The global aim of this thesis was to study the use of microarray technology for the screening and identification of biocompatible polymers, to understand physiological phenomena, and the design of biomaterials, implant surfaces and tissue-engineering scaffolds. This work was based upon the polymer microarray platform developed by the Bradley group. Polymer microarrays were successfully applied to find the best polymer supports for: (i) mouse fibroblast cells and used to evaluate cell biocompatibility and cell morphology. Fourteen polyurethanes demonstrated significant cellular adhesion. (ii) Analysis of the adhesion of human erythroleukaemic K562 suspension cells onto biomaterials with particular families of polyurethanes and polyacrylates identified. A DNA microarray study (to access the global gene expression profiles upon cellular binding) demonstrated that interactions between cells and some polyacrylates induced a number of transcriptomic changes. These results suggested that, during these interactions, a chain of cellular changes is triggered, most notably resulting in the downregulation of membrane receptors and ligands. (iii) Identification of polymers with potential applications in the field of stem cell biology. Polymers were identified that showed attachment, promotion and stabilisation of hepatocyte-like cells. A polyurethane support (PU-134) was pinpointed, which significantly improved both hepatocyte-like cell function and “lifespan”. A second project investigated biomaterials that promoted adhesion, growth and function of endothelial progenitor cells. A new polymer matrix was identified which contained the necessary signals to promote endothelial phenotype and function. This has potential application in the creation of blood vessels and the endothelialisation of artificial vessel prostheses and stent coatings for improving angioplasty therapy. (iv) The study of bacterial adhesion, focusing on the adhesion of food-borne pathogenic bacterium Salmonella enterica serovar typhimurium, strain SL1344, and the commensal bacterium Escherichia coli, strain W3110. Several polymers were found to support selective bacterial enrichment, as well as others that minimised bacterial adhesion.

O objetivo deste trabalho é apresentar uma técnica automática baseada em morfologia matemática para medida de sinal em imagens de cDNA desenvolvida no BIOINFO,em parceria com o Instituto Ludwig de Pesquisa contra o Câncer. A tecnologia de lâminas de cDNA é um processo baseado em hibridização que possibilita observar a concentração relativa de mRNA de amostras de tecidos analisando a luminosidade de sinais fluorescentes ou radioativos. Hibridização é o processo bioquímico onde duas fitas de ácido nucleico com seqüências complementares se combinam. A técnica apresentada permite o cálculo da expressão gênica com alto grau de automação, podendo o usuário corrigir com facilidade eventuais erros de segmentação. O usuário interage com o programa apenas para selecionar as imagens e inserir os dados de geometria da lâmina. A estratégia de solução usada tem três fases: gradeamento dos blocos, gradeamento dos spots e segmentação dos spots. Todas as fases utilizam filtros morfológicos e as fases de gradeamento possuem um passo final de correção baseado nos dados de geometria da lâmina o que aumenta a robustez do processo, que funciona bem mesmo em imagens ruidosas. / The objective of this work is to present the automated technique for measuring signal from cDNA images developed in BIOINFO, associated with the Ludwig Institute for Cancer Research. Microarray technology is a hybridization based process that makes possible to quantify the relative abundance of mRNA in two tissue samples analysing the luminosity of fluorescent or radioactive signals. Hybridization is a biochemical process where a strand of nucleic acid matches up its counterpart. The developed technique permits the calculation of gene expression with a high level of automation. Besides that, the user can easily correct eventual segmentation mistakes. The user interacts with the program only to select the images and to set the slide geometry parameters. The solution strategy has three main steps: subarray griding, spots gridding and spots detection. All the steps use morphological filters, and the two gridding steps have a final correction substep based on the slide geometry, increasing the process robustness, that works well even in noisy images.

Neste trabalho desenvolvemos um novo algoritmo para clustering para dados de expressão gênica. As abordagens tradicionais utilizam um conjunto de dados na forma de uma tabela de duas dimensões, onde as linhas são os genes e as colunas são as condições experimentais. Nós utilizamos uma estrutura de três dimensões, acrescentando fatias de tempo. Implementamos nosso algoritmo e testamos com conjuntos de dados sintéticos e dados reais, usando índices de validação para comparar os resultados obtidos pelo nosso algoritmo com os resultados produzidos pelo algoritmo TriCluster. Os resultados mostraram que o nosso algoritmo é bom para dados de expressão gênica em três dimensões e pode ser aplicado a dados de outros domínios / In this study we developed a new clustring algorithm for gene expression data. Previous solutions use a dataset in the form of a table, where the rows are the genes and the columns are the experimental conditions. We used a three-dimensional structure adding time-slices. We implemented this algorithm and tested it with synthetic and real data, using validation index to compare our results with the results obtained by the TriCluster algotithm. Results show that our solution is good for three dimensional gene expression data and can be employed to other domains

