Dissertations / Theses on the topic 'CDNA microarray'

Microarrays allow the expression level of thousands of genes to be measured simultaneously. This study will address analytical issues predominantly concerned with eDNA arrays. These include normalisation and data preprocessing, leading to an assessment of cluster analysis and the integration of database information to elucidate functional classes of biological relevance. This is then extended further to classify genes of unknown function using Markov Random Fields. I Modelling of uneven surface trends were considered in a new 2D-normalisation method which outperformed the popular loess method which concentrated on printing pin effects. With respect to cluster analysis, a new method to identify the number of clusters in higher dimension da~a is proposed which provides a visual way of determining at which point over-fitting of the data will occur. Once partitioned, functional information was incorporated to find enrichment of classes in clusters using a new application of X2 bootstrapping, which provides a very robust way of identifying these groups. A novel use of correspondence analysis was applied to the contingency tables produced from the cluster over class analysis which was used to show that functionally related groups were acting in concert when scrutinising the projection' of these classes onto three dimensions. The last part of this study attempted the use of Markov Random Fields to assign function to genes of unknown function using M. tuberculosis and E. coli data. The ability to determine function from the data used in this work was limited, but the method implemented in this study showed improvement over previous attempts.

Microarray technology enables simultaneous gene expression level monitoring for thousands of genes. While this technology has now been recognized as a powerful and cost-effective tool for large-scale analysis, the many systematic sources of experimental variations introduce inherent errors in the extracted data. Data is gathered by processing scanned images of microarray slides. Therefore robust image processing is particularly important and has a large impact on downstream analysis. The processing of the scanned images can be subdivided in three phases: gridding, segmentation and data extraction. To measure the gene expression levels, the processing of cDNA microarray images must overcome a large set of issues in these three phases that motivates this study. This study presents automatic gridding methods and compares their performances. Two segmentation techniques already used, the Seeded Region Growing Algorithm and the Mann-Whitney Test, are examined. We present limitations of these techniques. Finally, we studied the data extraction method used in MicroArray Suite (MS), a microarray analysis software, via synthetic images and explain its intricacies.
Master of Science

Many tests have been developed for comparing means in a two-sample scenario. Microarray experiments lead to thousands of such comparisons in a single study. Several multiple testing procedures are available to control experiment-wise error or the false discovery rate. In this dissertation, individual two-sample tests are compared based on accuracy, correctness, and power. Four multiple testing procedures are compared via simulation, based on data from the lab of Dr. Rajesh Miranda. The effect of sample size on power is also carefully examined. The two sample t-test followed by the Benjamini and Hochberg (1995) false discovery rate controlling procedure result in the highest power.

The advent of cDNA microarray technology makes it possible to measure theexpression level of thousands of genes simultaneously. This creates large volumes of data that require computational analysis.

One application of microarray technology is cancer studies, where supervised learning may be used on microarray data to predicting tumour subtypes and other clinical parameters. The number of available objects (microarrays) is much smaller than the number of attributes (genes). Hence it is necessary to determine which attributes are important for predicting a parameter. Feature selection methods determine which parameters are related to the predicted parameter, and classifiers are then trained on data sets consisting only of the selected attributes.

This thesis examines the performance of several feature selection methods on real life data sets. The implementation is based on ROSETTA, a toolkit that contains several rough set learning algorithms as well as discretization methods, but lacks algorithms for performing features selection. These missing algorithms are written in the C++ programming language as part of this thesis.

The conclusion is that even though it appears to perform well in binary classification, the current implementation of multi-class classification does not perform as well the other methods studied as part of this thesis. If multi-class classification using binary classifiers and additional optimization was implemented, then it would be possible to compare the performance of rough set based classifiers to other method in a fair and meaningful way.

A microarray is a powerful tool for surveying the expression levels of many thousands of genes simultaneously. It belongs to the new genomics technologies which have important applications in the biological, agricultural and pharmaceutical sciences. In this thesis, we focus on the dual channel cDNA microarray which is one of the most popular microarray technologies and discuss three different topics: optimal experimental design; estimating the true proportion of true nulls, local false discovery rate (lFDR) and positive false discovery rate (pFDR) and dye effect normalization. The first topic consists of four subtopics each of which is about an independent and practical problem of cDNA microarray experimental design. In the first subtopic, we propose an optimization strategy which is based on the simulated annealing method to find optimal or near-optimal designs with both biological and technical replicates. In the second subtopic, we discuss how to apply Q-criterion for the factorial design of microarray experiments. In the third subtopic, we suggest an optimal way of pooling samples, which is actually a replication scheme to minimize the variance of the experiment under the constraint of fixing the total cost at a certain level. In the fourth subtopic, we indicate that the criterion for distant pair design is not proper and propose an alternative criterion instead. The second topic of this thesis is dye effect normalization. For cDNA microarray technology, each array compares two samples which are usually labelled with different dyes Cy3 and Cy5. It assumes that: for a given gene (spot) on the array, if Cy3-labelled sample has k times as much of a transcript as the Cy5-labelled sample, then the Cy3 signal should be k times as high as the Cy5 signal, and vice versa. This important assumption requires that the dyes should have the same properties. However, the reality is that the Cy3 and Cy5 dyes have slightly different properties and the relative efficiency of the dyes vary across the intensity range in a "banana-shape" way. In order to remove the dye effect, we propose a novel dye effect normalization method which is based on modeling dye response functions and dye effect curve. Real and simulated microarray data sets are used to evaluate the method. It shows that the performance of the proposed method is satisfactory. The focus of the third topic is the estimation of the proportion of true null hypotheses, lFDR and pFDR. In a typical microarray experiment, a large number of gene expression data could be measured. In order to find differential expressed genes, these variables are usually screened by a statistical test simultaneously. Since it is a case of multiple hypothesis testing, some kind of adjustment should be made to the p-values resulted from the statistical test. Lots of multiple testing error rates, such as FDR, lFDR and pFDR have been proposed to address this issue. A key related problem is the estimation of the proportion of true null hypotheses (i.e. non-expressed genes). To model the distribution of the p-values, we propose three kinds of finite mixture of unknown number of components (the first component corresponds to differentially expressed genes and the rest components correspond to non-differentially expressed ones). We apply a new MCMC method called allocation sampler to estimate the proportion of true null (i.e. the mixture weight of the first component). The method also provides a framework for estimating lFDR and pFDR. Two real microarray data studies plus a small simulation study are used to assess our method. We show that the performance of the proposed method is satisfactory.

Goals of this dissertation were to 1) use custom-made cDNA microarrays to identify genes in the bovine pituitary gland that are differentially expressed during the estrous cycle and 2) characterize their patterns of gene expression. The estrous cycle is a dynamic process that requires coordination between the hypothalamus, pituitary gland, and ovaries. The anterior pituitary gland synthesizes and secretes the gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), which regulate steroidogenesis and follicular development. Currently, intrapituitary factors that modulate gonadotropin synthesis, storage, and release are not well described, thus, requiring investigation. To investigate the validity of the microarray results, we performed real-time PCR on 35 genes identified by cDNA microarray as being differentially regulated. Overall, microarray and real-time PCR results were consistent among our experiments suggesting that cDNA microarray is an efficacious tool for profiling gene expression in the bovine pituitary gland. Our first experiment was designed to identify genes that were regulated during the early luteal phase. This period is characterized by steadily increasing concentrations of progesterone (P4) from nadir to maximum. Samples from three different time points, d 2, d 6, and d 10 following initiation of the first follicular wave, were compared. One hundred and sixty nine genes were determined to be differentially expressed. Ten of these genes were validated using real-time PCR. The other two studies were designed to identify genes that were regulated during the preovulatory period as induced by the administration of prostaglandin F2α (PGF2α). This period is characterized by a decrease in circulating concentrations of P4 coincident with an increase in circulating concentrations of estradiol. Prior to the surge, FSH and LH are disconcordinately released but the underlying mechanisms regulating their release is unknown. The second study identified 1406 genes to be differentially regulated during the 72 h following administration of PGF2α. Twenty-seven of these transcripts were validated by real-time PCR. The third study identified 503 genes to be differentially regulated during the 48 h following administration of PGF2α. Twenty two of these transcripts were validated by real-time PCR. Together these experiments have identified several genes as potential intrapituitary factors that may function to regulate the reproductive axis.

