Le développement du membre passe par des étapes complexes de polarisation, dans les axes dorso-ventral et antéro-postérieur, qui ont lieu en parallèle de la croissance proximo-distale du bourgeon. Le déterminisme génétique de ces étapes est encore imparfaitement connu. Il implique des facteurs de transcription dont l’expression est temporo-spatiale spécifique, compliquant leur identification. L’étude de cohortes homogènes de patients porteurs d’anomalies développementales des membres est l’un des moyens d’identifier de nouveaux mécanismes impliqués dans leur modélisation. A cet effet, nous avons étudié des patients atteints de Syndrome Nail-Patella correspondant à un défaut de polarisation dorso-ventrale ; une grande famille de polydactylie préaxiale syndromique et une série de patients atteints de Syndrome de Nager comme modèles d’étude de la polarisation antéro-postérieure ; et enfin une cohorte de patients présentant des pieds et mains fendus correspondant à un défaut de la signalisation proximo-distale. Ces travaux nous ont permis de souligner le rôle crucial de la régulation d’expression génique dans la modélisation du bourgeon de membre et plus généralement dans le développement embryonnaire. / Limb development requires complex patterning along dorso-ventral, antero-posterior and proximo-distal axes. The molecular mechanisms underlying these stages are not fully delineated yet. Identification of the transcription factors involved is challenging because of their spatio-temporally restricted expression during limb bud development. Analysis of carefully selected series of patients affected with limb malformations is a clue to identify new mechanisms involved in this patterning. For this purpose, we studied several families presnsenting with Nail-Patella Syndrome corresponding to a disorder of dorso-ventral polarization; a large family affected with syndromic preaxial polydactyly and a series of Nager Syndrome cases as models of antero-posterior polarization; eventually, a cohort of patients affected with split hand/foot malformations corresponding to a defect in the signalisation center of proximo-distal growth and differenciation. This work has led us to emphasize the crucial role of gene expression regulation during limb bud patterning and more generally during embryological development.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-04-20T13:30:09Z No. of bitstreams: 1 FelipeAlbuquerqueMarquesTese2015.pdf: 5827285 bytes, checksum: 3dfbabb913ebff137b6f41b2cd1ba5fc (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-04-20T13:30:28Z (GMT) No. of bitstreams: 1 FelipeAlbuquerqueMarquesTese2015.pdf: 5827285 bytes, checksum: 3dfbabb913ebff137b6f41b2cd1ba5fc (MD5) / Made available in DSpace on 2017-04-20T13:30:28Z (GMT). No. of bitstreams: 1 FelipeAlbuquerqueMarquesTese2015.pdf: 5827285 bytes, checksum: 3dfbabb913ebff137b6f41b2cd1ba5fc (MD5) Previous issue date: 2015-09-25 / Genetic diseases of the skeleton affect the genesis of skeletal system. They are caused by mutations in genes which act on the cartilage and/or growth plate. The current classification of skeletal anomalies describes more than 456 distinct phenotypes organized into 40 groups. Of this total, 360 phenotypes are associated with defects in 336 genes (thus, 90 diseases remain to have their cause elucidated). The development of high resolution techniques for genomic analysis has enabled more genetic diseases, including skeletal phenotypes, to have molecular basis clarified. This research aimed to identify genes or regions in the human genome associated with genetic diseases of the skeleton. To this end, a pipeline was developed involving experiments and data analysis. Investigation of single nucletoide variation (SNV) was carried out using whole exome sequencing (WES) and submicroscopic structural variations were analyzed by Chromossomal Microarray Analysis (CMA). Moreover, Sanger sequencing, Fluorescence in situ Hybridization, in vitro functional tests (cell culture, qRT-PCR, Western Blot, Immunofluorescence and transcriptome), Immunohistochemistry and histochemistry were employed. 14 patients with rare diseases of skeleton (Craniosynostosis, s. FATCO sindrome, Catel-Manzke-like sindrome, Nager Syndrome and Rodriguez Syndrome) were selected. Craniosynostosis: of three patients, two had their molecular diagnoses elucidated, one with mutation in FGFR3 and the other with a translocation involving chromosomes 17q and 20q. s. FATCO: it wasn't possible to identify the causative mutation for this disease. Catel-Manzke-like Syndrome: initially this patient was diagnosed as Catel-Manzke Syndrome, and there was found a mutation in EXT2. Thus, this patient was reclassified as a new syndrome recently reported as seizures-scoliosis-macrocephaly. Nager and Rodriguez Syndrome: two of four have been diagnosed with mutation in SF3B4. For these patients, the results of qRT-PCR, Western Blot and Immunofluorescence together suggested that the phenotype is caused by SF3B4 haploinsuficiency. Immunohistochemistry and