O objetivo deste trabalho é apresentar uma técnica automática baseada em morfologia matemática para medida de sinal em imagens de cDNA desenvolvida no BIOINFO,em parceria com o Instituto Ludwig de Pesquisa contra o Câncer. A tecnologia de lâminas de cDNA é um processo baseado em hibridização que possibilita observar a concentração relativa de mRNA de amostras de tecidos analisando a luminosidade de sinais fluorescentes ou radioativos. Hibridização é o processo bioquímico onde duas fitas de ácido nucleico com seqüências complementares se combinam. A técnica apresentada permite o cálculo da expressão gênica com alto grau de automação, podendo o usuário corrigir com facilidade eventuais erros de segmentação. O usuário interage com o programa apenas para selecionar as imagens e inserir os dados de geometria da lâmina. A estratégia de solução usada tem três fases: gradeamento dos blocos, gradeamento dos spots e segmentação dos spots. Todas as fases utilizam filtros morfológicos e as fases de gradeamento possuem um passo final de correção baseado nos dados de geometria da lâmina o que aumenta a robustez do processo, que funciona bem mesmo em imagens ruidosas. / The objective of this work is to present the automated technique for measuring signal from cDNA images developed in BIOINFO, associated with the Ludwig Institute for Cancer Research. Microarray technology is a hybridization based process that makes possible to quantify the relative abundance of mRNA in two tissue samples analysing the luminosity of fluorescent or radioactive signals. Hybridization is a biochemical process where a strand of nucleic acid matches up its counterpart. The developed technique permits the calculation of gene expression with a high level of automation. Besides that, the user can easily correct eventual segmentation mistakes. The user interacts with the program only to select the images and to set the slide geometry parameters. The solution strategy has three main steps: subarray griding, spots gridding and spots detection. All the steps use morphological filters, and the two gridding steps have a final correction substep based on the slide geometry, increasing the process robustness, that works well even in noisy images.

Neste trabalho desenvolvemos um novo algoritmo para clustering para dados de expressão gênica. As abordagens tradicionais utilizam um conjunto de dados na forma de uma tabela de duas dimensões, onde as linhas são os genes e as colunas são as condições experimentais. Nós utilizamos uma estrutura de três dimensões, acrescentando fatias de tempo. Implementamos nosso algoritmo e testamos com conjuntos de dados sintéticos e dados reais, usando índices de validação para comparar os resultados obtidos pelo nosso algoritmo com os resultados produzidos pelo algoritmo TriCluster. Os resultados mostraram que o nosso algoritmo é bom para dados de expressão gênica em três dimensões e pode ser aplicado a dados de outros domínios / In this study we developed a new clustring algorithm for gene expression data. Previous solutions use a dataset in the form of a table, where the rows are the genes and the columns are the experimental conditions. We used a three-dimensional structure adding time-slices. We implemented this algorithm and tested it with synthetic and real data, using validation index to compare our results with the results obtained by the TriCluster algotithm. Results show that our solution is good for three dimensional gene expression data and can be employed to other domains

Les mammites sont des inflammations de la mamelle provoquées principalement par des bactéries. Elles représentent un problème majeur en élevage laitier. Elles sont caractérisées par de fortes augmentations de la concentration des cellules somatiques (CCS) dans le lait. Le score des CCS (SCS) est fortement corrélé à la présence d'infections mammaires, ainsi il est utilisé en sélection pour améliorer la résistance aux mammites. Pour comprendre les mécanismes mis en place lors d'une sélection sur les SCS, deux lignées divergentes de brebis ont été produites à partir de géniteurs ayant des valeurs extrêmes de cet index. Le principal objectif de ce travail est d'identifier et de comprendre les mécanismes qui confèrent une plus grande résistance ou sensibilité aux mammites provoquées par des staphylocoques. Nos travaux ont porté principalement sur des analyses transcriptomiques de trois types cellulaires majeurs, après contact avec des staphylocoques : les cellules inflammatoires du lait recueillies après infection, principalement composées de neutrophiles, les cellules dendritiques, comme cellules présentatrices d'antigènes et les cellules épithéliales mammaires, première barrière de défense contre l'infection. Nous avons montré que la migration des cellules immunitaires et les processus inflammatoires se traduisent par des activations de voies différentes chez les brebis des deux lignées divergentes. Nous avons aussi identifié des gènes fonctionnels candidats pour expliquer la différence de sensibilité aux mammites. En parallèle, nous avons étudié l'association entre le polymorphisme de l'ADN et la présence d'abcès mammaires liés aux mammites dans un dispositif cas-contrôle visant à détecter des gènes à effet majeur. Les études ont permis de mettre en évidence une zone chromosomique d'intérêt située sur le chromosome OAR5. Les deux approches de génétique moléculaire (analyses du transcriptome et du polymorphisme du génome) ont permis d'identifier des gènes fonctionnels et positionnels candidats pour comprendre les mécanismes associés à la résistance aux mammites / Mastitis is udder inflammation. It is one of the major health issues in dairy cattle and sheep as they produce economic losses for the dairy industry. Mastitis is characterized by an increase in milk inflammatory somatic cells. The somatic cell score (SCS) is well correlated to clinical and subclinical mastitis. To enhance insights into the genetic mechanisms involved in such a selection on SCS, two divergent lines of sheep were generated based on extreme breeding values for SCS. The main objective of this work was to identify and improve the understanding of mechanisms that are responsible for a better resistance to Staphylococcus mastitis. Our study was based on transcriptomic analysis of three cell types after Staphylococcus contact: milk inflammatory cells, mainly composed of neutrophils, dendritic cells and mammary epithelial cells. We showed that immune cell migration and inflammatory processes were activated by different pathways in the two divergent lines. We also identified functional candidate genes that may partly explain the differences of mastitis susceptibility. In parallel, we analysed the association of DNA polymorphism with the presence of mammary abscesses caused by mastitis. The study highlighted a chromosomal region on OAR5 that is associated to the presence of mammary abscesses. Both approaches of molecular genetics as transcriptomics and genomics analyses enabled identification of candidate functional and positional genes for the better understanding of the mechanisms associated to mastitis resistance