This dissertation includes three topics. First topic: Regularized estimation in the AFT model with high dimensional covariates. Second topic: A novel application of quantile regression for identification of biomarkers exemplified by equine cartilage microarray data. Third topic: Normalization and analysis of cDNA microarray using linear contrasts.

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.

Marine bivalves of the genus Mytilus are intertidal filter-feeders commonly used as biosensors of coastal pollution. Indeed, mussels readily bioaccumulate both organic and metal pollutants, and react to changes with physiological and genetic mechanisms. However, one main problem in using mussels as bio-sensors is the poor characterization of their functional and defence reactions. The currently used biomarkers provide insufficient understanding of mussel physiology status or stressor-induced effects, and knowledge of mussel genome structure, function and evolution are still lacking. Therefore, genomic approaches are needed to increase our knowledge of physiological processes and to better understanding molecular and cellular mechanisms involved in the stress responses. Based on the production and massive sequencing of 3'ESTs library from main mussel tissues, we arrayed, in collaboration of C.R.I.B.I., University of Padova, the first collection of selected transcript 1714 tags on glass slides as MytArray 1.0. In order to use this molecular platform defined in 2003, it has been necessary to refresh the physical collection of the bacterial clones bearing cDNA inserts in recombinant plasmids. Then, microarrays have been spotted and hybridisation experiments realized. The potential use of this novel tool was evaluated by analyzing gene expression changes in mussels exposed to mixture of toxic and genotoxic metals in laboratory and in their natural environment (Venice lagoon, Italy). Among the potential toxic contaminants, heavy metals can damage cell components, disturb cell signalling and are expected to modulate the expression of many genes. After competitive ibridisation experiments on MytArry 1.0, we found gene expression changes in gill and digestive gland in mussels treated with increasing micromolar doses of a metal mixture (Cd, Cu, Hg). Results appear instructive and consistent with the enhance of chromosomal damage in gill cells of same mussels (evaluated as increases of micronuclei and other nuclear abnormalities). The transcriptional changes raised in dose-dependent manner and transcripts showing consistent expression trends revealed the complexity of the induced cellular response, with the most evident changes referring to: ion homeostasis (i.e metallothionein 10IV isoform, ferritin), protein turnover (sequestosome 1 and proteasome subunits) and chaperones (hsp70, hsp90, shsp24), regulation of apoptosis and DNA damage-inducible transcripts (gadd45, apoptosis inhibitor 2), cell motility and adhesion. The subsequent real-time PCR performed supports further these results. To assess the potential use of the mussel microarray in environment, I evaluated the transcriptional digestive gland profiles of mussels living in the Venice lagoon. Venice lagoon is a unique case and significant concentrations of cadmium, mercury, PAHs, PCBs and dioxin-like compounds are recurrently detected in theindustrial area near the town. Native mussels were sampled in the early summer in 2005, 2006, 2007, from zones affected differently by chemical pollution: from industrial district channels and from Lido lagoon inlet relatively uncontaminated. Offshore mussel farm, was chosen as a source of reference. I performed the competitive hybridisation experiments on DG samples and detected differentially expressed genes are grouped into different functional categories. In mussels of the industrial canals (Marghera, Venice) microarray analysis performed on individual mussels indicated a general profile similarity which make them distinguishable from mussels living in less polluted sites. Chemical data support this work hypothesis. The number of biological replicates influence the study size but, how much the individual variability can influence our studies? To try to answer to this question, I performed new hybridisation experiments by using the pool from the same samples. The overall clustering of transcriptional profiles can be compared with data already obtained from individual mussel tissues even though only transcripts with significant expression values are found. In all three years, the suggestive presence of gene markers, tracing organic contaminants and heavy metals in mussels from the industrial district is consistent with reported trends of chemical contamination. Finally, I contributed to the preparation of samples generating new primary cDNA libraries and a unique normalized library from mussels treated with with chemical and biological contaminants in order to enlarge the transcript collection and better understanding transcriptional mussel responses (work in collaboration with C.R.I.B.I and UniTrieste). Massive sequencing of the primary and normalized libraries yielded positive results and information obtained are going to be organized in the first intergrated Mytilus databatase.

Cultivated chickpea (Cicer arietinum) has a narrow genetic base making it difficult for breeders to produce new elite cultivars with durable resistance to major biotic and abiotic stresses. As an alternative to genome mapping, microarrays have recently been applied in crop species to identify and assess the function of putative genes thought to be involved in plant abiotic stress and defence responses. In the present study, a cDNA microarray approach was taken in order to determine if the transcription of genes, from a set of previously identified putative stress-responsive genes from chickpea and its close relative Lathyrus sativus, were altered in chickpea by the three abiotic stresses; drought, cold and high-salinity. For this, chickpea genotypes known to be tolerant and susceptible to each abiotic stress were challenged and gene expression in the leaf, root and/or flower tissues was studied. The transcripts that were differentially expressed among stressed an d unstressed plants in response to the particular stress were analysed in the context of tolerant/susceptible genotypes. The transcriptional change of more than two fold was observed for 109, 210 and 386 genes after drought, cold and high-salinity treatments, respectively. Among these, two, 15 and 30 genes were consensually differentially expressed (DE) between tolerant and susceptible genotypes studied for drought, cold and high-salinity, respectively. The genes that were DE in tolerant and susceptible genotypes under abiotic stresses code for various functional and regulatory proteins. Significant differences in stress responses were observed within and between tolerant and susceptible genotypes highlighting the multiple gene control and complexity of abiotic stress response mechanism in chickpea. The annotation of these genes suggests that they may have a role in abiotic stress response and are potential candidates for tolerance/susceptibility.

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.

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.

Microarray experiments are used to perform gene expression profiling on a large scale. E- and A-optimality of mixed designs was established for experiments with up to 26 different varieties and with the restriction that the number of arrays available is equal to the number of varieties. Because the IBD setting only allows for a single blocking factor (arrays), the search for optimal designs was extended to the Row-Column Design (RCD) setting with blocking factors dye (row) and array (column). Relative efficiencies of these designs were further compared under analysis of variance (ANOVA) models. We also compared the performance of classification analysis for the interwoven loop and the replicated reference designs under four scenarios. The replicated reference design was favored when gene-specific sample variation was large, but the interwoven loop design was preferred for large variation among biological replicates. We applied mixed model methodology to detection and estimation of gene differential expression. For identification of differential gene expression, we favor contrasts which include both variety main effects and variety by gene interactions. In terms of t-statistics for these contrasts, we examined the equivalence between the one- and two-step analyses under both fixed and mixed effects models. We analytically established conditions for equivalence under fixed and mixed models. We investigated the difference of approximation with the two-step analysis in situations where equivalence does not hold. The significant difference between the one- and two-step mixed effects model was further illustrated through Monte Carlo simulation and three case studies. We implemented the one-step analysis for mixed models with the ASREML software.
Ph. D.

