Thesis (M.S.)--Rochester Institute of Technology, 2006. / Typescript. Includes bibliographical references (leaf 58).

Thesis (Ph.D.) -- Virginia Commonwealth University, 2006. / Thesis lacks signature page. Title from title-page of electronic thesis. Prepared for: Dept. of Chemical Engineering. Bibliography: p. 99-113.

Thesis (Ph.D.)-- University of Texas at Dallas, 2007. / Includes vita. Includes bibliographical references (leaves 76-81)

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: DNA; hybridization; microfluidics; kinetics. Includes bibliographical references (leaves 145-148).

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

No description available.

Microarrays make it possible to monitor the expression of thousands of genes in parallel thus generating huge amounts of data. So far, several databases have been developed for managing and analyzing this kind of data but the current state of the art in this field is still early stage. In this paper, we comprehensively analyze the requirements for microarray data management. We consider the various kinds of data involved as well as data preparation, integration and analysis needs. The identified requirements are then used to comparatively evaluate eight existing microarray databases described in the literature. In addition to providing an overview of the current state of the art we identify problems that should be addressed in the future to obtain better solutions for managing and analyzing microarray data.

A expressão de antígenos restritos a tecidos (TRAs do inglês tissue restricted antigens) no timo pelas células epiteliais medulares (mTECs de medullary thymic epithelial cells) é essencial para a tolerância central das células T. Devido à sua heterogeneidade em termos de representação de autoantígenos, esse fenômeno foi denominado como expressão gênica promíscua (PGE de promiscuous gene expression), no qual o gene Aire (de autoimmune regulator) desempenha um papel como principal regulador transcricional positivo sobre um grande conjunto de TRAs dependentes de Aire. A proteína Aire tem a capacidade de interagir com seqüências específicas de DNA desempenhando um papel como regulador direto. Neste estudo utilizamos o método dos cDNA microarrays para acessar a PGE em células mTEC CD80+ murinas cultivadas in vitro. O agrupamento hierárquico dos dados permitiu a observação de que os genes de TRAs foram diferencialmente expressos. Para testar essa hipótese, inicialmente silenciamos o gene Aire pelo método de RNA interferente (RNAi) nas células mTEC. O agrupamento hierárquico dos dados de cDNA microarray mostrou um conjunto de genes de TRAs dependentes de Aire, os quais foram reprimidos após o silenciamento deste último. Redes gênicas reconstruídas desses dados permitiram a identificação de um nó gênico (Gucy2d) estabelecendo regulação positiva sobre genes downstream nas células mTEC normais. Entretanto, sob efeito do silenciamento de Aire, Gucy2d passou a ser um repressor. Esses resultados evidenciaram que genes da PGE estão conectados em rede, que um nó gênico pode atuar como intermediário no seu controle e que Aire na rede PGE desempenha seu controle como regulador upstream. / The expression of tissue restricted antigens (TRAs) in thymus by medullary thymic epithelial cells (mTECs) is essential for the central selftolerance of T cells. Due to heterogeneity of autoantigen representation this phenomenon has been termed promiscuous gene expression (PGE), in which the autoimmune regulator (Aire) gene plays a role as main positive transcriptional regulator on a large set of Aire-dependent TRAs. Aire protein is able in binding to specific DNA sequence motifs and plays a role as a direct regulator. Here we used the cDNA microarray method to access PGE in murine CD80+ mTECs cultured in vitro. Hierarchical clustering of the data allowed observation that TRA genes were differentially expressed. To further investigate the control of PGE, we hypothesize that TRA genes establish networks contributing it selves to modulate their transcriptional levels. Aire in this case plays a role as upstream positive modulator. To test this hypothesis, initially we silenced Aire by gene knockdown (RNA interference) in mTECs. Hierarchical clustering of cDNA microarray data showed a set of Airedependent TRAs genes, which were down regulated after Aire silencing. Gene networks reconstructed from these data allowed the identification of a gene node (Gucy2d) establishing positive regulation upon downstream genes in normal mTECs. Nevertheless, under silencing of Aire, Gucy2d has become a repressor. These finding evidentiate that, genes features in PGE are connected in network; a gene node may act as intermediate in their control and that Aire in PGE network plays a role as an upstream regulator.

