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.

Barley and wheat are the two most important crop species in Turkey. Molecular studies for increasing crop yield of these species are very important for the economic benefits of Turkey. Powdery mildew and yellow rust are the two main pathogens, infecting barley and wheat, respectively in our country and causing a great amount of yield loss each year. Till now, classical genetics studies were performed in order to develop resistant barley and wheat cultivars, but these studies have not been succesful. Therefore, molecular plant-pathogen interactions studies are starting to become the new tool to fight against pathogens. In this thesis, two important aspects of plant microbe interactions were investigated. In the first part, the role of microRNAs (miRNAs) in powdery mildew-barley pathosysytem, and yellow rust-wheat pathosystem were studied. The expression levels of miRNAs and their putative targets were investigated via miRNA microarray analysis and qRT-PCR, respectively, in response to virulent and avirulent pathogen infections. These data were used to establish a new model for powdery mildew-barley and yellow rust-wheat pathosystems. In the second part, functional analysis of a novel F-box gene, which was a ZTL-type F-box, was performed by using Barley Stripe Mosaic Virus mediated Virus Induced Gene Silencing. This F-box gene (HvDRF) (Hordeum vulgare Disease Related F-box) was induced upon yellow rust infection and we studied its role in powdery mildew infection. The results confirmed HvDRF as a positive regulator of race specific immunity and enlarged the roles of ZTL-type F-box proteins to biotic stress responses.

[Truncated abstract] The enlargement of the heart, also known as myocardial hypertrophy, is thought to be a compensatory process that maintains the mechanical function of the heart in response to stress factors such as pressure or volume overload. Although this process is initially compensatory, it frequently results in heart failure and death. Cardiac hypertrophy is a complex process involving changes in the individual cardiac muscle cells, cardiac myocytes. As well as the morphological changes that result from hypertrophy, there are molecular changes within each cell that regulate the hypertrophic process. These molecular changes involve many different pathways within the cardiac myocytes and remain poorly understood . . . Both STAT3α and β overexpression resulted in the upregulation of the VEGF, MnSOD and SOCS-3 genes. This indicates that in the heart, STAT3β is able to activate the gene expression of these genes in a similar manner to STAT3α. However, STAT3α or β activation alone is not enough to induce cardiac hypertrophy. In conclusion, the results presented in this thesis determined a novel role for ERK in the induction of cell death in the heart and revealed many changes in cardiac gene expression following ERK activation. These genes may be the mediators of ERK responses and their identification provides valuable information and direction for further research in this area. One consequence of ERK activation was the negative regulation of the STAT3 pathway. Further investigation revealed for the first time that the STAT3 proteins themselves may not be involved in the induction of cardiac hypertrophy and that STAT3β, initially thought to be a transcriptional repressor, can induce the expression of genes that are known to be activated by STAT3α in the heart. Therefore, these results help to better understand the roles of these two signalling pathways in the heart.

O diagnóstico das neoplasias cutâneas do folículo piloso, particularmente do tricoepitelioma (TE), frequentemente representa dificuldade diagnóstica com o carcinoma basocelular (CBC). As semelhanças clínicas e histopatológicas somadas aos artefatos de amostragem (amostras exíguas por biopsias incisionais ou parcialmente danificadas por esmagamento ou fulguração) podem provocar situações de dificuldade na diagnose diferencial entre as duas neoplasias. O diagnóstico de certeza é importante, pois o CBC tem caráter agressivo local e, quando não totalmente excisado, infiltra os tecidos adjacentes. O TE é uma lesão benigna, sem capacidade de invasão local, não havendo recomendação de excisão com margem cirúrgica. Vários marcadores imunoistoquímicos têm sido propostos na literatura médica para auxiliar no diagnóstico diferencial entre o TE e o CBC. Esses estudos, entretanto, têm resultados conflitantes que podem estar relacionados à pequena casuística avaliada, que geralmente não excede 50 casos de TE. A técnica do arranjo em matriz de amostras teciduais, tissue microarray (TMA), permite a avaliação de um número grande de amostras teciduais, que podem ser submetidas de modo simultâneo aos procedimentos das reações imunoistoquímicas. O objetivo do presente estudo foi o de submeter uma ampla amostra de TE e CBC, obtida através da técnica de TMA, aos marcadores imunoistoquímicos