Análise de expressão gênica em larga escala é de fundamental importância para a biologia molecular atual pois possibilita a medida dos níveis de expressão de milhares de genes simultaneamente, o que torna viável a realização de trabalhos voltados para biologia de sistemas (systems biology). Dentre as principais técnicas experimentais disponíveis para esta finalidade, a tecnologia de microarray tem sido amplamente utilizada. Este procedimento para medida de expressão gênica é bastante complexo e os dados obtidos são freqüentemente observacionais, o que dificulta a modelagem estatística. Não existe um protocolo padrão para a geração e avaliação desses dados, sendo portanto necessário buscar procedimentos de análise que sejam adequados para cada caso. Assim, os principais métodos matemáticos e estatísticos aplicados para a análise desses dados deveriam estar disponíveis de uma forma organizada, coerente e simples em um ambiente computacional que confira robustez, confiabilidade e reprodutibilidade às análises realizadas. Uma forma de garantir estas características é através da representação (e documentação) de todos os algoritmos utilizados na forma de um grafo direcionado e acíclico que descreva todo o conjunto de transformações, ou operações, aplicadas seqüencialmente ao conjunto de dados. De acordo com esta filosofia, um ambiente foi implementado neste trabalho incorporando diversos procedimentos disponíveis na literatura atual, além de outros que foram aprimorados ou propostos nesta tese. Dentre os métodos de análise já disponíveis que foram incorporados destacam-se aqueles para a construção de agrupamentos, busca de genes diferencialmente expressos e classificadores, construção de redes de relevância e classificação funcional de grupos gênicos. Além disso, o método de construção de redes de relevância foi revisto e aprimorado e um modelo estatístico para a classificação funcional de redes de regulação gênica foi proposto e implementado. Esses dois últimos métodos surgiram a partir de problemas biológicos para os quais não existiam procedimentos de análise adequados na literatura. Finalmente, são apresentados dois conjuntos de dados que foram analisados utilizando diversas ferramentas disponíveis neste ambiente computacional.
High throughput gene expression analysis has a great importance to molecular biology nowadays because it can measure expression profiles for hundreds of genes, and this turn possible studies focused in systems biology. Between the main experimental techniques available in this direction, the microarray technology has been widely used. This experimental procedure to quantify gene expression profiles is very complex and the data obtained is frequently observational, what difficult the statistical modelling. There is not a standard protocol for the generation and evaluation of microarray data, therefore it is necessary to search by adequate analysis methods for each case. Thus, the main mathematical and statistical methods applied to microarray data analysis would have to be available in an organized, coherent and simple way in a computational environment that confer robustness, reliability and reproducibility to the data analysis. One way to guarantee these characteristics is through the representation (and documentation) of all used algorithms as a directed and acyclic graph that describes the set of transformations, or operations, applied sequentially to the dataset. According to this philosophy, an environment was implemented in this work aggregating several data analysis procedures already available in the literature, beyond other methods that were improved or proposed in this thesis. Between the procedures already available that were incorporated we can distinguish that ones for cluster analysis, differentially expressed genes and classifiers search, construction of relevance networks and functional classification of gene groups. Moreover, the method for construction of relevance networks was revised and improved and an statistical model was proposed and implemented for the functional classification of gene regulation networks. The last two procedures was born from biological problems for which adequate data analysis methods didn?t exist in the literature. Finally, we presented two datasets that were evaluated using several data analysis procedures available in this computational environment.

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

Autism is a pervasive developmental disorder (PDD) that&rsquo
s incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman®
SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman®
study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples.

Neste trabalho realizamos a análise das variações na expressão gênica global do fungo aquático Blastocladiella emersonii submetido ao estresse de carência de oxigênio (hipóxia), utilizando a técnica de microarranjos de cDNA em lâminas contendo 3773 genes distintos. Nos experimentos de hipóxia gradual (diminuição gradual da concentração de oxigênio dissolvido, seguido de reoxigenação) e hipóxia direta (diminuição direta da concentração de oxigênio dissolvido, seguido de reoxigenação) observamos que 650 genes foram diferencialmente expressos em pelo menos uma das condições de estresse e que 534 deles mostraram-se afetados (direta ou indiretamente) pela disponibilidade de oxigênio, uma vez que apresentaram recuperação (ou tendência à recuperação) da sua expressão aos níveis normais, quando as células foram reoxigenadas. Além de modular a expressão de diversos genes sem função conhecida, B. emersonii responde à hipóxia reajustando a expressão de genes responsáveis pela produção e consumo de energia. Pelo menos transcricionalmente, este fungo favorece o metabolismo anaeróbico, através da indução de genes que codificam enzimas da via glicolítica e lactato desidrogenase, ao passo que no ciclo do ácido cítrico, a maioria dos genes encontram-se reprimidos ou não sofrem alteração na expressão. Processos dispendiosos em energia como síntese protéica, metabolismo de aminoácidos, enovelamento de proteínas e transporte por membrana apresentaram perfis predominantemente de repressão gênica quando em carência de oxigênio. Ainda utilizando a técnica de microarranjos, mostramos semelhanças entre os perfis transcricionais nos experimentos hipóxia e de carência de Fe2+ (tratamento com quelante de Fe2+ 2,2´-dipyridyl) sugerem que estes estresses estão de alguma forma relacionados, fornecendo bons indícios de que o íon Fe2+ possa ter um papel importante no mecanismo sensor de oxigênio e/ou de resposta a hipóxia em B. emersonii. Além disso, o tratamento prévio de células submetidas à hipóxia com o antibiótico geldanamicina, um conhecido inibidor da proteína de choque térmico HSP90, levou à diminuição da indução de certos genes de hipóxia, indicando que este fungo pode possuir algum mecanismo semelhante ao do fator de transcrição de hipóxia HIF1-α de mamíferos, uma vez que este fator também é afetado por geldanamicina. Adicionalmente, desenvolvemos um protocolo para transformação de B. emersonii mediada por Agrobacterium tumefasciens que se mostrou promissor. A transferência do T-DNA contendo um gene de resistência a higromicina B, presente no vetor binário pBINPLUS-Hph, foi evidenciada pelo crescimento normal e esporulação das células transformadas, na presença do antibiótico e pela amplificação do gene de resistência no DNA genômico de células transformadas.
In this work we analyzed global gene expression changes in the aquatic fungus Blastocladiella emersonii submitted to oxygen deprivation (hypoxia), using cDNA microarrays containing 3,773 distinct genes. In gradual hypoxia (gradual decrease in dissolved oxygen concentration, followed by reoxygenation) and direct hypoxia (direct decrease of dissolved oxygen concentration, followed by reoxygenation) we observed 650 differentially expressed genes in at least one of the stress conditions tested, 534 of them being affected (directly or indirectly) by oxygen availability, since they showed recovery of normal expression levels or a tendency to recover, when cells were reoxygenated. Besides modulating many genes with no previously assigned function, B. emersonii responds to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favour anaerobic metabolism through the induction of genes encoding glycolytic enzymes and lactate dehydrogenase, while in the TCA-cycle, most genes were repressed or unchanged. Energy-costly processes like protein synthesis, amino acid metabolism, protein folding and transport had their gene expression profiles predominantly repressed during oxygen deprivation. Microarray experiments also showed similarities between the transcriptional profile of genes in hypoxia and iron (II) deprivation (treatment with the iron (II) chelator 2,2\'-dipyridyl), suggesting that these stresses are somehow related, giving good evidence that Fe2+ ion could have a role in the mechanism of oxygen sensing and/or response to hypoxia in B. emersonii. Furthermore, pretreatment of cells subjected to hypoxia with the antibiotic geldanamycin, a known inhibitor of the heat shock protein HSP90, caused a significant decrease in the induction of certain hypoxic genes, indicating that this fungus could have a mechanism similar to that of the mammalian hypoxia transcription factor HIF-1α, which is also affected by geldanamycin. Additionally, we developed an Agrobacterium tumefasciens-mediated protocol for transformation of B. emersonii that has shown to be promising. The capacity to transfer the T-DNA containing a hygromycin B resistance gene, present in the pBINPLUSHph binary vector, was evidenced by the normal growth and sporulation of the transformed cells in the presence of antibiotic and by amplification of the resistance gene from the genomic DNA of transformed cells

