Übergewicht und Adipositas führen zu Insulinresistenz und erhöhen deutlich das Risiko für die Entwicklung von Typ-2-Diabetes und kardiovaskulären Erkrankungen. Sowohl Adipositas als auch die Suszeptibilität gegenüber Diabetes sind zu einem erheblichen Teil genetisch determiniert. Die relevanten Risikogene, deren Interaktion mit der Umwelt, insbesondere mit Bestandteilen der Nahrung, und die Pathomechanismen, die zur Insulinresistenz und Diabetes führen, sind nicht vollständig aufgeklärt. In der vorliegenden Arbeit sollte durch Genexpressionsanalysen des weißen Fettgewebes (WAT) und der Langerhansschen Inseln die Entstehung und Progression von Adipositas und Typ-2-Diabetes untersucht werden, um relevante Pathomechanismen und neue Kandidatengene zu identifizieren. Zu diesem Zweck wurden Diät-Interventionsstudien mit NZO- und verwandten NZL-Mäusen, zwei polygenen Mausmodellen für das humane metabolische Syndrom, durchgeführt. Eine kohlenhydrathaltige Hochfett-Diät (HF: 14,6 % Fettanteil) führte in beiden Mausmodellen zu früher Adipositas, Insulinresistenz und Typ 2 Diabetes. Eine fettreduzierte Standarddiät (SD: 3,3 % Fettanteil), welche die Entstehung von Adipositas und Diabetes stark verzögert, sowie eine diabetesprotektive kohlenhydratfreie Hochfett-Diät (CHF: 30,2 % Fettanteil) dienten als Kontrolldiäten. Mit Hilfe der Microarray-Technologie wurden genomweite Expressionsprofile des WAT erstellt. Pankreatische Inseln wurden durch laserbasierte Mikropräparation (Laser Capture Microdissection; LCM) isoliert und ebenfalls hinsichtlich ihres Expressionsprofils analysiert. Differenziell exprimierte Gene wurden durch Real-Time-PCR validiert. Im WAT der NZO-Maus bewirkte die HF-Diät eine reduzierte Expression nukleärer Gene der oxidativen Phosphorylierung und von lipogenen Enzymen. Dies deutet auf eine inadäquate Fettspeicherung und -verwertung in diesen Tieren hin. Die Reduktion in der Fettspeicherung und -oxidation ist spezifisch für das adipöse NZO-Modell und konnte bei der schlanken SJL Maus nicht beobachtet werden, was auf eine mögliche Beteiligung an der Entstehung der Insulinresistenz hinweist. Zusätzlich wurde bestätigt, dass die Expansion des Fettgewebes bei der adipösen NZO-Maus eine zeitlich verzögerte Infiltration von Makrophagen in das WAT und dort eine lokale Immunantwort auslöst. Darüber hinaus wurde die Methode der LCM etabliert und zur Gewinnung hochangereicherter RNA aus den Langerhansschen Inseln eingesetzt. In erstmalig durchgeführten genomweiten Expressionsanalysen wurde zu einem frühen Zeitpunkt in der Diabetesentwicklung der Einfluss einer diabetogenen HF-Diät und einer diabetesprotektiven CHF-Diät auf das Expressionsprofil von pankreatischen Inselzellen verglichen. Im Gegensatz zum WAT bewirkt die diabetogene HF-Diät in Inselzellen einerseits, eine erhöhte Expression von nukleären Genen für die oxidative Phosphorylierung und andererseits von Genen, die mit Zellproliferation assoziiert sind. Zudem wurden 37 bereits annotierte Gene identifiziert, deren differenzielle Expression mit der Diabetesentwicklung korreliert. Das Peptidhormon Cholecystokinin (Cck, 11,8-fach erhöht durch die HF) stellt eines der am stärksten herauf regulierten Gene dar. Die hohe Anreicherung der Cck-mRNA in Inselzellen deutet auf eine bisher unbekannte Funktion des Hormons in der Regulation der Inselzellproliferation hin. Der Transkriptionsfaktor Mlxipl (ChREBP; 3,8-fach erniedrigt durch die HF) stellt in Langerhansschen Inseln eines der am stärksten herunter regulierten Gene dar. Ferner wurde ChREBP, dessen Funktion als glucoseregulierter Transkriptionsfaktor für lipogene Enzyme bislang in der Leber, aber nicht in Inselzellen nachgewiesen werden konnte, erstmals immunhistochemisch in Inselzellen detektiert. Dies deutet auf eine neue, bisher unbekannte regulatorische Funktion von ChREBP im Glucosesensor-Mechanismus der Inselzellen hin. Eine durchgeführte Korrelation der mit der Diabetesentwicklung assoziierten, differenziell exprimierten Inselzellgene mit Genvarianten aus humanen genomweiten Assoziationsstudien für Typ-2-Diabetes (WTCCC, Broad-DGI-T2D-Studie) ermöglichte die Identifizierung von 24 neuartigen Diabetes-Kandidatengenen. Die Ergebnisse der erstmals am polygenen NZO-Mausmodell durchgeführten genomweiten Expressionsuntersuchungen bestätigen bisherige Befunde aus Mausmodellen für Adipositas und Diabetes (z.B. ob/ob- und db/db-Mäuse), zeigen in einigen Fällen aber auch Unterschiede auf. Insbesondere in der oxidativen Phosphorylierung könnten die Ergebnisse relevant sein