<p>Streptococcus pneumoniae is a gram-positive bacterium that can be found in both healthy carriers as well as in people that have developed disease. One of the major virulence factors of pneumococci is their polysaccharide capsule. Based on the capsule that surrounds the bacteria, pneumococci are divided into at least 90 different serotypes. Some serotypes seem to be more related to virulence than others.</p><p>I have with comparative genome hybridization microarray technique, studied gene differences between 18 epidemiological well-characterised pneumococcal strains with different potential to cause disease. A microarray chip based on two sequenced pneumococcal genomes, R6 and TIGR4, has already been designed. According to Hierarchical clustering, both the serotype and the genetic type as assessed by MLST (sequence type or ST) seem to have impact on the relationship of clinical isolates. Most clinical isolates of the same serotype are clustered together except for serotype 14 isolates that seem to be more divergent. Further more the number of genes that are divergent between clinical isolates compared to R6 and TIGR4 differ from 65 to 289. Preliminary results indicate that although there is diversity among clinical isolates some are more closely related to each other then others. Absent genes seem to be evenly distributed among all 18 clinical isolates tested but hypothetical genes and genes for cell envelope are two groups of role categories that are absent to the largest extent in all isolates.</p><p>According to results from microarray analysis, a gene region, spr0112-spr1015- is present in all type 9V isolates and absent in many isolates of serotype 14, 19F and 7F. These results have been confirmed by polymerase chain reaction (PCR).</p><p>Conserved genes in a region around the capsule genes have been sequenced to identify marker genes for a capsulular switch between serotype 9V and 14. Preliminary results of the sequencing showed that as much as 750kb might have been transferred in the event of capsular switch.</p>

No description available.

Influenza infection remains constant threat to human health and results in huge financial loss every year. Rapid and accurate detection of influenza can help governments and health organizations monitor influenza activity and take measurements when necessary. In addition, influenza detection in a timingly manner can help doctors make diagnosis and provide effective treatment. On the other hand, novel inhibitors of influenza virus are in high demand because circulating strains have started to develop resistance to currently available anti-viral drugs. Influenza virus has two surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA), which play important roles in the influenza infection. The binding of HA to sialic acid-containing carbohydrates on cell surface initiates virus internalization, while cleavage of terminal sialic acid by NA facilitates viral particle release. In this dissertation, we focus on the development of glycan microarray that is comprised of a panel of NA resistant sialosides, and demonstrate the application of microarray to capture influenza virus at ambient temperature without the addition of NA inhibitors. We also describe a novel electrochemical biosensor for the detection of influenza virus. In addition, we have developed a new class of bivalent NA inhibitors that show promising inhibitory activities against influenza viruses.