descritos, com a finalidade de identificar um marcador, ou painel de marcadores, capaz de auxiliar a diferenciação do TE do CBC. Cortes histológicos de quatro blocos de TMA representando espécimes de 162 TE e 328 CBC foram submetidos às reações imunoistoquímicas com os anticorpos CD34, BCL-2, CD 10, antígeno de membrana epitelial (EMA), citoqueratinas (CK) 20 e 15, D2-40 e 34 E12. A fim de facilitar a avaliação dos resultados e padrões de expressão antigênica, os espécimes foram digitalizados para obtenção de lâminas histológicas virtuais. Estas foram analisadas por meio de um programa de computador. Fez-se inicialmente a análise dos resultados de 85 TE e 62 CBC representados no primeiro bloco de TMA. Esta verificação identificou a expressão dos marcadores CD34, CD10, EMA, CK15, CK20 e D2-40 com diferença significativa entre os TE e os CBC. Procedeu-se a seguir a avaliação da imunomarcação de toda a casuística. As análises estatísticas de regressão linear multifatorial e regressão logística multifatorial indicaram os marcadores e padrões de expressão em ordem decrescente de importância: D2 40 positivo em células tumorais periféricas, CK 15 positivo em células tumorais periféricas, CD10 positivo no estroma tumoral, CK 20 positivo em células tumorais periféricas e positividade estromal de CD 34. A regressão logística evidenciou ainda que, na amostra examinada, a presença de três ou quatro desses marcadores, com exceção do CD 34, pode identificar 35,9% dos TE. Nossos resultados, obtidos pelo estudo de casuística expressiva, são concordantes com os achados de outros trabalhos que sugerem que o TE e o CBC são neoplasias que estão em diferentes pontos da mesma linhagem de diferenciação dos tumores basalóides foliculares e que, por este motivo, podem expressar os mesmos marcadores/perfil antigênico epitelial e estromal. Embora o painel de quatro anticorpos acima relatado possa ser de grande ajuda, e até mesmo identificar 35,9% dos TE, os critérios histopatológicos clássicos e clínicos ainda devem ser os principais guias para o diagnóstico diferencial entre o TE e o CBC / Trichoepithelioma is a benign neoplasm that shares both clinical and histological features with basal cell carcinoma. It is important to distinguish these neoplasms because they have different clinical behavior and require proper therapeutic planning. Many studies have addressed the use of immunohistochemistry to improve the differential diagnosis of these tumors. These studies present conflicting results when addressing the same markers, probably due to the small number of basaloid tumors that comprised their studies, which generally did not exceed 50 cases. We built a tissue microarray with 162 trichoepithelioma and 328 basal cell carcinoma biopsies and tested a panel of immune markers composed of CD34, CD10, epithelial membrane antigen, BCL-2, cytokeratins 15 and 20 and D2-40. The results were analyzed using multiple linear and logistic regression models. This analysis revealed a model that could differentiate trichoepithelioma from basal cell carcinoma in 35,9% of the cases. The panel of immunohistochemical markers required to differentiate between these tumors was composed of CD10, cytokeratin 15, cytokeratin 20 and D2-40. The results obtained in this work were generated from a large number of biopsies and resulted in the confirmation of overlapping epithelial and stromal immunohistochemical profiles from these basaloid tumors. The results also corroborate the point of view that trichoepithelioma and basal cell carcinoma tumors represent two different points in the same line of differentiation. Despite the use of panels of immune markers, histopathological criteria associated with clinical data certainly remain the best guideline for the differential diagnosis of trichoepithelioma and basal cell carcinoma

Orientadores: Renata de Oliveira Mattos-Graner, Jose Francisco Hofling / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-15T12:50:41Z (GMT). No. of bitstreams: 1 Stipp_RafaelNobrega_D.pdf: 11660668 bytes, checksum: b140f6b3e8ab94a99f86a4eee7101635 (MD5) Previous issue date: 2010 / Resumo: S. mutans são os principais patógenos da cárie, pois expressam diversas proteínas importantes para a colonização e aumento em proporção nos biofilmes dentais na presença de sacarose. Estas incluem as glicosiltransferases (Gtf), que sintetizam uma matriz extracelular de glucano a partir da sacarose, e proteínas ligadoras de glucano (Gbps), as quais participam da interação entre S. mutans e matriz de glucanos. Há evidências de que diversos genes de virulência são regulados por sistemas reguladores de transcrição de dois componentes, dentre os quais CovR (de Control of Virulence) e VicRK (de Virulence Control). Propomos neste estudo descobrir novos alvos e/ou funções dos sistemas CovR e VicKR e avaliar características celulares e fenotípicas decorrentes da ausência desses sistemas. Para isso construímos cepas S. mutans mutantes knockout dos