für das Verständnis der Pathogenese des polygen-bedingten humanen metabolischen Syndroms. / Overweight and obesity cause insulin resistance and increase the risk of developing type 2 diabetes and cardiovascular diseases. Both, obesity and susceptibility to diabetes, are to a major part genetically predisposed. The relevant genes, their interaction with the environment – especially with food components – and the pathomechanisms causing insulin resistance and diabetes are not fully known yet. In the present study the development and progression of obesity and type 2 diabetes should be investigated by the means of gene expression analyses of the white adipose tissue (WAT) and the islets of Langerhans to identify underlying pathomechanisms and new causative candidate genes. For this purpose diet intervention studies on NZO- and related NZL-mice – two polygenic mouse models for the human metabolic syndrome – were performed. A carbohydrate containing high fat-diet (HF: 14.6 % fat) caused early obesity, insulin resistance and type 2 diabetes in both mouse models. A fat reduced standard chow (SD: 3.3 % fat) which strongly delayed the onset of obesity and diabetes, and a diabetes protective carbohydrate free high fat-diet (CHF: 30.2 % fat) served as control diets. Using microarray technology genome wide expression profiles of the WAT were generated. Pancreatic islets were isolated by the means of laser capture microdissection (LCM) and expression profiles of them were created, too. Differentially expressed genes were validated by quantitative real time PCR. The HF-diet reduced the expression of nuclear genes of the oxidative phosphorylation and lipogenic enzymes in the WAT of the NZO-mouse. This suggests an inadequate storage and utilization of fat in these animals. This is specific for the obese NZO-model and wasn’t observed for the lean SJL-mouse, indicating a role in the development of insulin resistance. Additionally, there was proof that the enlargement of the WAT triggers a retarded infiltration of macrophages into the WAT and there a local immune response. Moreover, the LCM technique was established and used for the isolation of highly enriched RNA from islets of Langerhans. For the first time the influence of carbohydrates in a high fat-diet on the expression profile of pancreatic islets was investigated by the use of genome wide expression analyses at an early time point at the onset of diabetes. Contrary to the WAT the diabetogenic HF-diet in islets cells increased the expression of both nuclear genes coding for the oxidative phosphorylation and genes associated with cell proliferation. Furthermore 37 already annotated genes correlated with diabetes progression were identified. The peptide hormone cholecystokinin (Cck: 11.8-fold enriched by the HF-diet) is one of the most up-regulated genes. The strong enrichment of Cck-mRNA in islets suggests a previously unknown function of the hormone in the regulation of the islet cell proliferation. The transcription factor ChREBP (Mlxipl: 3.8-fold reduced by the HF-diet) is one of the most down-regulated genes in the islets of Langerhans. Moreover, ChREBP, which has been already identified as a glucose regulated transcription factor for lipogenic enzymes in the liver but not in islets of Langerhans, was detected for the first time in islet cells, using immunohistochemistry. This points to an until now unknown regulatory function of ChREBP in the glucosesensor mechanism of the islet cells. Correlation of the differentially expressed genes associated with diabetes progression with gene variants from human genome wide association studies for type 2 diabetes (WTCCC, Broad-DGI-T2D-study) made the identification of 24 new diabetes candidate genes possible. The results of the genome wide expression analyses, which were done for the first time on a polygenic mouse-model, corroborated previous results for monogenic mouse-models for obesity and diabetes (e.g. ob/ob- and db/db-mice), however also demonstrated differences in some instances. Especially the results concerning the oxidative phosphorylation could be relevant for the comprehension of the pathogenesis of the polygenic human metabolic syndrome.

Background: Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling. Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression. Methods: We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors. Results: High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers. Conclusions: Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.