The Nuclear Factors of Activated T cells (NFATs) are critical transcription factors that direct gene expression in immune and non-immune cells. Interaction of T cells with Ag-presenting cells results in the clustering of T-cell antigen receptor (TCR), co-receptors and integrins. Subsequent signal transduction resulting in NFAT activation leads to cytokine gene expression. Among the NFATs expressed in T cells, NFATc1 shows a unique induction property, which is essential for T cell differentiation and activation. It was revealed before that 3 major isoforms of NFATc1 are generated in activated T cells – the inducible short NFATc1/A, and the longer isoforms NFATc1/B and C. However, due to alternative splicing events and the existence of two different promoters and two alternative polyadenylation, we show here that 6 isoforms are synthesized in T cells which differ in their N-terminal and C-terminal peptides. In these experiments, we have identified these 6 isoforms by semi-quantitative long distance RT-PCR in several T cells subsets, and the inducible properties of 6 isoforms were investigated in those cells. The short NFATc1/&#61537;A which is under control of the P1 promoter and the proximal pA1 polyadenylation site was the most prominent and inducible isoform in T effector cells. The transcription of the longer NFATc1/&#61538;B and &#61538;C isoforms is constitutive and even reduced in activated T lymphocytes. In addition to NFATc1 autoregulation, we tried to understand the NFATc1 gene regulation under the control of PKC pathways by microarray analysis. Compared to treatment of T cells with ionomycin alone (which enhances Ca++ flux), treatment of cells with the phorbolester TPA (leading to PKC activation) enhanced the induction of NFATc1. Microarray analysis revealed that PKC activation increased the transcription of NF-&#61547;B1, Fos and JunB, which are important transcription factors binding to the regulatory regions of the NFATc1 gene. Besides the promoting effect of these transcription factors, we provided evidence that p53 and its targeting gene, Gadd45&#61537;, exerted a negative effect on NFATc1 gene transcription. Summarizing all these results, we drew novel conclusions on NFATc1 expression, which provide a more detailed view on the regulatory mechanisms of NFATc1 transcription. Considering the high transcription and strong expression of NFATc1 in various human lymphomas, we propose that similar to NF-&#61547;B, NFATc1/&#61537;A plays a pivotal role in lymphomagenesis. / NFATs (Nuclear Factors of Activated T cells) sind entscheidende Transkriptionsfaktoren für die Genexpression in Immun- und Nichtimmunzellen. Die Interaktion von T-Zellen mit Antigen-präsentierenden Zellen hat eine Klusterbildung von T-Zellrezeptoren, Korezeptoren und Integrinen zur Folge. Die nachfolgende Signaltransduktion führt zur NFAT-Aktivierung und dies wiederum zur spezifischen Zytokin-Genexpression. Von den in T-Zellen exprimierten NFATs weist NFATc1 eine besondere Regulation auf. Diese ist wichtig für die T-Zell-Aktivierung und -Differenzierung. Frühere Daten zeigten, dass drei Isoformen von NFATc1 in aktivierten T-Zellen gebildet werden: die induzierbare kurze Isoform NFATc1/A und die längeren Isoformen NFATc1/B und C. In dieser Arbeit wird nachgewiesen, dass in aktivierten T-Zellen sechs Isoformen exprimiert werden. Diese Isoformen unterscheiden sich N- und C-terminal infolge der Aktivität von zwei verschiedenen Promotoren, dem Vorhandensein von zwei unterschiedlichen Polyadenylierungsstellen sowie alternativer Spleißereignisse. Die Identifikation der sechs Isoformen erfolgte mittels semi-quantitativer “long distance” RT-PCR. Ferner konnte mit dieser Methode die induzierbaren Eigenschaften der Isoformen untersucht werden. Die prominenteste und am stärksten induzierbare Isoform in T-Effektorzellen ist NFATc1/&#61537;A. NFATc1/&#61537;A wird unter Kontrolle des P1-Promoters und der proximalen pA1-Polyadenylierungsstelle gebildet. Die Transkription von NFATc1/&#61538;B und &#61538;C erfolgt hingegen konstitutiv, wobei eine Reduktion ihrer Synthese in aktivierten T-Lymphozyten zu beobachten ist. Neben der NFATc1-Autoregulation interessierte uns die NFATc1-Genregulation unter der Kontrolle des PKC-Weges. Durchgeführte Microarray-Analysen zeigten, dass T-Zellen, die mit dem Phorbolester TPA stimuliert wurden, eine stärkere Induktion von NFATc1 aufweisen als Zellen, die nur mit Ionomycin stimuliert wurde. Es ist bekannt, dass TPA die Aktivierung von PKC stimuliert, und wir beobachteten eine verstärkte Transkription von NF-&#61547;B1, Fos und JunB, die an die regulatorischen Region von NFATc1 binden können. Für p53 und seinem Zielgen Gadd45&#61537; zeigen wir unterstützende Beweise für einen negativen Einfluß auf die NFATc1-Transkription. Zusammenfassend erlauben die Resultate neue Schlussfolgerungen für die NFATc1-Genexpression und ermöglichen damit eine detailiertere Sicht auf den Regulationsmechanismus der NFATc1-Transkription. Bezug nehmend auf die hohe Expressionsrate von NFATc1 in verschiedenen humanen Lymphomen vermuten wir, dass - ähnlich NF-&#61547;B - NFATc1/&#61537;A eine entscheidende Rolle bei der Lymphomagenese spielt.