A expressão de antígenos restritos a tecidos (TRAs do inglês tissue restricted antigens) no timo pelas células epiteliais medulares (mTECs de medullary thymic epithelial cells) é essencial para a tolerância central das células T. Devido à sua heterogeneidade em termos de representação de autoantígenos, esse fenômeno foi denominado como expressão gênica promíscua (PGE de promiscuous gene expression), no qual o gene Aire (de autoimmune regulator) desempenha um papel como principal regulador transcricional positivo sobre um grande conjunto de TRAs dependentes de Aire. A proteína Aire tem a capacidade de interagir com seqüências específicas de DNA desempenhando um papel como regulador direto. Neste estudo utilizamos o método dos cDNA microarrays para acessar a PGE em células mTEC CD80+ murinas cultivadas in vitro. O agrupamento hierárquico dos dados permitiu a observação de que os genes de TRAs foram diferencialmente expressos. Para testar essa hipótese, inicialmente silenciamos o gene Aire pelo método de RNA interferente (RNAi) nas células mTEC. O agrupamento hierárquico dos dados de cDNA microarray mostrou um conjunto de genes de TRAs dependentes de Aire, os quais foram reprimidos após o silenciamento deste último. Redes gênicas reconstruídas desses dados permitiram a identificação de um nó gênico (Gucy2d) estabelecendo regulação positiva sobre genes downstream nas células mTEC normais. Entretanto, sob efeito do silenciamento de Aire, Gucy2d passou a ser um repressor. Esses resultados evidenciaram que genes da PGE estão conectados em rede, que um nó gênico pode atuar como intermediário no seu controle e que Aire na rede PGE desempenha seu controle como regulador upstream. / The expression of tissue restricted antigens (TRAs) in thymus by medullary thymic epithelial cells (mTECs) is essential for the central selftolerance of T cells. Due to heterogeneity of autoantigen representation this phenomenon has been termed promiscuous gene expression (PGE), in which the autoimmune regulator (Aire) gene plays a role as main positive transcriptional regulator on a large set of Aire-dependent TRAs. Aire protein is able in binding to specific DNA sequence motifs and plays a role as a direct regulator. Here we used the cDNA microarray method to access PGE in murine CD80+ mTECs cultured in vitro. Hierarchical clustering of the data allowed observation that TRA genes were differentially expressed. To further investigate the control of PGE, we hypothesize that TRA genes establish networks contributing it selves to modulate their transcriptional levels. Aire in this case plays a role as upstream positive modulator. To test this hypothesis, initially we silenced Aire by gene knockdown (RNA interference) in mTECs. Hierarchical clustering of cDNA microarray data showed a set of Airedependent TRAs genes, which were down regulated after Aire silencing. Gene networks reconstructed from these data allowed the identification of a gene node (Gucy2d) establishing positive regulation upon downstream genes in normal mTECs. Nevertheless, under silencing of Aire, Gucy2d has become a repressor. These finding evidentiate that, genes features in PGE are connected in network; a gene node may act as intermediate in their control and that Aire in PGE network plays a role as an upstream regulator.

The development and identification of new biomaterials that can replace specific tissues and organs is desirable. In the presented PhD thesis polymer microarrays were applied for the screening of polyacrylates and polyurethanes and evaluation for material discovery for applications in the life sciences. In the first part of the thesis, the largest polymer microarray ever made with more than 7000 features was fabricated and subsequently used for the screening of polyacrylates that can control the fate of human embryonic stem cells. As stem cells have unique properties that offer the potential of replacing damaged or diseased tissue in future, the identification of cultivation substrates that can replace current biological and animal derived products was desirable. The water contact angle, roughness and cell doubling time of the cells on the identified polymers was determined and the stem cells characterised after 5 passages and compared to the currently most widely used animal derived substrate MatrigelTM. In the second part of the thesis, the development of a new polymer gradient microarray is presented. Initial studies involved the optimisation of printing parameters for the generation of linear polymer gradient lines and confirmed by XPS analysis. Cellular binding studies with the suspension cell line K562 and the adherent cell line HeLa were carried out and compared to previous binding studies to confirm the success of the concept. In further studies, the polymer gradients were functionalised with small molecules and proteins, allowing the generation of a protein gradient microarray with Semaphorin 3F. In binding studies with neuron cells it could be shown that the binding of the cells was concentration-dependent. The identification of polyacrylates for the effective and rapid activation and aggregation of platelets is described in the third part of the presented thesis. Here, polymer microarrays were applied for the binding of platelets in human blood samples. The amount of bound platelets as well as their activation state was compared to the natural agonist collagen by employing fluorescence intensity studies and scanning electron microscopy. In shear studies, the activation of the platelets by the polymers was evaluated under physiological conditions. The mechanism by which the polymer triggered the activation was further explored by protein binding studies. It was shown that the initial adsorption of fibrinogen and von Willebrand factor on the polymers lead to the adherence and aggregation of platelets. In the final part of the presented thesis, polymer microarrays were used to identify polymers that can sort and collect the precursor cells of platelets (megakaryocytes). For this purpose, the cell lines K562 and MEG-01 were used as cellular models. The identified polymers and the effect on the immobilised cells was further investigated by scanning electron microscopy, flow cytometry and miRNA studies. The adsorbed proteins on the different polymers were found to influence the cellular morphology on the different substrates.