O extraordinário potencial regenerativo do complexo dentino-pulpar enfatiza a importância da caracterização dos processos celulares e moleculares envolvidos na regeneração dentinária. O avanço da pesquisa com células-tronco desencadeou um grande interesse de cultivá-las na presença de sinais de indução odontogênica. O objetivo do presente trabalho foi avaliar comparativamente células indiferenciadas da polpa (OD-21) e odontoblásticas (MDPC-23) através da avaliação do estímulo celular e do perfil de expressão gênica. As células OD-21 e MDPC-23 foram cultivadas em garrafas de cultura até a subconfluência e, em seguida, cultivadas em placas de 24 poços na concentração de 104 células /poço (n = 5). Os parâmetros analisados foram: (1) proliferação, viabilidade celular e atividade de fosfatase alcalina após 3, 7 e 10 dias; além de detecção e quantificação de matriz mineralizada após 17 dias (o teste estatístico utilizado foi o de Mann-Whitney para p≤0,05); (2) imunofluorescência para proteínas não-colágenas (DSPP e osteopontina) após 1, 3 e 7 dias; (3) análises de expressão transcricional através da tecnologia de cDNA microarray e PCR em tempo real. Os dados de microarrays foram analisados com o auxílio de programas de bioinformática especializados como SAM (significance analysis of microarrays), Cluster-TreeView e GeneNetwork. A expressão gênica foi avalidada pela reação em tempo real em cadeia da polimerase (PCR). Os resultados mostraram que a viabilidade celular ficou acima dos 80% em ambas as células, e que a proliferação celular e a atividade de fosfatase alcalina foram maiores nas células MDPC-23. Foram observados nódulos de mineralização somente na cultura de células odontoblásticas. A osteopontina apresentou-se igualmente presente em ambas as células, enquanto a sialofosfoproteína dentinária foi expressa em maior quantidade nas células MDPC-23. Os resultados demonstraram genes com comportamento semelhante nos dois tipos de células, tais como Bad (morte celular), Erf e Btg1 (proliferação celular), Cxcl10 e Il13 (resposta imune) e Arfgef1 (comunicação celular). Além disso, regiões no heatmap mostraram diferenças na indução e repressão de genes, como C1qb (resposta imune), Jak2 (morte, comunicação e proliferação celular), Col4a1 (adesão celular), Rpl6 e Rpl26 (processo metabólico celular). Concluímos que as células OD-21, embora indiferenciadas, compartilham muitos genes com comportamento semelhante à células odontoblásticas MDPC-23, sugerindo seu potencial para se diferenciar em odontoblastos. / The extraordinary regenerative potential of pulp-dentin complex leads to the importance of the characterization of cell and molecular processes involved in regeneration of dentin. The advancement of stem cell research sparked great interest in cultivating them in the presence of signs of odontogenic induction. The purpose of the present investigation was to evaluate comparatively undifferentiated pulp cells (OD-21) and odontoblastic cells (MDPC-23) through the assessment of cell stimulation and gene expression profiling. OD-21 and MDPC-23 cells were grown in culture flasks until subconfluence, and then cultured in 24-well plates at a concentration of 104 cells/well (n=5). The parameters analyzed were: (1) proliferation, cell viability and alkaline phosphatase activity after 3, 7 and 10 days, in addition to detection and quantification of mineralized matrix after 17 days (the statistical test used was the Mann-Whitney p≤0.05), (2) immunofluorescence for non-collagen proteins (DSPP and osteopontin) at 1, 3 and 7 days, (3) transcriptional expression analysis using cDNA microarray technology and real-time PCR. The microarray data were analyzed with the aid of specialized bioinformatics programs such as SAM (significance analysis of microarrays), Cluster, TreeView and GeneNetwork. Gene expression was avalided by real-time polymerase chain reaction (PCR). The results showed that cell viability was above 80% in both cells, and cell proliferation and alkaline phosphatase activity were higher in MDPC-23 cells. Mineralization nodules were observed only in the odontoblastic cell cultures. Osteopontin was present equally in both cells, whereas dentin sialophosphoprotein was higher expressed in MDPC-23 cells. The results showed genes with similar behavior in two cell types, such as Bad (cell death), Erf and Btg1 (cell proliferation), Il13 and Cxcl10 (immune response) and Arfgef1 (cell communication). Moreover, regions of the heatmap showed differences in induction and repression of genes, such C1qb (immune response), Jak2 (death, communication and cell proliferation), Col4a1 (cell adhesion), Rpl26 and Rpl6 (cellular metabolic process). We conclude that the OD-21 cells, although undifferentiated, share many genes with similar behavior to the odontoblastic MDPC-23 cells, suggesting their potential to differentiate into odontoblasts.

O hepatoblastoma é uma neoplasia embrionária hepática que ocorre na faixa pediátrica, rara, sendo bastante heterogênea devido aos seus diferentes componentes epiteliais e mesenquimais. Pouco ainda se sabe a respeito de sua patogênese. Utilizando um microscópio de captura a laser foram dissecadas áreas de componente epitelial do hepatoblastoma e áreas de tecido hepático adjacente não acometido. Destas amostras obtidas de pacientes não submetidos ao tratamento prévio, foram extraídos RNA e confeccionados cDNA microarrays, para análise comparativa da expressão gênica, seguida de validação por método imunohistoquímico de alguns genes selecionados. Comparando neoplasia e tecido hepático em duas amostras criopreservadas foram identificados 70 genes diferencialmente expressos, sendo 19 hiperexpressos e 51 hipoexpressos no tumor. A maioria dos genes encontrados foi mapeada nos cromossomos 1 e 2. Dos genes selecionados para validação por método imuno-histoquímico, destacaram-se o receptor de Insulina e o TFE3 (genes hipoexpressos no cDNA microarray). A imunomarcação para o receptor de insulina foi positiva tanto no tecido hepático não acometido quanto no componente epitelial fetal do hepatoblastoma , mas foi consistentemente negativa nas amostras de componente embrionário (9/9). A imunomarcação para o TFE3 foi positiva no tecido hepático não acometido, e nos componentes epitelial fetal e embrionário, em proporção variável das células com expressão mais intensa no componente embrionário. As reações imuno-histoquímicas para os outros genes selecionados não permitiram interpretação conclusiva. A alta proporção dos genes diferencialmente expressos localizados nos cromossomos 1 e 2 reflete os achados citogenéticos relatados na literatura relacionada ao hepatoblastoma . Achados de imunoexpressão de proteínas relacionadas aos genes TFE3 e receptor de insulina no tecido hepático e nos diferentes componentes do hepatoblastoma são inéditos e sugerem participação da via sinalizadora da insulina na patogênese destes tumores / Hepatoblastoma is a rare tumor, and little is known about its pathogenesis and genetic alterations. Using a laser capture microdissection microscope