Histochemistry showed the expression of SF3B4 in cartilage tissue and the disorganization of hypertrophic cells in growth plate, respectively. The transcriptome result from cartilage tissue of one patient with SF3B4 mutation showed 12 underexpressed genes involved in skeletogeneses. The combination of techniques like classical cytogenetics and molecular cytogenetics as well as sequencing and in vitro assays were effective to achieve a diagnosis. Although there was an investigative core common to all diseases, investigations were customized to each case, seeking greater efficiency in the detection of the molecular basis and cost optimization of molecular research. / As doen??as gen??ticas do esqueleto s??o anomalias que envolvem a g??nese do sistema esquel??tico, causadas por altera????es em genes que atuam principalmente na cartilagem e/ou no n??cleo de crescimento. Na classifica????o atual h?? 456 doen??as do esqueleto categorizadas em 40 grupos. Destes, 360 patologias esquel??ticas est??o associadas a defeitos em 336 genes (portanto, existem 90 displasias esquel??ticas sem causa definida). O surgimento de t??cnicas de alta resolu????o de an??lise gen??mica tem permitido que cada vez mais doen??as gen??ticas, incluindo as doen??as gen??ticas do esqueleto, possam ter suas bases moleculares elucidadas. Esta pesquisa teve como objetivo a identifica????o e caracteriza????o de genes e regi??es do genoma humano associado a doen??as gen??ticas raras do esqueleto. Para isso, implantou-se um pipeline envolvendo experimentos e an??lise de dados. Esta tese investigou varia????es de nucleot??deo ??nico (SNVs) pelo uso Whole Exome Sequencing (WES) e varia????es estruturais submicrosc??picas, pelo uso Chromossomal Microarray Analysis (CMA). Al??m disso, fez-se uso de Sequenciamento de Sanger, Fluorescence in situ Hybridization, testes funcionais in vitro (cultura celular, qRT-PCR, Western Blot, Imunofluoresc??ncia e Transcritoma), Imunohistoqu??ca e histoqu??mica. Foram selecionados 14 pacientes com doen??as raras do esqueleto (Craniossinostose, s??ndrome FATCO, s??ndrome Catel-Manzke-like, s??ndrome de Nager e s??ndrome Rodriguez). Craniossinostose: dos quatro pacientes, dois foram diagnosticados com muta????o em FGFR3 e outro com uma transloca????o envolvendo os cromossomos 17q e 20q. S??ndrome de FATCO: n??o foi poss??vel identificar as bases moleculares da doen??a. ???Catel-Manzke-Like???: inicialmente diagnosticado com Sindrome de Catel-Manzke, o paciente teve uma muta????o detectada em EXT2, sendo reclassificado como uma nova s??ndrome (S??ndrome Convuls??o-Escoliose-Microcefalia). S??ndromes de Nager e Rodriguez: foram diagnosticado muta????es no gene SF3B4 em dois dos quatro pacientes. Nestes mesmos pacientes foram realizados qTR-PCR, Western Blot e Imunofluoresc??ncia que juntos sugeriram que estas patologias sejam causadas pela haploinsufici??ncia do SF3B4. A imunohistoqu??mica e histoqu??mica mostraram a express??o de SF3B4 na placa de crescimento e desorganiza????o de condr??citos hipertr??ficos, respectivamente. O transcritoma de um dos pacientes, com muta????o em SF3B4, evidenciou 12 genes ligados a esqueletog??nese com express??o diminu??da no tecido cartilaginoso. A combina????o de t??cnicas seja citogen??tica cl??ssica, citogen??tica molecular, sequenciamento, bem como an??lises in vitro se mostraram eficientes para se alcan??ar o diagn??stico. Embora houvesse um cerne investigativo em comum a todas as doen??as, as investiga????es foram personalizadas para cada caso, visando maior efici??ncia na detec????o das bases moleculares e otimiza????o dos custos da investiga????o molecular.

Depuis la fin des années 1990, la littérature québécoise pour la jeunesse aborde des thématiques délicates auparavant taboues, dont le suicide. Le traitement d’un tel sujet est grandement facilité par l’attention qu’accordent les auteurs à la forme de leur récit, ce que nous observons dans Une vie en éclats de Maryse Pelletier, Le long silence de Sylvie Desrosiers, Le parfum des filles de Camille Bouchard ainsi que Ma vie ne sait pas nager d’Élaine Turgeon. La quête des personnages, désormais plus englobante, est centrée sur le rapport à l’Autre, qui se transforme au cours du processus de deuil. Les protagonistes traversent en fait un long moment d’errance où ils n’ont plus de repères et tentent de fuir, jusqu’à ce que l’apparition d’un nouveau personnage qui comprend leur douleur les aide à établir ce nouveau rapport avec le défunt. La déconstruction formelle des oeuvres vient faire écho à la fragmentation vécue par les personnages, ce qui rend fort significatives l’attention portée à la temporalité ainsi que la multiplicité des narrateurs, voire l’hybridité générique au sein des récits. Les oeuvres semblent remettre en question certaines conventions de la littérature jeunesse afin de faire vivre au lecteur la confusion vécue par les protagonistes : le lecteur confronté à un texte d’apparence déconstruite doit ainsi effectuer un travail de reconstruction semblable à celui opéré par les personnages.