Natural diseases caused by keratin mutations are rare and have only been reported in humans. We have recently identified a heritable skin disorder in Norfolk terriers caused by a mutation in KRT10. Affected dogs have a tendency to form shallow erosions or blisters following mild trauma, which is first noted after the birthing process. As the dogs age, they display generalized hyperpigmentation and scaling that is most severe in the axillary and inguinal regions. The main histologic and ultrastructural features include: marked hyperkeratosis, epidermal hyperplasia, prominent vacuolation of the upper suprabasal layers, eosinophilic intracytoplasmic aggregates (keratin bundles), numerous and frequently enlarged keratohyaline granules, and epidermal hyperplasia. Analysis of an extended pedigree through seven generations confirmed an autosomal recessive mode of inheritance. The keratin 10 mutation was defined as a G-T point mutation in intron 5 that affected splicing at the boundary of exon 4 and intron 5. The primary outcome of the mutation was a 35 bp deletion in exon 4 caused by use of a cryptic splice site. Real-time PCR quantitation of KRT10 confirmed that this mutation led to premature mRNA decay and an average 35-fold decrease in KRT10 message. Organotypic cell culture techniques were used to establish in vitro models for normal and affected Norfolk terriers. After 21 days of culture, normal epidermis was cornified with a compact and multifocally parakeratotic stratum corneum. Affected epidermis largely reproduced the expected morphologic alterations. Immunoblotting and immunohistochemistry for keratin 10 protein and real-time PCR quantitation of KRT10 message showed significantly less keratin expression in vitro than in vivo suggesting that the differentiation program in vitro underwent significant alterations. A diagnostic PCR assay was established for detection of the carrier state. Global analysis of gene expression between normal, carrier and affected dogs was performed with DermArray cDNA microarrays. Affected and carrier dogs showed differential regulation of 320 and 298 genes, respectively, between normal dogs. In affected dogs, 217 were upregulated and 103 were downregulated. In carrier dogs, 222 were upregulated and 76 were downregulated. 72 genes (65 upregulated, 7 downregulated) were altered in both affected and heterozygous dogs.

To establish a better understanding of the neonatal mammalian digit tip regeneration response, a custom cDNA microarray consisting of regeneration specific genes was created. Combinations of several normalizations were used to account for biases inherent in the array. From this subset of clones, one gene found to be differentially expressed in non-regenerating digits was Keratin 6b. Keratin 6b mRNA was seen in the entire wound edge of non-regenerating digits but only on the ventral side of the regenerating digit. Further analysis found that Keratin 6b is expressed in a symmetrical pattern in embryonic regenerating digits, suggesting that wound healing is different between the neonate and embryonic regeneration responses. During the embryonic analysis it was also shown that Keratin 6b is present in the digit pulp of developing and mature digits Analysis of the genes up regulated in regenerating digits as opposed to non-regenerating digits also identified several genes, two of which were Keratin 1--4 and Keratin Associated Protein 6.1, as differentially expressed. Further analysis of the embryonic expression pattern of these genes showed that they are early markers for dorsal nail bed and nail plate Since Msx genes play a critical role in the digit regeneration response, the 3 keratins found in the microarray analysis were examined in the Msx-/- mutant mice. All gene expression remained unmodified, suggesting that none of these genes are downstream of the Msx genes This study demonstrated the validity of the custom cDNA microarray. There are 17 clones yet to be sequenced from the microarray analysis and could lead to critical genes in the regeneration process being found
acase@tulane.edu

碩士
國立臺灣大學
農藝學研究所
90
In general,the experiment data collected from the two-dye cDNA microarrays can be classified into the single-array data and the replicated-array data.And the statistical approach to analyzing microarray data is not the same for these two types of data.Regardless of single or replicated microarray experiments,there are many sources of systematic variation which may affect the really expression values of cDNA.Normalization is the term used to describe the process of removing such systematic variation.Our research is based on a publicly available cDNA microarray data set on E.coli from Richmond et al.(1999).In this study,we compare the efficiency of various normalization methods based on this real data set and some simulation studies.One of the main purposes of the microarray experiments is to find significant differential gene expression between two distinct cell populations.We present a single-array analyzing method by using two normal mixture model. Then, we compare the proposed method with another three methods given in Chen et al.(1997),Sapir and Churchill (2000) and Newton et al.(2001).It is shown that method of two normal mixture model is not inferior to the others.Regarding the replicated-array data,we observe that variation of individual genes on the differential array is very large,which are also discussed in the recent groundbreaking works on the data analysis of microarray experiments by Kerr et al.(2001) and Wolfinger et al. (2001).This observation is the motivating reason to use the tolerance interval procedure for analyzing the replicated-array data based on the some ANOVA models. We find that our analysis method is superior to Mg and tg statistical analysis method discussed in Lonnstedt and Speed (2002).Also,statistical analysis of Sg discussed in Lonnstedt and Speed (2002) may determine the possibly significant genes with small mean and small variation.On the other hand, our tolerance interval analysis method may determine the possibly significant genes with large mean and large variation.