we sampled areas of epithelial component of hepatoblastoma prior chemotherapy and their normal liver counterpart in order to perform the comparative gene expression analysis followed by the validation of selected genes by immunohistochemistry. Comparing tumor and non-diseased liver in two frozen samples, 70 differentially expressed genes were found, 19 overexpressed and 51 underexpressed in the tumor. Most of the genes were located at chromosomes 1 and 2. Of the genes selected for validation by immunohistochemistry, the most interesting findings came from Insulin receptor and TFE3 (both underexpressed genes). Insulin receptor was positive in non diseased liver and in the fetal component of the Hepatoblastoma but was consistently negative in the embryonal component (9/9 cases). The TFE3 staining was positive in the normal liver and fetal and embryonal components of the tumor in variable proportion of the cells, more marked in the embryonal component. The higher proportion of genes located at chromosomes 1 and 2 corroborates the cytogenetics findings reported in the literature related to Hepatoblastoma . The immunohistochemistry findings of different expression of insulin receptor in the fetal and embryonal components of Hepatoblastoma and the positivity of TFE3 in normal liver and in the tumors epithelial components deserves further investigation regarding the role of these genes to the pathogenesis of Hepatoblastoma

Doenças tireoideanas são bastante comuns, sendo sua maioria benigna. A relação entre os diversos tipos de doenças tireoideanas, bem como seus aspectos moleculares, são pouco conhecidos. O bócio (hiperplasia), por exemplo, é descrito por alguns como relacionado com carcinoma (tumor maligno) papilífero, enquanto que outros afirmam não haver relação causal entre as duas doenças. A questão mais desafiante, porém, refere-se à distinção entre adenoma (tumor benigno) e carcinoma folicular, que atualmente é feita apenas após à cirurgia, não permitindo tratamento diferenciado para os dois tipos de tumor. Este trabalho buscou identificar genes diferencialmente expressos entre tecido tireoideano normal, bócio, adenoma e carcinoma papilífero utilizando microarrays. Carcinomas foliculares não foram incluídos devido ao número e tamanho reduzidos das amostras. Dois tipos de array foram utilizados: arrays em membranas de nylon, contendo 213 clones obtidos por DDRT-PCR de amostras de tireóide, e arrays em vidro, contendo 3800 clones ORESTES. Experimentos utilizando o primeiro tipo de array identificaram três genes diferencialmente expressos, cuja expressão foi analisada por RT-PCR em 10 amostras de cada tipo de tecido. Dois deles foram capazes de diferenciar carcinomas papilíferos de tecido normal e bócio com 89% de precisão para o tumor maligno e 80% para os tecidos não malignos. Os arrays em vidro foram utilizados para avaliar o perfil de expressão de aproximadamente 10 amostras de cada tipo de tecido tireoideano. Foram identificados 160 clones diferencialmente expressos entre quaisquer dois tipos de tecido, cujas seqüências foram determinadas e comparadas com as dos bancos de dados. Dentre os genes mais interessantes destacam-se o correspondente à ATPase Na/K, cuja expressão está reduzida nos carcinomas em relação a tecidos normais e adenomas, o da proteína PDCD4, envolvida em morte celular programada, mais expresso em adenomas e tecidos normais em comparação com carcinomas e bócios, e os da calgizzarin (S100A11) e da α1-anti-tripsina, ambos mais ativos nos carcinomas do que nos demais tecidos. Todos esses genes já foram descritos como diferencialmente expressos em algum tipo de tumor. Este trabalho levou à padronização da metodologia de microarray em lâminas de vidro em nosso laboratório, bem como à identificação de genes que podem elucidar as alterações envolvidas na formação do bócio, adenoma e carcinoma papilífero. A implantação da técnica de amplificação de mRNA em nosso laboratório viabilizou a utilização de 10 amostras de carcinoma folicular, cuja massa de RNA total era insuficiente para as hibridizações. Essas amostras serão hibridizadas, juntamente com 10 amostras de adenoma, com microarrays contendo 4800 genes humanos conhecidos para a busca de genes diferencialmente expressos, de grande interesse diagnóstico. / Thyroid diseases are very common and are usually benign. The causal relationships among the different types of disease, as well as their molecular aspects, are not well understood. The goiter (hyperplasia), for instance, is described by some as related to papillary carcinoma (a malignant tumor), while others say there is no causal relationship between the two diseases. The most defying question, however, concerns the distinction between adenoma (benign tumor) and follicular carcinoma, which is currently made only after surgery, not allowing distinct treatments for the two kinds of tumor. This work aimed to identify differentially expressed genes among normal thyroid tissue, goiter, adenoma and papillary carcinoma using microarrays. Follicular carcinomas were not included due to the reduced number and size of the samples. Two kinds of array were used: arrays in nylon membranes, with 213 clones isolated from thyroid samples by differential display (DDRT-PCR); and glass slide arrays containing 3800 ORESTES clones.Experiments using the first type of array identified three differentially expressed genes, whose expression was analyzed by RT-PCR in 10 samples of each kind of tissue. Two of these genes were able to differentiate papillary carcinomas from goiters and normal tissues with precisions of 89% for the malignant tumor and 80% for the non-malignant tissues. Glass slide arrays were used to evaluate gene expression profile of approximately 10 samples of each type of thyroid tissue. 160 clones differentially expressed between any two tissues were identified, and their sequences were determined and compared with databases. Among the most interesting genes are Na/K ATPase gene, whose expression is reduced in carcinomas compared to normal tissues and adenomas, the gene corresponding to PDCD4 protein, involved in program cell death, with elevated expression in adenomas and normal tissues than carcinomas and goiters, and the genes of calgizzarin (S100A11) and α1-antitrypsin, both more active in carcinomas than the other tissues. All these genes have already been described as differentially expressed in at least one type of human cancer. This work led to the standardization of glass slide microarray technology in our laboratory, and to the identification of genes that may clarify the alterations involved in the formation of goiter, adenoma and follicular carcinoma. The implementation of mRNA amplification technique in our laboratory allowed the utilization of 10 samples of follicular carcinoma, whose mass was insufficient for microarray hybridizations. These samples will be hybridized along with 10 samples of adenomas, with microarrays containing 4800 known human genes to search for differentially expressed genes, of great diagnostic interest.

