A análise da expressão gênica através de dados gerados em experimentos de microarrays de DNA vem possibilitando uma melhor compreensão da dinâmica e dos mecanismos envolvidos nos processos celulares ao nível molecular. O aprimoramento desta análise é crucial para o avanço do conhecimento sobre as bases moleculares das neoplasias e para a identificação de marcadores moleculares para uso em diagnóstico, desenho de novos medicamentos em terapias anti-tumorais. Este trabalho tem como objetivos o desenvolvimento de modelos de análise desses dados, propondo uma nova forma de normalização de dados provenientes de microarrays e dois modelos para a construção de redes regulatórias de expressão gênica, sendo uma baseada na conectividade dinâmica entre diversos genes ao longo do ciclo celular e a outra que resolve o problema da dimensionalidade, em que o número de experimentos de microarrays é menor que o número de genes. Apresenta-se, ainda, um pacote de ferramentas com uma interface gráfica de fácil uso contendo diversas técnicas de análise de dados já conhecidas como também as abordagens propostas neste trabalho. / The analyses of DNA microarrays gene expression data are allowing a better comprehension of the dynamics and mechanisms involved in cellular processes at the molecular level. In the cancer field, the improvement of gene expression interpretation is crucial to better understand the molecular basis of the neoplasias and to identify molecular markers to be used in diagnosis and in the design of new anti-tumoral drugs. The main goals of this work were to develop a new method to normalize DNA microarray data and two models to construct gene expression regulatory networks. One method analyses the dynamic connectivity between genes through the cell cycle and the other solves the dimensionality problem in regulatory networks, meaning that the number of experiments is lower than the number of genes. We also developed a toolbox with a user-friendly interface, displaying several established statistical methods implemented to analyze gene expression data as well as the new approaches presented in this work.

This work concerns with data analysis of DNA microarrays by using cluster analysis. It explains biological terms - gene expression and DNA microarray. Next, it contains mathematical and informatical description of clustering methods and describes a way to apply these methods to microarrays data. Next, the work contains implementation's detail of clustering methods k-means, DBSCAN and introduces an original clustering algorithm Strom++. Then, description of implementation and application manual follow. Finally, accomplished results are evaluated.