The expression of genetic information, in all organisms, might be characterized as in a constant state of flux with only a fraction of the gene within a genome being expressed at any given time. The genes’ expression pattern reflects the response of cells to stimuli that control growth, development and signal environmental changes. Understanding genes’ expression at the level of transcription and/or other stages of gene regulation at the mRNA level (half life of mRNA, RNA production from primary transcript) might reveal insights into the genes expression mechanisms that control these changes. With the DNA microarray technology researchers are now able to determine, in a single experiment, the gene expression profiles of hundreds to tens of thousands of genes in tissue, tumors, cells or biological fluids. Accordingly, and since the patterns of gene expression are strongly functionally correlated, microarrays might provide unprecedented information both on basic research (e.g. expression profiles of different tissues) and on applied research (e.g. human diseases, drug and hormone action etc). While the simultaneous measurement of thousands of gene expression levels potentially serves as source of profound knowledge, genes quantification (i.e. extraction of the genes expression levels) is confounded by various types of noise originating both from the microarray experimental procedure (e.g. sample preparation) and the probabilistic characteristics of the microarray detection process (e.g. scanning errors). The “noisy” nature of the measured gene expression levels obscures some of the important characteristics of the biological processes of interest. The latter, as a direct effect, renders the extraction of biological meaningful conclusions through microarray experiments difficult and affects the accuracy of the biological inference. Thus, as a major challenge in DNA microarray analysis, and especially for the accurate extraction of genes expression levels, might be considered the effective separation of “true” gene expression values from noise. Noise reduction is an essential process, which has to be incorporated into the microarray image analysis pipeline in order to minimize the “errors” that propagate throughout the microarray analysis pipeline and, consequently, affect the extracted gene expression levels. A possible solution, as proposed in previous studies, for addressing microarray image noise is image enhancement. Results of these studies have indicated a superior quality of the enhanced images, without however examining whether enhancement leads to more accurate spot segmentation or reduces the variability of the extracted gene expression levels. As foresaid, noise also complicates the extraction of meaningful biological conclusions. While more advanced methods have been introduced [28-32] that attempt to prevent the noisy set of genes from being grouped, there is a lack of consensus among experts on the selection of a single method for determining meaningful clusters of genes. The latter, directly affects the biological inference, since different number of clusters are produced when different clustering techniques or either different parameters in the clustering algorithms are utilized. Thus, it turns up that it is not only important to assess the performance of each analysis stage independently (i.e. whether the techniques employed in the microarray analysis pipeline provide accurate extracted gene expression levels or the clustering techniques group biologically related genes) but it is also necessary to ensure an acceptable performance of all steps, as a whole, in terms of biologically meaningful information. This thesis has been carried out towards the development of a complete microarray image processing and analysis framework in order to improve the extraction and, consequently, the quantification of gene expression levels on spotted complementary DNA (cDNA) microarray images. The aims of the present thesis are: a) to model and address the effects of cDNA microarray image noise in such a way that it will increase the accuracy of the extracted gene expression levels, b) to investigate the impact of noise and facilitate genes expression data analysis in order to allow biologists to develop an integrated understanding of the process being studied, c) to introduce a semi-supervised biologically informed criterion for the detection of meaningful biological clusters of genes that answer specific biological questions, d) to investigate the performance and the impact of various state-of-art and novel cDNA microarray image segmentation techniques in the quantification of genes expression levels For exploring all of these aspects, a complete and robust framework of microarray image processing and analysis techniques was designed, built and implemented. The framework incorporated in the microarray analysis pipeline a novel combination of image processing and analysis techniques originating from the comprehensive quantitative investigation of the impact of noise on spot segmentation, intensity extraction and data mining. Additionally, novel formulations of known image segmentation techniques have been introduced, implemented and evaluated in the task of microarray image segmentation. The usefulness of the proposed methods has been validated experimentally on both simulated and real cDNA microarray images.
Η έκφραση της γενετικής πληροφορίας, σε όλους τους οργανισμούς, χαρακτηρίζεται από μια σταθερή κατάσταση «ροής» στην οποία όμως μόνο ένα μέρος του γονιδίου μέσα στο γονιδίωμα (genome) εκφράζεται ανά χρονική στιγμή. Το γονιδιακό μοτίβο έκφρασης (gene expression pattern or gene expression profile) θα μπορούσαμε να πούμε ότι αντανακλά την αντίδραση των κυττάρων στα διάφορα εξωτερικά ερεθίσματα. Για να μπορέσουν να απαντηθούν ερωτήματα σχετικά με τους μηχανισμούς που επηρεάζουν και μεταβάλλουν τη γονιδιακή έκφραση ανάλογα με το εξωτερικό ερέθισμα είναι απαραίτητη η μελέτη της γονιδιακής έκφρασης σε μεταγραφικό επίπεδο (transcription level) ή/και άλλα στάδια (παράγοντες) που ρυθμίζουν τη γονιδιακή έκφραση (gene regulation) σε επίπεδο mRNA. Με τη χρήση της τεχνολογίας των μικροσυστοιχιών, οι ερευνητές έχουν πλέον τη δυνατότητα να μελετήσουν ταυτόχρονα την γονιδιακή έκφραση δεκάδων ή και εκατοντάδων χιλιάδων γονιδίων σε ιστούς, κύτταρα όγκους κλπ με τη χρήση ενός και μόνο πειράματος. Κατά συνέπεια, και από τη στιγμή που τα γονιδιακά μοτίβα έκφρασης συσχετίζονται έντονα λειτουργικά (functionally correlated), η τεχνολογία των μικροσυστοιχιών παρέχει ανεκτίμητης αξίας πληροφορίες που μπορούν να δώσουν ώθηση τόσο στην ανάπτυξη της βασικής έρευνας π.χ. μελέτη των γονιδιακών προφίλ έκφρασης διαφορετικών ιστών όσο και στην ανάπτυξη της εφαρμοσμένης έρευνας π.χ. μελέτη ασθενειών, δράση φαρμάκων και ορμονών κλπ. Παρά τη δυνατότητα που παρέχει η τεχνολογία των μικροσυστοιχιών για την ταυτόχρονη μέτρηση των επιπέδων έκφρασης χιλιάδων γονιδίων, η ποσοτικοποίηση της γονιδιακής έκφρασης (δηλ. η εξαγωγή των επιπέδων έκφρασης των γονιδίων), επηρεάζεται από τους διάφορους τύπους θορύβου που υπεισέρχονται τόσο κατά την πειραματική διαδικασία κατασκευής των μικροσυστοιχιών (π.χ. προετοιμασία δειγμάτων) όσο και από τα πιθανοκρατικά χαρακτηριστικά που διέπουν τη διαδικασία ανίχνευσης (microarray scanning procedure) των μικροσυστοιχιών (π.χ. λάθη ανίχνευσης). Η «θορυβώδης» φύση των γονιδίων και κατά συνέπεια των μετρούμενων γονιδιακών εκφράσεων «κρύβει» (obscure) μερικά από τα πιο σημαντικά χαρακτηριστικά των βιολογικών διαδικασιών ενδιαφέροντος και καθιστά δύσκολη την εξαγωγή χρήσιμων βιολογικών συμπερασμάτων. Από τα παραπάνω διαφαίνεται ότι η μείωση του θορύβου είναι μια πολύ σημαντική διαδικασία η οποία θα πρέπει να ενσωματωθεί στην αλγοριθμική μεθοδολογία που μέχρι στιγμής χρησιμοποιείται για την εξαγωγή των γονιδιακών εκφράσεων από τις εικόνες μικροσυστοιχιών. Με αυτό τον τρόπο θα ελαχιστοποιηθούν τα πιθανά «λάθη» τα οποία μεταφέρονται (propagate) κατά τη διαδικασία εξαγωγής των εντάσεων (μέσω της χρησιμοποιούμενης αλγοριθμικής μεθοδολογίας) και τελικά επηρεάζουν την «ακριβή» εξαγωγή των γονιδιακών εκφράσεων. ‘Ως πιθανή λύση για την αντιμετώπιση του θορύβου στις εικόνες μικροσυστοιχιών, έχει προταθεί στη διεθνή βιβλιογραφία η χρήση τεχνικών αναβάθμισης εικόνας. Τα αποτελέσματα αυτών των επιστημονικών εργασιών συμπεραίνουν ότι με τη χρήση τεχνικών αναβάθμισης η ποιότητα των επεξεργασμένων εικόνων είναι σαφώς καλύτερη. Ωστόσο, καμία από αυτές τις εργασίες δεν μελετάει εάν οι τεχνικές αναβάθμισης οδηγούν στον ακριβέστερο προσδιορισμό των παρυφών των κουκίδων (spot) από τις οποίες εξάγονται οι γονιδιακές εκφράσεις ή εάν βοηθάνε στη μείωση της μεταβλητότητας (variability) των εξαγόμενων γονιδιακών εκφράσεων. Επιπρόσθετα, όπως έχει ήδη προαναφερθεί, ο θόρυβος παρεμποδίζει την εξαγωγή χρήσιμων βιολογικών συμπερασμάτων. Παρά το μεγάλο πλήθος εξελιγμένων μεθόδων που έχουν προταθεί στη διεθνή βιβλιογραφία για την αποτροπή της ομαδοποίησης γονιδίων που χαρακτηρίζονται ως «θορυβώδη», δεν έχει καθοριστεί ακόμα (από τους ειδικούς) μια ενιαία μέθοδος που να βρίσκει και να ομαδοποιεί γονίδια τα οποία θα παρέχουν βιολογικά χρήσιμες πληροφορίες. Αποτέλεσμα αυτής της «ασυμφωνίας» μεταξύ των ειδικών αποτελεί η εξαγωγή διαφορετικών βιολογικών συμπερασμάτων ανάλογα α) με τον αριθμό των δημιουργούμενων γονιδιακών ομάδων (που εξαρτάται άμεσα από τη διαφορετική μέθοδο ομαδοποίησης (clustering)) και β) με τις διαφοροποιήσεις που μπορεί να έχουμε στις παραμέτρους των διαφόρων μεθόδων ομαδοποίησης. H παρούσα διατριβή στοχεύει στη δημιουργία ενός ολοκληρωμένου πλαισίου για την επεξεργασία και ανάλυση εικόνων μικροσυστοιχιών με σκοπό την βελτιστοποίηση της εξαγωγής και κατά συνέπεια της ποσοτικοποίησης των γονιδιακών εντάσεων από εικόνες μικροσυστοιχιών κουκίδων (spotted cDNA microarray images). Οι στόχοι της παρούσας διατριβής συνοψίζονται ως εξής: α) μοντελοποίηση και περιορισμός των επιδράσεων του θορύβου σε εικόνες μικροσυστοιχιών κουκίδων κατά τέτοιο τρόπο ώστε να αυξηθεί η ακρίβεια των εξαγόμενων γονιδιακών εκφράσεων, β) μελέτη της επίδρασης του θορύβου και βελτιστοποίηση των μεθόδων ανάλυσης των γονιδιακών εκφράσεων με σκοπό τη διευκόλυνση των βιολόγων στην εξαγωγής βιολογικών συμπερασμάτων και την καλύτερη κατανόηση της βιολογικής διεργασίας που μελετάται, γ) εισαγωγή ενός ημιεποπτευόμενου (semi-supervised) κριτηρίου που στηριζόμενο σε βιολογικές πληροφορίες θα αποσκοπεί στην ανεύρεση βιολογικά σημαντικών ομάδων γονιδίων τα οποία ταυτόχρονα θα απαντούν σε συγκεκριμένα βιολογικά ερωτήματα ,δ) μελέτη της επίδρασης και της απόδοσης διαφόρων τεχνικών κατάτμησης εικόνων μικροσυστοιχιών κουκίδων, τόσο ανωτάτου επιπέδου (state-of-art) όσο και νέων, στην ποσοτικοποίηση γονιδιακών εκφράσεων. Για την πραγματοποίηση των παραπάνω στόχων σχεδιάστηκε και κατασκευάστηκε μια πλήρως δομημένη μεθοδολογία (a complete and robust framework) που περιελάμβανε αλγοριθμους επεξεργασίας και ανάλυσης εικόνας κουκίδων μικροσυστοιχιών Η προτεινόμενη μεθοδολογία ενσωμάτωσε στην ήδη υπάρχουσα αλγοριθμική μεθοδολογία (microarray analysis pipeline) έναν πρωτότυπο συνδυασμό τεχνικών επεξεργασίας και ανάλυσης εικόνας βασισμένο στην εις βάθος ποσοτική έρευνα της επίδρασης του θορύβου στην κατάτμηση κουκίδων (spot segmentation), στην εξαγωγή εντάσεων και στην εξόρυξη δεδομένων (data mining). Επιπρόσθετα, κατά την παρούσα διατριβή προτάθηκαν, κατασκευάστηκαν και αξιολογήθηκαν νέες τεχνικές κατάτμησης εικόνας από μικροσυστοιχές κουκίδων. Η χρησιμότητα των προτεινόμενων μεθοδολογιών αξιολογήθηκε τόσο σε εικονικές (simulated) όσο και σε πραγματικές εικόνες μικροσυστοιχιών κουκίδων.