Pseudomonas aeruginosa (PA) est la troisième bactérie impliquée dans les maladies nosocomiales et est la principale cause de mortalité des patients atteints de la mucoviscidose. PA est résistante à la plupart des traitements antibiotiques. Trouver de nouvelles stratégies thérapeutiques est devenu un enjeu majeur de santé publique, l’une d’entre elles est l’inhibition de facteurs de virulence. Parmi ceux-ci, les lectines sont des protéines impliquées dans l’adhésion et la formation de biofilm via des interactions avec des sucres (PA-IL, PA- IIL, FliC, FliD, PilA, PilY1 et CupB6).Le but de ce travail est donc de trouver des leurres moléculaires ayant une forte affinité pour ces lectines. Ceux-ci sont des motifs saccharidiques présentés de façon multivalente : glycoclusters. De part leur grande diversité structurale et leur faible quantité, un outil de criblage innovant a été développé qui consiste en une lame de verre microstructurée : le glycocluster-microarray. Les glycoclusters sont immobilisés de manière ordonnée par DNA Directed Immobilization (DDI). Deux méthodes de criblage ont été développées grâce à cet outils : 1) le criblage en solution et par compétition d’une bibliothèque de motifs saccharidiques et 2) le criblage d’une bibliothèque de glycoclusters immobilisés sur le microarray. Avec cet outil, des protocoles de mesures d’IC50 et de Kd ont aussi été fiabilisés pour caractériser les meilleurs candidats inhibiteurs des lectines. Le glycocluster- microarray présente l’avantage de n’utiliser qu’une très faible quantité de matériel (quelques picomoles) et permet de réaliser diverses analyses en parallèle.Afin de valider cet outil, une étude sur l’impact de la densité de surface en glycocluster a été menée. Le criblage de plus de 150 motifs saccharidiques a permis de sélectionner ceux ayant une forte affinité pour les lectines. L’analyse sur microarray complétée par de la modélisation moléculaire d’une bibliothèque de glycoclusters, possédant ces motifs et différentes topologies, valences et propriétés (aromaticité, charge,…), a permis d’identifier les paramètres clés dirigeant les relations structure-affinité. Une activité anti-biofilm chez PA a été démontrée avec les meilleurs glycoclusters ciblant PA-IL.Tester l’activité in vivo, chez l’animal, des meilleurs candidats est une voie à explorer. Cibler d’autres lectines comme celles présentes sur le flagelle et les pili de PA et notamment impliquées dans son adhésion précoce est aussi une voie à développer. Pour cela, des tests préliminaires ont été présentés et d’autres sont en cours faisant appel à l’utilisation de bactéries entières ainsi qu’à une détection sans marquage des lectines. / Summary: Pseudomonas aeruginosa (PA) is the third pathogen involved in nosocomial diseases and the major cause of mortality of cystic fibrosis patients. PA develops resistance to antibiotics treatments. And so, developing new therapeutic strategies is a public health issue. One of the promising strategies is to inhibit virulence factors involved in the adhesion and the biofilm formation of PA. Some of these virulence factors are lectins which interact with sugars (PA-IL, PA-IIL, FliC, FliD, PilA, PilY1 and CupB6).The goal of this work is to find molecular decoys which have a strong affinity for these lectins. These are saccharidic units with a multivalent display: glycoclusters. An innovative screening tool has been developed: the glycocluster-microarray, to study lectin/glycocluster interactions. It is a microstructured glass slide where glycoclusters are immobilized by DNA Directed Immobilization (DDI). Two screening methods have been developed with this microarray: 1) the screening in solution and by competition of a saccharidic units library and2) the screening of a glycoclusters library immobilized on the microarray. Protocols of IC50 and Kd measurements have also been developed with this tool to characterize the best lectins inhibitors. This tool allows to use few amount of material (few picomoles) and to do parallel analysis.To validate the microarray, a study of the impact of glycoclusters surface density has been done. The screening of more than 150 saccharidic units allowed the selection of the ones that display the best affinity forlectins. The analysis, on microarray and molecular simulations, of the glycoclusters library displaying thesesaccharidic units and several topologies, valences and properties (aromaticity, charge,…) enable to identify key parameters of structure-affinity relationships. An anti-biofilm activity has been observed for the best glycoclusters targeting PA-IL.Testing in vivo activity of these best candidates will be explored. Targeting others lectins such as the ones on the flagella and pili of PA and involved in the early adhesion needs also to be developed. To this end, preliminary tests have been showed and some are in progress.