genes covR (SMU.1924) ou vicK (SMU.1517) e realizamos comparações com as respectivas cepas selvagens (WT). A caracterização dos transcriptomas foi triada por microarray e quantificada por qPCR, demonstrando que vinte e três genes foram super expressos na ausência de CovR, enquanto trinta genes foram reprimidos na ausência de VicK. Para demonstrar a regulação direta, as regiões promotoras destes genes foram submetidas a ensaios de retardamento da mobilidade eletroforética com as proteínas recombinantes CovR ou VicR. Além dos genes já conhecidos, os ensaios mostraram CovR como regulador negativo de quatro novos genes e VicR como regulador de cinco novos genes, dentre os quais há, em ambos os casos, genes com provável função na biogênese da parede celular. Nos ensaios fenotípicos, os mutantes covR- demonstraram diminuição no crescimento total em cultura, enquanto que os mutantes vicK observados por microscopia eletrônica de varredura apresentaram formação de cadeias extremamente longas com células atípicas. A formação de biofilme in vitro por mutantes covR- foi de 3 a 17 vezes maior comparado ao WT, enquanto mutantes vicK- demonstraram uma redução de 3,5 a 6,5 vezes. Foi observado aumento de 40% na hidrofobicidade celular nos mutantes covR- quando comparados a cepa WT, enquanto os mutantes vicK- tiveram aumento de 10 vezes, tornando-se altamente hidrofóbicos, provavelmente em decorrência das alterações na superfície da parede celular. Os mutantes covR- foram 30% mais resistente a autólise que as cepas WT, enquanto que os mutantes vicK- possuem o dobro de resistência. Em conclusão, os regulons controlados por CovR e VicKR incluem diversos genes com provável função na biogênese de parede celular, além de genes importantes para a formação de biofilmes. A inativação destes sistemas promoveu alterações fenotípicas notáveis, que realçam os achados daparticipação destes sistemas na biogênese das estruturas celulares. Palavras-chave: Análise em Microsséries, Parede Celular, Cárie Dental, Regulação Bacteriana da Expressão Gênica, Virulência. / Abstract: Streptococcus mutans, a major dental caries pathogen, expresses several virulence genes that mediate its growth and accumulation on tooth surfaces. In this process, GtfB and GtfC catalyze the extracellular synthesis of water-insoluble glucan matrix from sucrose, and are essential for accumulation of S. mutans in the dental biofilm. Glucan binding protein B might also mediate cell surface interaction with glucan. Two component transduction systems, such as CovR (Control of Virulence) and VicRK (Virulence Control), regulate, at least, part of S. mutans virulence genes. In this study we characterized CovR and VicRK functions and some phenotypic traits related to their absences. Knockouts strains covR- (SMU.1924) and vicK- (SMU.1517) strains were constructed and compared by differential microarray against wild type. Quantitative RT-PCR confirmed twenty-three up-regulated genes in covR-, while thirty down-regulated genes in vicKmutant. Recombinant CovR and VicR proteins were used in protein:DNA-promoter electrophoretic mobility shift assay (EMSA) to confirm direct regulator-promoter interaction and transcription. In addition to already-know CovR and VicR targets, qPCR and EMSA revealed that CovR acts as a negative regulator of four additional genes, and VicR controls five undescribed promoters. In both cases, genes controlled are mainly involved in biofilm growth and in cell wall biogenesis. Phenotypically, covR- mutants showed lower yield in culture, while vicK- mutants had altered cell morphology and long chains formation. Inactivation of covR significantly increased in vitro biofilm formation in all strains (3 to 17-fold increase, p<0.01), while vicK mutants showed poor biofilm growth (3.5 to 6.5-fold reduction; p<0.01). Cell hydrophobicity was increased by 40% in covR- mutants and by 10- fold in vicK- mutants, possibly due cell surface changes. Both knockoutsalso increased the autolysis resistance, in about 30% for the covR- mutants and in 2- fold for the vicK- mutant. It is concluded that CovR and VicR systems have important participation on S. mutans physiology, regulating genes that not only are involved in biofilm formation but that have functions in cell wall biogenesis. / Doutorado / Microbiologia e Imunologia / Doutor em Biologia Buco-Dental