碩士
國立成功大學
統計學系碩博士班
94
Microarray experiment is a useful way to observe the display for thousands of genes simultaneously. To improve the reliability of dowe-stream data analysis, a quantity to evaluate the quality of microarray chip is established. Motivated by the MA-plot of Dudoit et al. (2002), we develop a statistic E(|I|) to measure the quality of one microarray chip. Simulation study shows that E(|I|) is able to distinguish the chip quality effectively. Furthermore, we suggest one threshold value |I|* for purpose of diagnosis. The quality of one chip is acceptable if the E(|I|) value for the chip is smaller than |I|*. Three sets of microarray chips are presented to illustrate the application of E(|I|), with RT-PCR being used to validate the efficacy of the suggested quality statistic.

The advent of microarray imaging technology has lead to enormous progress in the life sciences by allowing scientists to analyze the expression of thousands of genes at a time. For complementary DNA (cDNA) microarray experiments, the raw data are a pair of red and green channel images corresponding to the treatment and control samples. These images are contaminated by a high level of noise due to the numerous noise sources affecting the image formation. A major challenge of microarray image analysis is the extraction of accurate gene expression measurements from the noisy microarray images. A crucial step in this process is denoising, which consists of reducing the noise in the observed microarray images while preserving the signal information as much as possible. This thesis deals with the problem of developing novel methods for reducing noise in cDNA microarray images for accurate estimation of the gene expression levels. Denoising methods based on the wavelet transform have shown significant success when applied to natural images. However, these methods are not very efficient for reducing noise in cDNA microarray images. An important reason for this is that existing methods are only capable of processing the red and green channel images separately. In doing so. they ignore the signal correlation as well as the noise correlation that exists between the wavelet coefficients of the two channels. The primary objective of this research is to design efficient wavelet-based noise reduction algorithms for cDNA microarray images that take into account these inter-channel dependencies by 'jointly' estimating the noise-free coefficients in both the channels. Denoising algorithms are developed using two types of wavelet transforms, namely, the frequently-used discrete wavelet transform (DWT) and the complex wavelet transform (CWT). The main advantage of using the DWT for denoising is that this transform is computationally very efficient. In order to obtain a better denoising performance for microarray images, however, the CWT is preferred to DWT because the former has good directional selectivity properties that are necessary for better representation of the circular edges of spots. The linear minimum mean squared error and maximum a posteriori estimation techniques are used to develop bivariate estimators for the noise-free coefficients of the two images. These estimators are derived by utilizing appropriate joint probability density functions for the image coefficients as well as the noise coefficients of the two channels. Extensive experimentations are carried out on a large set of cDNA microarray images to evaluate the performance of the proposed denoising methods as compared to the existing ones. Comparisons are made using standard metrics such as the peak signal-to-noise ratio (PSNR) for measuring the amount of noise removed from the pixels of the images, and the mean absolute error for measuring the accuracy of the estimated log-intensity ratios obtained from the denoised version of the images. Results indicate that the proposed denoising methods that are developed specifically for the microarray images do, indeed, lead to more accurate estimation of gene expression levels. Thus, it is expected that the proposed methods will play a significant role in improving the reliability of the results obtained from practical microarray experiments.

碩士
臺北醫學大學
醫學研究所
91
Part I: To Study the Pathological Mechanisms and Markers for Endometriosis Endometriosis is one of the most common gynecological diseases and about 40 % of infertile women have endometriosis. The gonadotropin releasing hormone analog (GnRHa) has been widely used in the treatment of endometriosis for many years. However, the genetic mechanisms and diagnostic marker of endometriosis are still unclear. In this study, the global gene expression profiles in endometric tissues (chocolate cysts) which have been treated with or without GnRHa were analyzed by using cDNA microarray technology. Institutional review board approval was obtained before initiation of this investigation by the Taipei Medical University Hospital (Taipei, Taiwan). Endometric tissues were obtained from endometriosis patient have been treated with or without GnRHa (n=4). The mRNA was extracted for cDNA microarray analysis. The human cDNA microarray system (9,600 genes, including known regulatory genes and expressed sequence tag, EST) with colorimetric detection system was used to identify the differentially expressed genes. The real time Q RT-PCR, Immunohistochemistry and Western blotting were used to confirm the microarray data. According to cDNA microarray analysis, we have identified 75 genes whose expression was down regulated in endometric tissues with GnRHa treatment, and 216 genes were up regulated. The genes related to cell growth (PCNA, CDC2 delta T, and topoisomerase II alpha), cell transformation (pituitary tumor transforming), and cell invasion (Enolase 1 alpha) were highly expressed in the endometric tissues without GnRHa treatment. Immunohistochemistry was used to confirm the cell proliferating marker, PCNA, was decreased following GnRHa treatment. According to the results from microarray, real time Q RT-PCR, and Western blotting, we defined that Enolase 1 alpha, Keratin 19, pituitary tumor-transforming gene (PTTG), and H-cadherin were consisted expressed differentially in endometriotic tissues, PF, and/or serum from patients with or without GnRHa treatment. To identify these differentially expressed genes globally by microarray add to our understanding of the pathological mechanisms about endometriosis- induced infertility and might provide the information to find the diagnostic markers or therapeutic targets for endometriosis. Part II: To Study the Gene Regulation during Blastocyst Hatching Blastocyst hatching process is very important for implantation during early embryo development. Several factors are highly regulated and cross-talked with maternal endometrium during this stage. Previous studies were focused on the endometrium and relatively little known about blastocysts. In this study, the global gene expression profiles in blastocysts before and after hatching were analyzed and compared by integrating the technologies of T7-based RNA amplification (in vitro transcription) and cDNA microarray. ICR-mouse embryos (unhatched and hatched blastocysts) were collected for RNA extraction (twenty-five blastocysts were used in each group in the triplicate experiments). The RNA was amplified by in vitro transcription for microarray analysis. The mouse cDNA microarray system (6,144 genes, including known regulatory genes and expressed sequence tags, ESTs) with colorimetric detection system was used. Real time Q RT-PCR was used to confirm the gene expression differences. According to cDNA microarray analysis, we have identified 2,087 genes were detectable during blastocyst stage, 13 genes whose expression was higher in pre-hatched blastocyst, and 85 genes were higher in hatching stage. The differentially expressed genes were further grouped into categories by their putative functions, including: cell adhesion molecules, hormones/cytokines, immuno-regulators, extracellular matrix and related enzymes, and some ESTs. The different expressed ratio greater than 2.5-folds (including, INF- receptor-2, IL-4 receptor, IL-7 receptor, neurotrophin-3, copine III, type II keratin, and some ESTs) were selected to confirm their mRNA expression levels from early blastocyst stage to hatched blastocyst stage by real time RT-PCR. We find that copine III was up-regulated in hatching stage and was reported to be capable of interacting with MEK1 and the CDC42 regulated kinase, which indicate that copine III may be involved in cell division and growth in the hatching stage of blastocyst. Using immuno-staining with specific antibodies, IL-4 receptor is highly expressed in hatching blastocyst which may regulate the immunoresponse and blastocysts growth during implantation. This work adds to our understanding in the mechanisms of blastocyst hatching and provides the information for studying the cross-talk of blastocyst and endometrium by reporting the global gene expression profiles of blastocyst during hatching process.