La santé digestive du lapin est difficile à maîtriser en élevage, notamment en période de sevrage où des troubles digestifs sont fréquents et peuvent entraîner des pertes importantes. Bien que l’utilisation d’antibiotiques diminue, afin d’éviter l’apparition de problèmes de résistance, des solutions alternatives doivent être trouvées. Nos principaux objectifs étaient : i) d’étudier l’influence de l’incorporation de fibres rapidement fermtentescibles (FRF) dans un aliment distribué précocement dès 15 jours d’âge, en comparaison d’un aliment avec antibiotiques (tiamuline et apramycine) utilisés pour lutter contre l’entéropathie épizootique du lapin ; ii) de déployer des méthodologies en génomique ciblant l’espèce lapin (amélioration et réannotation d’un microarray) et son microbiote digestif (pipeline d’analyse du gène de l’ARNr 16S), pour du phénotypage moléculaire. L’originalité de notre travail réside ainsi dans l’acquisition de données par trois approches complémentaires : phénotypique, transcriptomique, et métagénomique. Au niveau phénotypique, la stimulation de l’activité fermentaire par les FRF est vérifiée avec une hausse de la concentration caecale en AGV (+20%) et une plus forte acidité du biotope caecal. De plus, les FRF tendent à réduire la mortalité en post-sevrage et améliorent l’efficacité alimentaire. Au niveau transcriptomique, nous avons tout d’abord apporté de nouvelles connaissances sur l’immunité du lapin adulte avec la stimulation in vitro de cellules mononuclées du sang périphérique par du LPS et de la PMA-ionomycine. Cette étude confirme l’importance du lapin comme animal modèle pour la recherche biomédicale. Chez le jeune lapin, la nouvelle version du microarray nous a permis de montrer que les fonctions biologiques sont plus rapidement mises en place avec les FRF qu’avec un aliment standard, et que les antibiotiques nivellent l’expression génique. De plus, les FRF contribueraient à limiter l’inflammation intestinale. Au niveau métagénomique et en ciblant l’ARNr 16S, nous montrons qu’une augmentation de l’activité microbienne caecale n’est pas associée à une modification majeure de la composition du microbiote, à l’exception de la stabilisation de l’abondance des Campylobacteraceae, qui limiterait l’inflammation digestive. Nos travaux, encore préliminaires, n’ont pas permis d’identifier des corrélations significatives entre l’expression génique dans le sang et/ou l’iléon, et les profils taxonomiques (OTUs) majoritaires du microbiote digestif. L’ensemble de nos résultats suggère d’une part que les FRF peuvent être proposées comme une nouvelle stratégie nutritionnelle péri-sevrage, avec une amélioration de l’efficacité alimentaire et une protection accrue de la santé digestive, et d’autre part que des approches génomiques sont prometteuses pour qualifier finement les phénotypes d’intérêt chez le lapin. / Digestive health of rabbits is difficult to manage in breeding systems, especially around weaning where digestive problems are very common and can result in significant losses. The use of antibiotics decreases in order to avoid the emergence of problems with antibiotic resistance, but alternatives must be found. The objectives of this work were: i) to study effects of the incorporation of rapidly fermentable fiber (FRF) in diets fed to rabbits from 15 days of age, and compared to a feed with antibiotics (tiamulin and apramycin), used to fight against epizootic rabbit enteropathy; ii) to deploy genomics methodologies targeting the rabbit species (improvement and re-annotation of a microarray) and the digestive microbiota (pipeline analysis of 16S rRNA gene), for molecular phenotyping. An innovative approach was used in this work through the combination of three complementary approaches: phenotypic, transcriptomic, and metagenomic. At the phenotypic level, the stimulation of the fermentation activity by FRF is verified with higher cecal VFA concentrations (+ 20%) and higher cecal acidity. Moreover, the FRF tend to reduce mortality post-weaning and improve feed efficiency. At the transcriptomic level, we obtained new data on the immunity of the adult rabbit with in vitro stimulation of peripheral blood mononuclear cells with LPS and PMA-ionomycin. This study confirms the importance of the rabbit as an animal model for biomedical research. In young rabbits, the new version of the microarray allowed us to show that biological functions are more quickly implemented with FRF than a standard diet, and that antibiotics level out the gene expression. In addition, FRF help to limit intestinal inflammation. At the metagenomic level, the targeting of the 16S rRNA, we showed that an increase in cecal microbiota activity is not associated with major changes of microbiota composition, with the exception of the stabilization of the relative abundance of Campylobacteraceae, which limits gastrointestinal inflammation. Our preliminary results did not identify significant correlations between gene expression in blood and/or ileum, and principal taxonomic profiles of the digestive microbiota. Our results suggest that FRF can be proposed as a new nutrition strategy peri-weaning, with improved feed efficiency and increased protection of digestive health, and secondly that genomic approaches are promising to describe more precisely phenotypes of interest in rabbits.