La choroïde ou uvée, située entre la rétine et la sclérotique, est une membrane vasculaire qui tapisse la paroi de l’œil, son rôle est d’assurer l’apport en nutriment de la rétine et de l’iris. Ce tissu peut être le siège de nombreuses tumeurs, bénignes ou malignes. Le mélanome de la choroïde est la tumeur intra-oculaire la plus fréquente de l’adulte mais les facteurs de risque sont mal connus: l’exposition aux ultraviolets n’est pas clairement établi dans la genèse de la tumeur, de même que l’âge ou le sexe.L’énucléation a longtemps été considérée comme la seule option thérapeutique, mais depuis de nombreuses années, des techniques dites conservatrices de l’œil se sont développées. Des études ont montré qu’il n’y a pas de différence significative de survie entre les patients ayant subis une énucléation et les patients traités avec des méthodes conservatrices. De plus, à ce jour, aucune thérapie adjuvante n’a montré son efficacité après le traitement du mélanome oculaire primaire. En effet, malgré un traitement initial bien adapté, la moitié des patients va récidiver sur le mode métastatique. Environ 30\% des patients récidivent dans les 5 ans, ce chiffre augmente jusqu’à 50\% à 15 ans.L’œil étant dépourvu de structures lymphatiques, la diffusion métastatique du melanome uvéal se fait par voie hématogène. Le foie est le site privilégié de développement de métastases, faisant toute la gravité du pronostic. La médiane de survie après apparition de métastases est de 2 à 6 mois en l’absence de traitement. Il peut exister de façon plus anecdotique des métastases pulmonaires, ganglionnaires, osseuses ou cutanées.Sur un plan génétique, les critères les plus fréquemment détectés pour le mélanome uvéal sont la perte du chromosome 3 et le gain du 8q. Plusieurs études montrent dans beaucoup de cas des aberrations chromosomiques non aléatoires sur les chromosomes 1, 3, 6 et 8 et que la perte du chromosome 3 et le gain du 8q sont associés significativement à une survie réduite et au développement de métastase. Plusieurs rapports suggèrent deux entités distinctes de mélanome uvéal (avec et sans monosomie du chromosome 3) qui ne peuvent pas être différenciées du fait de leur aspect clinico-pathologique similaire.Afin d’améliorer le diagnostic et le traitement du mélanome de la choroïde, nous proposons d’effectuer des analyses d’expression et du nombre de copie d’ADN de ce mélanome particulier, avec pour objectifs : l’identification des gènes liés à l’apparition de métastase pour classer les patients à haut risque afin qu’ils puissent bénéficier d’une immunothérapie adjuvante spécifique, la caractérisation de ces gènes au niveau moléculaire, et l’étude du potentiel de ces gènes en tant que cibles thérapeutiques.Dans ce manuscrit je décrirai en détail le lignage mélanocytaire afin de comprendre les particularités du mélanome de la choroïde par rapport au mélanome cutané, puis j'aborderai l'importance des approches haut débit dans l'étude des cancers et les techniques d'analyse bioinformatiques utilisées. Je présenterai ensuite les différents résultats obtenus comme la mise en évidence d'une phosphatase, PTP4A3, qui semble avoir de l'importance dans le développement métastatique du mélanome de la choroïde. / The choroid is a layer of highly vascularised tissue surrounding the eye. Choroidal blood nourishes the retinal pigment epithelium and the photoreceptors on the outer layer of the retina. Uveal melanoma occurs to the detriment of uveal melanocytes (located in the iris, ciliary body and choroid) and is the most common intraocular malignancy in adults. The etiological factors involved in the process of malignant transformation are poorly understood. There is a doubtful role of environmental factors such exposure to sunlight, age or sexe in the emergence of uveal melanoma.The management of uveal melanomas has greatly evolved, moving towards more focused and conservative treatments (such as observation, photocoagulation, thermotherapy, radiotherapy). According to literature, there is no significant difference in survival between patients treated with enucleation and those treated with conservative methods. To date, no adjuvant therapy has proven effective following the initial treatment of ocular melanoma. The metastatic pattern for uveal melanoma differs from that of skin melanoma and is usually located in the liver. About 50\% of patients will develop metastases after a median time of three years, and will ultimately die of their disease. Once the disease becomes metastatic, median survival ranges from two to six months, and only 15\% of the patients survive more than one year. Surgical resection of metastases is feasible only if occurring in limited areas. Genetic differences may be the origin of the various types of melanoma and their different features. Multivariate analyses of genomic imbalances, showed that cutaneous and uveal melanomas presnted different copy number changes. The most frequently detected imbalances in uveal melanoma is the loss of chromosome 3 and gain of 8q. Further studies revealed that most cases show non-random chromosomal aberrations of chromosomes 1, 3, 6 and 8 and that the loss of chromosome 3 and gain of 8q were significantly associated with overall survival and the development of metastases. Some reports suggested two distinct entities of uveal melanoma (with or without chromosome 3 monosomy) previously unrecognized because of their similar clinicopathological features. In order to improve diagnosis and treatment of uveal melanoma, we propose to perform transcriptome and DNA copy number analysis with following objectives : identify genes linked to metastasis behaviour to identify high risk patients who could take advantage of specific adjuvant therapy ; characterize these genes at molecular level ; study if these genes could be powerful therapeutic target.In this thesis, I will describe the melanocyte lineage in order to understand differences observed between cutaneous and uveal melanoma, then I will discuss the importance of high-throughput approaches in the study of cancer and bioinformatics analysis techniques used. I will finally present the different results as the significance of a phosphatase, PTP4A3, which seems to be relevant in metastatic behaviour in uveal melanoma.

