recent skin injuries in this population which are unusual by their number, appearance or distribution, should raise at least the same level of suspicion for physical abuse as in children without disabilities.

Genetic factors of obesity and eating disorders: Copy number variations (CNV) involving SH2B1

Context : It is now clearly shown that genetic factors in association with environment play a key role in obesity and eating disorders. This project studies the clinical symptoms and molecular abnormalities in patients carrying a strong hereditary predisposition to obesity and eating behavior disorders. We have previously published the association between the 16:29.5-30.1 deletion and a very penetrant form of morbid obesity and macrocephaly. We have also demonstrated the association between the reciprocal 16:29.5-30.1 duplication and underweight and small head circumference. These 2 studies demonstrate that gene dosage of one or several genes in this region regulates BMI as well as brain growth. At present, there are no data pointing towards particular candidate genes. We are currently investigating a second non-overlapping recurrent CNV encompassing SH2B1, upstream of the aforementioned rearrangement. SNPs in this gene have been associated with BMI in GWAS studies and mice models confirmed this association. Bokuchova et al have reported an association between deletions encompassing this gene and severe early onset obesity, as well as insulin resistance. We are currently collecting and analyzing data to fully characterize the phenotype and the transcriptional patterns associated with this rearrangement. Aims : 1. Identify carriers of any CNVs in the greater 16p11.2 region (between 16:28MB and 32MB) in the EGG consortium. 2. Perform association studies between SNPs in the greater 16p11.2 region (16:28-32MB) and anthropometric measures with adjusted "locus-wide significance", to identify or prioritize candidate genes potentially driving the association observed in patients with the CNVs (and thus worthy of further validation and sequencing). 3. Explore associations between GSV genome-wide and brain volume. 4. Explore relationship between brain volumes (whole brain and regional for those who underwent brain MRI), head circumference and BMI. 5. Extrapolate this procedure to other regions covered by the Metabochip. Methods : - Examine and collect clinical informations, as well as molecular informations in these patients. - Analysis of MRI data in children and adults with BMI > 2SD. Compare changes to MRI data obtained in patients with monogenic forms of obesity (data from Lausanne study) and to underweight (BMI<-2SD) individuals from EGG. - Test whether opposite extremes of the phenotypic distribution may be highly informative Expected results : This is a highly focused study, pertaining to approximately 1 0/00 of the human genome. Yet it is clear that if successful, the lessons learned from this study could be extrapolated to other segments of the genome and would need validation and replication by additional studies. Altogether they will contribute to further explore the missing heritability and point to etiologic genes and pathways underlying these important health burdens.

Improved three-dimensional free-breathing coronary magnetic resonance angiography using gadocoletic acid (B-22956) for intravascular contrast enhancement.

PURPOSE: To evaluate gadocoletic acid (B-22956), a gadolinium-based paramagnetic blood pool agent, for contrast-enhanced coronary magnetic resonance angiography (MRA) in a Phase I clinical trial, and to compare the findings with those obtained using a standard noncontrast T2 preparation sequence. MATERIALS AND METHODS: The left coronary system was imaged in 12 healthy volunteers before B-22956 application and 5 (N = 11) and 45 (N = 7) minutes after application of 0.075 mmol/kg of body weight (BW) of B-22956. Additionally, imaging of the right coronary system was performed 23 minutes after B-22956 application (N = 6). A three-dimensional gradient echo sequence with T2 preparation (precontrast) or inversion recovery (IR) pulse (postcontrast) with real-time navigator correction was used. Assessment of the left and right coronary systems was performed qualitatively (a 4-point visual score for image quality) and quantitatively in terms of signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), vessel sharpness, visible vessel length, maximal luminal diameter, and the number of visible side branches. RESULTS: Significant (P < 0.01) increases in SNR (+42%) and CNR (+86%) were noted five minutes after B-22956 application, compared to precontrast T2 preparation values. A significant increase in CNR (+40%, P < 0.05) was also noted 45 minutes postcontrast. Vessels (left anterior descending artery (LAD), left coronary circumflex (LCx), and right coronary artery (RCA)) were also significantly (P < 0.05) sharper on postcontrast images. Significant increases in vessel length were noted for the LAD (P < 0.05) and LCx and RCA (both P < 0.01), while significantly more side branches were noted for the LAD and RCA (both P < 0.05) when compared to precontrast T2 preparation values. CONCLUSION: The use of the intravascular contrast agent B-22956 substantially improves both objective and subjective parameters of image quality on high-resolution three-dimensional coronary MRA. The increase in SNR, CNR, and vessel sharpness minimizes current limitations of coronary artery visualization with high-resolution coronary MRA.

Effects of trimethyltin (TMT) on glial and neuronal cells in aggregate cultures: dependence on the developmental stage.

Long-term effects of trimethyltin (TMT) applied at concentrations below the cytotoxic level were examined in three-dimensional cell cultures of fetal rat telencephalon using biochemical, immunochemical and morphological criteria. It was found that in immature cultures low concentrations of TMT (10(-8) M) specifically induced a gliotic response in astrocytes, with increased immunoreactivity for glial fibrillary acidic protein, and a greater number of astrocytic processes. Significant changes in oligodendrocytic and neuronal parameters were found only at 10(-6) M of TMT. In differentiated cultures, distinct changes in cell type-specific parameters occurred at 10(-6) M of TMT (the lowest effective concentration). In addition, different patterns of responses were found for astrocytes and oligodendrocytes, as compared to immature cultures. These results suggest that among neural cells, astroblasts are most sensitive to TMT, and that the glial responses to this neurotoxicant are development-dependent.