O objetivo deste trabalho é apresentar os métodos originais em Bioinformática desenvolvidos para a análise estatística na interpretação dos dados de duas técnicas baseadas em imagens: a técnica de microarranjos de DNA e a técnica de ressonância magnética funcional. O interesse principal é abordar essas técnicas experimentais quando enfrenta-se uma situação clara de amostras escassas, isto é, quando existem relativamente poucas observações experimentais do fenômeno estudado, sendo a análise individual/personalizada o representante extremo desta situação, que tem que ser resolvida. Para tanto, opta-se pelo uso da Inferência Bayesiana no contexto da Teoria da Decisão sob Incerteza, implementada computacionalmente sob o arcabouço dos Sistemas de Suporte à Decisão. Ambas as tecnologias estudadas produzem dados complexos, baseados na interpretação das diferenças entre imagens obtidas da resposta do sistema a um estímulo e da resposta numa situação controle. O resultado deste trabalho é o desenvolvimento de dois sistemas de suporte à decisão, chamados HTself e Dotslashen, para a análise de dados de microarranjos e ressonância magnética funcional, respectivamente; e de seus métodos matemáticos/computacionais subjacentes. Os sistemas desenvolvidos extraem conhecimento racional de bancos-de-dados normativos, através de modelos matemáticos específicos, contornando então o problema de amostras escassas. Finalmente, neste trabalho são descritas aplicações a problemas reais, para destacar a utilidade dos sistemas de suporte à decisão desenvolvidos nas áreas de Biologia Molecular e Neuroimagem Funcional. / The goal of this work is to present the novel Bioinformatics methods that were developed aiming the statistical analysis of two image-based techniques: DNA microarrays and functional magnetic resonance imaging. The main interest is to approach these experimental techniques in small sample size situations, i.e., when there are relatively few experimental observations of the phenomena of interest, for which the case of single subject/datum analysis is its most extreme. In order to approach these problems we chose to use Bayesian Inference in the context of the Decision Theory under Uncertainty, computationally implemented under the Decision Support Systems framework. Both technologies produce complex data, based on the interpretation of differences between images from the response to a given stimulus and the control situation. The result of this work is the development of two decision support systems, called HTself and Dotslashen, to analyze microarray and functional magnetic resonance imaging data, respectively; and the underling mathematical and computational methods. These systems use the rational knowledge from normative databases implemented in specific mathematical models, overcoming the problem of small sample size. Finally, in this work it is described applications to real problems in order to stress the utility for Molecular Biology and Functional Neuroimaging of the developed decision support systems.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2015-07-27T14:33:27Z No. of bitstreams: 1 Erika Aparecida Felix de Barros.pdf: 3994975 bytes, checksum: 34211c9fc059d61aa8302afe5a482223 (MD5) / Made available in DSpace on 2015-07-27T14:33:27Z (GMT). No. of bitstreams: 1 Erika Aparecida Felix de Barros.pdf: 3994975 bytes, checksum: 34211c9fc059d61aa8302afe5a482223 (MD5) Previous issue date: 2014-12-18 / Prostate cancer (PCa) is the most common non-skin solid tumor in man in western countries. The prognosis is defined by the measurement of PSA, stage and Gleason score, however, none of these factors classics, even when combined, show good performance in prognosis definition, therefore molecular markers are being studied. Cytogenetic abnormalities, represented by chromosomal deletions and gains, are characteristic of oncogenesis and potentially can be used as a prognostic marker. Objectives: The study aimed to identify the cytogenetic alterations of aneuploidy in chromosomes 3 , 7 , 17 and 9p21 locus by FISH technique located in PCa underwent radical prostatectomy. As a secondary objective correlate the changes found with biochemical recurrence after surgical treatment and the classical prognostic factors of prostate adenocarcinoma. Methodology: For this case-control study 112 patients with clinically localized PCa who underwent radical prostatectomy were followed up postoperatively than ten years were analyzed. Samples of the primary tumor of each patient were available for tissue microarray matrix and cytogenetic changes were assessed by FISH technique. Results: Among the patients for the 9p21 locus, 6.4 % had lost one of the two alleles. In relation to the results obtained for chromosomes 3, 7 and 17 find deletion of one allele of 2.3, 1.2 and 1.8% of the cases respectively. No association between aneuploidy of these chromosomes with the Gleason score, pathological stage, serum PSA level or risk group . We observed that the loss of the 9p21 locus was associated with shorter time to recurrence (p 0.038 ). Conclusions: We found a low occurrence of aneuploidy detected by probes CEP 3 , 7 and 17 and LSI 9p in our series of tumors. We observed that the loss of the 9p21 locus was associated with worse prognosis in CaP located treated with surgery. / Introdução: O câncer de próstata (CaP) é o tumor sólido não cutâneo mais comum do homem em países ocidentais. O prognóstico é feito pela dosagem do PSA, estádio e escore de Gleason, porém, nenhum destes fatores clássicos, mesmo quando avaliados em conjunto, apresentam bom desempenho na determinação prognóstica, por isso marcadores moleculares estão sendo estudados. Alterações citogenéticas, representadas por ganhos e deleções cromossômicas, são características da oncogênese e potencialmente podem ser