A via AKT/PI3K apresenta-se geralmente alterada nos diversos tipos de cânceres humanos e a alteração dos componentes desta via ocorre através da ativação de oncogenes ou inativação de genes supressores tumorais levando a transformações celulares que podem promover a tumorigênese. No câncer de mama a via AKT/PI3K pode ser ativada por Erb-B2, receptores dos fatores de crescimento de insulina (IGF), receptores de estrógeno e perda da expressão do gene PTEN. mTOR (proteína alvo da rapamicina em mamíferos) é uma serina treonina quinase, membro da via AKT/PI3K que se encontra envolvida em múltiplas funções biológicas como controle da tradução, transcrição, degradação protéica e biogênese ribossomal. A ativação desta proteína resulta na fosforilação e ativação de seus principais substratos 4EBP1 e S6K1, requeridos para a biossíntese ribossomal e tradução de RNAms importantes para controle e progressão no ciclo celular. A rapamicina é uma droga com propriedades fungicidas, imunossupressoras e anticancerígenas que atua na inibição de mTOR afetando a expressão de genes envolvidos no metabolismo e síntese protéica. No nosso estudo avaliamos os elementos da via do AKT através de análise imunoistoquímica em fatias de tumores mantidos em cultura de órgão antes e depois do tratamento com rapamicina. A cultura de órgão mantém uma interação entre o epitélio mamário e estroma podendo-se preservar o microambiente que reconstitui o comportamento da célula tumoral. Nesta análise imunoistoquímica observamos uma diminuição significativa de 4EBP1 nas fatias dos tumores tratados com rapamicina em relação aos casos controles. Além disso, fizemos uma avaliação da mudança no perfil da expressão gênica nestas fatias tumorais sub-divididas em Erb-B2 positivos e negativos através da análise por microarray e observamos que a maioria dos genes afetados estavam envolvidos com as funções de transcrição e tradução celulares. Para confirmarmos os resultados obtidos por microarray fizemos uma análise por RT-PCR dos genes WWOX, EXT1 e GTF2E2 em amostras independentes e escolhidos aleatoriamente, conseguindo validá-los em 60% dos casos. Conclusão: A cultura de órgão representa um método simples para determinação dos efeitos da rapamicina. Utilizamos uma estratégia de análise do perfil gênico e novas proteínas que poderiam servir como possíveis marcadores de resposta aos inibidores da proteína mTOR foram identificadas / The AKT/ PI3K pathway are frequently disturbed in many human cancers and the alteration of the components of this pathway occurs through activation of oncogenes or inactivation of tumor suppresors leading to cellular transformation that can promove tumorigenesis. In breast cancer the AKT/PI3K pathway can be activated by ERb-B2, the insulin like growth factor (IGF), estrogen receptors and PTEN loss. mTOR (mammalian target of rapamycin) is a serine threonine kinase, member of the AKT/PI3K pathway, which is involved in multiple biologic functions such as transcription, translation, protein degradation and ribosome biogenesis. The activation of this protein results in phosphorilation and activation of S6K1 and 4EBP1, two downstream signaling elements that are required for ribosomal biosynthesis and mRNAs translation, which is important for cell cycle control and progression. Rapamycin is a potent fungicide, immunossupressive and anticancer agent that inhibits mTOR affecting the expression of genes involved in metabolism and protein synthesis. In the present study we examined some elements of AKT pathway by immunohistochemistry analysis in samples of breast cancer mantained in organ culture before and after treatment with rapamycin. The organ culture maintain an interaction between the mammary epithelium and stroma preserving the micro-environment and restoring the tumor cell behavior. In this immunohistochemistry analysis we noticed a significative decrease of 4EBP1 in the samples of tumors treated with rapamycin compared with the control cases. Besides this, we determined the variation of gene expression profile through microarray analysis in these samples subdivided in positive and negative Erb-B2 and we have identified that most part of the affected genes were mainly involved in cellular transcription and translation.To confirm the results obtained through microarray technique, we have performed the RT-PCR analysis of WWOX, EXT1, GTF2E2 genes and we were able to validate them in 60% of our cases. Conclusion: The organ culture represents a simple method to determine the effects of rapamycin. Using a strategic analysis of the gene profile, news proteins that possibly could be used as markers to the mTOR inhibitors were identifyied

La déficience intellectuelle (DI) est une affection fréquente à causes multiples et souvent inconnues. Durant les 30 dernières années, l’examen utilisé pour l’exploration des anomalies chromosomiques chez des patients libanais présentant une DI était le caryotype standard. Le but de ce projet de thèse était d'appliquer pour la 1ère fois au Liban les technologies d'hybridation sur puces à ADN dans la recherche de nouveaux microremaniements (CNV) impliquant des gènes susceptibles d'engendrer une DI.Ainsi, les ADNs de 99 contrôles et de 185 patients libanais présentant une DI inexpliquée ont été hybridés sur des puces à ADN. Nous avons, par la suite, classé les CNVs identifiés en groupes selon leur transmission, leur contenu en gènes et leur localisation.Nous avons identifié 29 CNVs pathogènes associés à des syndromes ou gènes morbides responsables du phénotype recherché et 90 variants de signification inconnue