碩士
臺北醫學大學
醫學資訊研究所
92
cDNA microarray is widely used to identify and quantify different gene-expression for large-scale analysis. In order to extract microarray data precisely from microarray images, robust image processing is indispensable. Most microarray image processing methods are still semi-automatic because of the variations between different arrayers and spots properties. Besides, in a research always use many cDNA microarrays, and a cDNA microarray allow the monitoring of expressions for tens of thousands of genes simultaneously.An automatic method to resolve these cDNA microarray images quickly and accurately is very important. This study attempts to propose an automatic spot detection method using mathematical morphology technology. Watershed transform is the main technique for our spot automatic detection method. Furthermore, Radon transform is used to correct the oblique images and projection technique is used to find the spot grids. To avoid the over-segment problem brought on the sensitive to noises of gradient image used in watershed algorithm, internal and external markers for allocating watershed immersion positions were also used to reduce the number of image segments. Two methods were tested in this research. The first method was designed as follows: first, find the spot grids; then, segment spot from a grid; last, calculate foreground and background intensity. The second method was designed to segment fluorescent region from image directly. Next, calculate foreground and background intensity of every segment regiong. Last, compare every segment region with spot grids to screen out non-spot regions. We compare our experimental results with those obtained from the popular software ScanAlyze and GenePix. Correlation coefficient and linear-regression statistical methods where employed to illustrate the relationship between our methods’ foreground intensity and popular software foreground intensity. Paired-samples T test was used to test the difference between our methods’ background intensity and popular software background intensity. The result showed that the foreground intensities of our methods and popular software have high correlation and the regression model and the coefficient of determination between our methods and popular software reached good suitability, especially in the second method. The background intensities of our methods were really lower than popular software background intensities. We present fully automatic methods for microarray image processing. It showed that our methods achieved automatic spot detection on non-supervised environment. The advantage of our methods is that it does not have any restrictions for the shape of spots. The methods automatically locate subarray grids, individual spots, and calculate distance between spots or blocks requiring no user identification of any image parameters.

碩士
國立成功大學
統計學系碩博士班
91
Microarray experiment involves many steps, and for each step there may exist systematic variation. In order to making correct decision, this systematic bias has to be identified and be removed before analyzing data. Based on the concept of lowess normalization process suggested by Dudoit et al. (2001), a more appropriate normalization method would be presented in this thesis to deal with genes with replicate expressions. Simulation study shows that the performance of the suggested normalization method is superior to the available approaches. We use a microarray data for bladder cancer supplied by National Cheng Kung University Hospital to illustrate the application, and use real-time PCR to evaluate the performance of our approach.

碩士
國立陽明大學
醫學生物技術研究所
90
The hepatitis B virus X protein (HBx) is thought to be involved in the development of hepatocellular carcinoma (HCC) , but its exact role in HCC remains unclear. HBx mainly acts as a transcriptional transactivator involving in multiple gene deregulations. To identify additional genes that may be affected by the HBx expression, we used an established inducible system HepG2-3x in which the expression of HBx in HepG2 cell line is inducible by the removal of doxycycline. We then performed a cDNA microarray analysis on an array (As-chip-TCL-03-Vo) contains 759 gene— specific DNA fragments. When comparing gene patterns that were differentially expressed in the absence or presence of HBx, many cellular genes that are involved in cell cycle regulators, apoptosis, oncogenes, signaling transducers and other cellular functions were observed. Some selected genes were chosen for further study by RT-PCR analysis. Our results indicate that the expression of p21, caspase-3 and FAST kinase was up regulated by HBx which was consistent with microarray data, whereas the expression of Bcl-2 remained unchanged. We also examined the protein expression of p21 and caspase-3 by western blotting, and it showed that both proteins were slightly elevated by HBx expression. Furthermore, we found that HBx enhanced the induction of caspase-3 and FAST kinase under UV irradiation, yet the induction of p21 was not apparent. These results suggest that HBx may play a pro-apoptotic role in HepG2 cell. The mechanism that HBx may mediate apoptosis and its role in pathogenesis of HCC remained to be determined.

Ziel der Arbeit war die Erstellung eines „Kaliumkanal-Chips“, die Entwicklung einer geeigneten Messmethode und Auswertungsstrategie, die Durchführung von Testmessungen und die Untersuchung eines Knockout-Mausstammes auf den Genexpressionsstatus und die auftretenden Kompensationsmechanismen. Am Beginn der Arbeit stand vor allem die Auswahl der zu untersuchenden Kaliumkanal-Gene und die Sammlung von Sequenz-Informationen. Ausgehend davon konnte die cDNAMicroarray-Technologie als Methode der Wahl bestimmt werden und die entsprechenden Vorbereitungen für die Umsetzung getroffen werden. Die ersten Messungen im Zuge der Methodenentwicklungen zeigten vor allem, dass jeder Microarray seine individuellen Probleme mit sich bringt, ließen jedoch auch schon erahnen, welche umfangreichen Möglichkeiten diese Technologie bietet. Dann folgten Versuchsmessreihen, wie die Untersuchung der lterspezifischen Expression und der Vergleich von bestimmten Gehirnabschnitten mit dem Gesamtgehirn. Den Abschluss bildete die Messung der TRESK-Knockout-Mauslinie im Vergleich zu ihrem Wildtyp. Hier stand die Frage nach möglichen Kompensationsmechanismen im Vordergrund. Mit kcnk16 haben die Messungen einen interessanten Kandidaten aus der gleichen Genfamilie geliefert, dessen Funktion und Kompensationsvermögen nun in weiteren Tests zu untersuchen ist. Die Arbeit hat gezeigt, dass der Einsatz der Microarray-Technologie zur Untersuchung von Genexpressionsdaten bei Ionenkanalfamilien geeignet ist. Das Fundament der Microarrayanalyse von Kaliumkanälen mit einem individuell entwickelten Microarray ist zum einen das Wissen um Genetik und Funktion der Kaliumkanäle und zum anderen die Technologie, die eine solche Analyse möglich macht. Die Tatsache, dass Säugerorganismen wie Maus und Mensch eine solch hohe Zahl an Kaliumkanälen entwickelt haben und im ständigen Zellstoffwechsel in umfassender Form einsetzen, zeigt die Bedeutung dieser Ionenkanalfamilie und macht die Forschung an diesen Kanälen so interessant und wichtig für die medizinische Grundlagenforschung. Eine Vielzahl von Krankheiten kann schon jetzt direkt oder indirekt auf Gendefekte bei Kaliumkanal-Genen zurückgeführt werden. Mit der Microarray-Analyse steht nun eine Technologie zu Verfügung, die es ermöglicht, die Expression dieser Gene direkt zu untersuchen und mögliche Kompensationsvorgänge aufzudecken. Damit können Zusammenhänge ermittelt werden, die die Grundlage für weitere Forschungen sein können, mit deren Hilfe wir Krankheiten wie Depression eines Tages wirklich verstehen und behandeln können
The aims of this dissertation were the creation of a „potassium channel chip“, the development of adequate measurement method and strategy of analysis, the performance of developmental experiments and the analysis of the status of genexpression and the occurring mechanisms of compensation in a knockout mouse stem. In beginning the selection of the potassium channel genes to be considered as interesting part of the microarray and the compilation of the sequence information was the main part of the work. Starting from this the choice of the adequate cDNA-microarray-technology and the preparation of the implementation was possible. The first experiments performed in the course of the method development have given a hint on the problems connected with every microarray. However they also have shown the great possibilities of the microarray technology. In the ollowing series of measurements like the investigation of variation of expression during the juvenile development and the comparison of different parts of the brain with the whole brain were performed. The studies were completed by the investigation of the TRESK-Knockout mouse stem in comparison to its wild type. The centre of these studies was the question for possible mechanisms of compensation. As a result kcnk16 - being part of the same gene family as TRESK - can be named as an interesting candidate to be investigated for its function and capacity of compensation in the future. In my dissertation I was able to show that the microarray technology is an adequate method for the comparison of genexpression between members of ion channel families. The bases of the microarray analysis of potassium channels with a individually designed microarray are on the one side the knowledge of the genetics and function of the potassium channels and on the other side the technology which allows this kind of analysis. The fact that mammalian organism like mouse and human have developed such a great number of potassium channels and are using these in the regular cell metabolism in a comprehensive way shows the importance of this ion channel family and makes the research on these channels so interesting and important for fundamental research. A multiplicity of diseases can be attributed indirectly or directly to gene malfunctions in potassium channels. With microarray a technology is available, which permits to investigate the expression of these genes directly and to discover possible ways of compensation. By this coherences can be identified being the basis for continuative research which one day will make it possible to really understand and treat diseases like depression