empregadas como marcador de prognóstico. Objetivos: O estudo teve como objetivo principal identificar as alterações citogenéticas de aneuploidia nos cromossomos 3, 7, 17 e lócus 9p21 pela técnica de FISH no CaP localizado submetido à prostatectomia radical. Como objetivo secundário correlacionamos as alterações encontradas com a recidiva bioquímica após tratamento cirúrgico e com os fatores prognósticos clássicos do adenocarcinoma de próstata. Metodologia: Para este estudo caso controle foram analisados 112 pacientes com diagnóstico de CaP clinicamente localizado submetidos à prostatectomia radical com seguimento pós operatório superior a dez anos. As amostras do tumor primário de cada paciente foram disponibilizadas em matriz de microarranjo tecidual e as alterações citogenéticas foram avaliadas através da técnica de FISH. Resultados: Dos pacientes avaliados para o lócus 9p21, 6,4% apresentaram perdas de um dos dois alelos. Em relação aos resultados obtidos para os cromossomos 3, 7 e 17 encontramos deleção de um dos alelos em 2,3; 1,2 e 1,8% dos casos respectivamente. Não observamos associação entre aneuploidia desses cromossomos com o escore de Gleason, estádio patológico, nível sérico de PSA ou grupo de risco. Observamos que a perda do lócus 9p21 esteve associado a menor tempo para recidiva (p 0,038). Conclusões: Encontramos baixa ocorrência de aneuploidia detectadas pelas sondas CEP 3, 7 e 17 e LSI 9p em nossa série de tumor. Observamos que a perda do lócus 9p21 esteve associado a pior prognóstico no CaP localizado tratado com cirurgia.

To understand transmissible human diseases, disciplines such as epidemiology and the surveillance of affected cases are as essential as the knowledge about the pathogenesis and the course of a disease. Epidemiologists categorize and estimate factors for public health risks by taking metadata into account including geographic aspects, health and social states to study a disease transmission and prevent further cases. In addition, a focus on the causative agents itself is necessary in order to understand their ecology and hence their virulence traits. The causative agents for a severe pneumonia named Legionnaires’ disease (LD) are bacteria of the genus Legionella. The putative sources of LD infection are any aerosol-generating natural or man-made fresh water systems. Due to this ubiquitous distribution of legionellae, it is difficult to find the source of infection. Therefore, it is necessary to isolate the bacterium from the suffering patients to further characterize it in the laboratory and to compare the clinical isolates with isolates obtained from probable environmental sources. The predominant species isolated from LD patients is Legionella pneumophila serogroup (Sg) 1. Intensive genotyping of L. pneumophila Sg1 isolates by using the current gold standard method, the sequence-based typing scheme (SBT), revealed limitations in the discrimination of several sequence types (ST) which could not be compensated for by additional phenotypic typing scheme. In practical terms, this means that several clones or STs are disproportional frequently found in both, patients and water systems, and cannot be distinguished by current methods. Therefore, a distorted picture of endemic and globally-spread clones is generated and current typing methods cannot add substantial information during the identification of the infectious source. The aim of this thesis is to develop and implement new typing methods for L. pneumophila isolates with a higher resolution than the gold standard methods. A DNA-DNA hybridization based microarray was designed and equipped with probes that target specifically L. pneumophila virulence factors and genes that are involved in the biosynthesis of lipopolysaccharide structures. Legionellae can be subgrouped on the basis of their lipopolysaccharide structures. Here, the usually phenotypic characterization of L. pneumophila Sg1 is successfully transmitted to a DNA-based genotypic method. Furthermore, the detailed validation of the DNA-microarray revealed a higher discriminatory power in comparison to the gold standard methods. It enables previously indistinguishable clones to be subdivided, providing valuable information about probable sources of infection. The second new tool for typing of L. pneumophila is based on the core genome of the bacteria. An extended SBT-scheme was extracted from the core genome and accordingly named core genome multilocus sequence typing (cgMLST). This genome wide gene-by-gene typing approach allows a high genomic resolution of L. pneumophila isolates by retaining epidemiological concordance. A major advantage of this genome-based method is the detection of large recombination events within the analysed genomes, which is, so far, reserved for whole genome sequencing. The population structure of legionellae is largely driven by recombination and horizontal gene transfer rather than by spontaneous mutations. Therefore, the detection of recombination events is essential for typing of L. pneumophila isolates. In addition, the cgMLST-scheme assigns a core genome sequence type to the analysed isolate and allows backwards compatibility with the current SBT-scheme. Both methods proved to be fast, reliable and robust typing methods through their application during outbreak investigations. Furthermore, both systems are particularly suited as routine molecular typing tools for the surveillance of single cases. The raw data are verified and translated into uniform portable codes, which enables the easy transfer and comparison of results. The standardized and portable quality of the results of both methods enables the establishment of a curated global database. This qualifies both methods as potential new gold standard methods for the genotyping of L. pneumophila isolates.