dont 25 ont été investigués. On a retrouvé 18 de ces derniers comme probablement bénins, 5 comme probablement pathogènes, et 2 à investiguer puisqu'ils sont rapportés comme pathogènes dans la littérature mais hérités d’un parent sain dans l’étude. Nous avons élaboré 4 cas des CNVs pathogènes, 3 des CNVs probablement pathogènes qui sont des microdélétions à l'état homozygote chez des sujets issus de mariages consanguins; et les 2 CNVs à investiguer.Finalement, nous avons discuté les avantages de cette technique permettant l'identification chez 8% des patients ayant une DI inexpliquée, des microremaniements non identifiables par caryotype standard. Cependant nous avons aussi souligné la complexité, les limites et certaines incertitudes d’interprétation des résultats. / Chromosomal imbalances are the most frequent cause of intellectual disability (ID). In Lebanon, during the past 30 years, screening of these imbalances was done using standard karyotyping. However, the resolution of this test was insufficient to detect submicroscopic chromosomal imbalances (CNV). The aim of this thesis was to apply, for the first time in Lebanon, advanced techniques like the chromosomal microarray analysis (CMA), capable of detecting CNVs. Therefore, we screened the DNAs of 185 Lebanese subjects having unexplained ID and those of 99 healthy controls.CNVs identified were classified into groups upon their inheritance status, gene/microRNA content, and their localization.We identified 29 pathogenic CNVs associated to known syndromes or to morbid genes responsible for the patient's phenotype. We also found 90 variants of unknown significance of which 25 were investigated. 18 of the latter were likely benign, 5 were considered as probably pathogenic, and 2 needed future investigations to be classified, as they were considered as pathogenic in the literature but were inherited from a normal parent in this study.We discussed interesting cases by developing 4 pathogenic CNVs, 3 probably pathogenic that were homozygous microdeletions found in patients issued from consanguineous parents, and the 2 CNVs that required further investigations.Finally, we discussed the advantage of this CMA technique that lead to the identification of microimbalances unseen by a standard karyotype in 8% (14/174) of the patients with unexplained ID. Moreover, we mentioned the complexity, limitation and difficulty of interpretation of some results.

Statisticians are used to model interactions between variables on the basis of observed<p>data. In a lot of emerging fields, like bioinformatics, they are confronted with datasets<p>having thousands of variables, a lot of noise, non-linear dependencies and, only, tens of<p>samples. The detection of functional relationships, when such uncertainty is contained in<p>data, constitutes a major challenge.<p>Our work focuses on variable selection and network inference from datasets having<p>many variables and few samples (high variable-to-sample ratio), such as microarray data.<p>Variable selection is the topic of machine learning whose objective is to select, among a<p>set of input variables, those that lead to the best predictive model. The application of<p>variable selection methods to gene expression data allows, for example, to improve cancer<p>diagnosis and prognosis by identifying a new molecular signature of the disease. Network<p>inference consists in representing the dependencies between the variables of a dataset by<p>a graph. Hence, when applied to microarray data, network inference can reverse-engineer<p>the transcriptional regulatory network of cell in view of discovering new drug targets to<p>cure diseases.<p>In this work, two original tools are proposed MASSIVE (Matrix of Average Sub-Subset<p>Information for Variable Elimination) a new method of feature selection and MRNET (Minimum<p>Redundancy NETwork), a new algorithm of network inference. Both tools rely on<p>the computation of mutual information, an information-theoretic measure of dependency.<p>More precisely, MASSIVE and MRNET use approximations of the mutual information<p>between a subset of variables and a target variable based on combinations of mutual informations<p>between sub-subsets of variables and the target. The used approximations allow<p>to estimate a series of low variate densities instead of one large multivariate density. Low<p>variate densities are well-suited for dealing with high variable-to-sample ratio datasets,<p>since they are rather cheap in terms of computational cost and they do not require a large<p>amount of samples in order to be estimated accurately. Numerous experimental results<p>show the competitiveness of these new approaches. Finally, our thesis has led to a freely<p>available source code of MASSIVE and an open-source R and Bioconductor package of<p>network inference. / Doctorat en sciences, Spécialisation Informatique / info:eu-repo/semantics/nonPublished

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