碩士
國立成功大學
統計學系碩博士班
95
With the prominent advance of microarray technology, people can observe thousands of gene expressed values simultaneously. To have valid results, quality control on array data becomes a necessary and important step before analysis is performed. In this thesis, motivated by the M-A plot of Dudoit et al. (2002), quality indexes, β1、σR'/σG'and ρR'G'are proposed to assess the array quality. Simulation study shows that σR'/σG'and ρR'G'both have large influence on array quality. In practice, theses arrays with high σR'/σG'and low ρR'G'could be considered as outliers and be excluded from further analysis. Through the two color model suggested by Rocke and Durbin (2001), we find out that σ2ηT and σ2ηC, the multiplicative error terms unique to control and treatment group, affect σR'/σG'and ρR'G'the most, and hence the gene expressed value. People can improve the quality of microarray data by controlling σ2ηT and σ2ηC.

碩士
國立清華大學
生醫工程與環境科學系
96
AbstractcDNA microarray contains densely packed spots with known DNA sequences. The deposited DNA sample is referred to as the probe. After the fluorescently-labeled target DNA is hybridized to its complementary probe strand, the microarray was scanned to measure the intensity of the fluorescent signals on each spot. The intensity of each spot represents the expression level of a specific gene. However, the DNA distribution within each spot will influence the analysis of the fluorescent signals. This research aims at applying the spectral images technology and other analytical methods to study the distribution of DNA within the probe spot and analyze the cause of its irregular distribution. The spectral-scanning system was applied in this study to scan the spectrum of probes labeled with Cy3 dye on the cDNA microarray. The characteristics of spectrum were used to distinguish the fluorescence, which may overlap with that of Cy3, emitted from the other materials on the cDNA microarray. SSC, which was added in the DNA spotting solution, do not produce fluorescent signal, therefore cannot be detected by the fluorescence and spectral imaging systems. The non- fluorescence morphologic image acquired by EMCCD camera represents the distribution of crystal, after the spotting solution was dry out. We compared the morphologic and fluorescent images of each spot. The pixel correlation between the distribution of DNA and crystal is higher than 0.9. Furthermore, data reveals that rehydration and surface blocking also affected the distribution of crystal and Cy3-labeled DNA on the glass surface. When microarray slides were processed by rehydration followed by snap-heating, UV cross-linking and baking, the SSC and DNA crystal diffused outward and crystallized out DNA on its surface. DNA molecules tend to gather inward, thus the spots become more rounded, enlarged and the DNA is more evenly distributed. After the surface blocking and washing, DNA molecules and SSC crystal that were loosely attached to the glass surface were washed away. Therefore, the distribution of DNA became less homogenized. The degree of homogenization of DNA distribution on the glass surface affects the reliability of cDNA microarray results.

碩士
國立臺灣大學
生物產業機電工程學研究所
94
In this research, we used circular Hough transform to be the core method to search circular spots in microarray images. Due to high sensitivity of circular Hough transform to noises in an image, noise removal plays a very important role in image pre-processing. At first, we used histogram equalization to enhance the contrast of images before image thresholding. As a result, it is easier to separate foreground pixels from the background using the Otsu’s method to find the best threshold value. Following image binarization, we used blob algorithm to filter noise pixels. Then we developed grids by vertical and horizontal histogram projections to determine the possible area of every spot in an image. The binary image was further processed with the Sobel operator to obtain the edge image that was further processed with the circular Hough transform to determine the position and boundary of each spot. The determination of circular spots was based on the number of pixels in the accumulating cells in the parameter space. To reduce computation complexity, it is important to avoid identification errors by decreasing redundant accumulations. Using the gradient angle information in an edge image, we were able to use only half circle, +90 and -90 degrees between gradient angles, in the circular Hough transform. With this approach, we can not only reduce the influences of noises but also improve the accuracy of spot identification. The computation time was also reduced in half. After identifying the spots, the intensities of foreground and background pixels were used in the subsequent statistical analysis of microarray data. Comparing the performance of SPOTCapture software developed in this research with the commercialized software GenePix Pro 6.0, SPOTCapture has the precision rate of 98.6% and the recall rate of 98.3% while the precision rate and recall rate of GenePix Pro 6.0 was 97.5% and 97.9, respectively. The performance difference between these two approaches was statistically significant as the results were tested with the chi-square test. As for the determination of position and area of spots in a microarray image, SPOTCapture has a higher accuracy than the GenePix Pro 6.0 by 2.4%. Our method is also sensitive in resolving the problems of donut spots frequently occurred in microarray images. In addition, the parameter settings of our method are less complicated and require less manual intervention. Thus, with all these advantages, the SPOTCapture can be used as an efficient platform for the analyses of microarray images.

碩士
國立清華大學
生醫工程與環境科學系
94
ABSTRACT The biological effects of microwave(300MHz~300GHz) have remained contentious. This study considers the subject from the following two aspects: (1) the non-thermal effects of microwave, (2) the interference of non-thermal effects from microwave on cell repair process. We used two biological experiments colony assay and cDNA microarray to explain cell proliferation and gene expression after 2.45GHz, 4mW/cm2 microwave exposure at 37°C. Firstly, the colony assay experiment showed that cell proliferation decreased significantly after cell was exposed under microwave for 6 hours. The finding of cDNA microarray study indicated that 82 gene altered their gene expression after microwave exposure. Their major biological functions related to protein folding, phosphorylation, cell cycle, DNA replication, and nucleotide biosynthesis. Secondly, the study of colony assay revealed that microwave could interfere with cell repair process and thus decrease cell survival rate after damaged by UVB. Besides, the cDNA microarray study presented that 104 genes, which mostly command the genomic functions of regulation of transcription, phosphorylation, proteolysis, and metal ion transport were affected. Judging from the above, the non-thermal effects of microwave can influence cell proliferation and regulate parts of genomic functions.