The genus Candida includes several opportunistically pathogenic species which are common causative agents of the yeast infections in humans. Although current medical research is focused mostly on cancer, AIDS or Alzheimer disease, the problem of systemic candidiases cannot be neglected. These infections represent a real threat to the immunocompromissed patients, they are connected with a high mortality rate and expensive medication with poor prognosis. Pseudomonas aeruginosa could be an inspiration in a way of how to eliminate the pathogenic yeasts. The bacterium can inhibit growth of the most common yeast species of the genus Candida, C. albicans. This effect is based on production of toxic substances by the bacterium and on interaction of the bacterium with the C. albicans cell wall, which leads to the lysis of the yeast cells and which is not fully understood. Nevertheless, coexistence of these microorganisms is also possible and their relationship is affected by various factors. Knowledge of these inter- microbial interactions was obtained from studies of diseases and pathologies, during which C. albicans + P. aeruginosa coinfections occur. In this thesis I studied mechanisms of interaction between pathogenic yeast C. albicans and bacterium P. aeruginosa by a) C. albicans gene expression...

Introduction: Methamphetamine is a drug frequently abused by drug-addicted pregnant women and also one of the mostcommonly used drugs in the CzechRepublic. This drug passes easily through a placental barrier into the fetus. Thus it can negatively affect not only the mother but also the prenatal development of her offspring. Objectives: In the framework of the grant project GA CR: 14-03708S, the long-term effects of prenatal exposure to methamphetamine were detected. It was determined whether the prenatal methamphetamine exposure affects the generation of offspring of exposed females at the level of gene expression of genes in specific regions of the brain, striatum, hippocampus and prefrontal cortex. Methods: In the selected parts of the brain, which were removed from the rat, the microarray hybridization and the real-time PCR to express changes in expression of selected genes were performed. Results: Statistical analysis of microarray hybridization did not show the significantly altered gene expression in tested genes significantly. Only boundary values for 13 genes were measured, which were further tested by real-time PCR.After a statistic evaluation of real-time PCR, the significantly altered expression was found in 2 genes. The significantly changed expression of DRD3 and TACR3 genes was found...

Introduction: Methamphetamine is a drug frequently taken by drug-addicted pregnant women and happens to be one of the most commonly used drugs in the Czech Republic. This drug passes easily through a placental barrier into the fetus. Thus it can negatively affect not only the mother but also the prenatal development of her offspring. Objectives: This research aims to provide a general screening of gene expression in selected regions of the F1 generation of the brain prenatally affected by methamphetamine, to verify whether exposure to methamphetamine affects the generation of offspring of exposed females at the level of gene expression in selected regions of the brain, and to valuate possible changes in gene expression. Methods: In selected parts of the brain, collected from a rat, the microarray hybridization and the real-time PCR were set to evaluate express changes in the expression of selected genes. Results: Statistical analysis of the microarray hybridization did not show a significantly altered gene expression in the tested genes. Only boundary values for 13 genes were measured, which were further tested by the real-time PCR. After a statistic evaluation of the real-time PCR, the significantly altered expression was found in 2 genes. The notably changed expression of DRD3 and TACR3 genes...

eDNA microarray technology has generated a tremendous amount of interest among biologists because of its promise to monitor the entire genome on a single chip, thus enabling researchers to have a better picture of the interaction among thousands of genes simultaneously. In the current study, this technology was used to print over 3,000 unknown genes from various silk glands of the black widow spider to profile their expression patterns and to identify novel candidates. Spiders are remarkable creatures because of their ability to make different silks, each with a specific function. Some of these silks have amazing mechanical properties, comparable to those of the finest synthetic materials. Several silk genes have been cloned from various spiders over the last few years, and the contribution of each of those genes in silk production has been identified. However, the majority of cellular and biochemical processes involved in silk manufacture remain a mystery. In our research, we attempt to identify genes that might be involved in silk assembly, on a global scale and investigate more about those genes and their interplay with other key biological molecules involved in silk manufacture. Our study showed that silking spiders for a certain period of time resulted in down-regulation of two important silk genes, ECP-1 and ECP-2. Both these genes are key molecules implicated for their role in maintaining the egg case architecture in the black widow spider.,-and we believe that these genes are also directly or indirectly involved in the manufacture of dragline silk. Microarray analyses also enable the discovery of several other interesting molecules, two of which could be accessory proteins involved in silk formation. Furthermore, in a separate study we also characterized a novel silk glue protein with unique ensemble repeats. In conclusion, we believe that the findings of this study will indeed be significant to silk researchers and material scientists alike and it will enhance our knowledge in understanding the mystery behind silk production.

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